Absiracts FEBS'99

sl 1

The Sir Hans Krebs Lecture

Structural and transgenetic studies on the mechanism
of prion formation
S. B. Prusiner
Institute for Neurodegenerative Diseases.
University o f California, San Francisco. CA 94143, USA
Prions cause neurodegenerative diseases including Creutzfeldt-Jacob disease
(CJD) of humans as well as bovine spongiform encephalopathy (BSE) and
scrapie of animals. The human prion diseases may be manifest as sporadic,
genetic or infectious disorders. More than 20 different mutations of the PrP
gene have been identified. A redacted prion protein (PrP) of 106 amino acids
with two large deletions was expressed in transgenic (Tg) mice deficient for
wild-type (wt) PrP (Prnp '~) and supported prion propagation. PrPI06 is
composed of PrP residues 89-140 and 177-231. RML prions containing fulllength PrPs~ produced disease in Tg(PrPl06)Pmp j mice after ~300 days
while transmission of RML(106) prions containing PrPs~106 created disease
in Tg(PrP106)Prnp ~ mice after only -66 days on repeated passage. This
artificial transmission barrier for the passage ofRML prions was diminished
by the coexpression of wt MoPrP in Tg(Prpl06)Prnp+/ mice that
developed serapie in ~165 days, suggesting that wt MoPrP acts in trans to
accelerate replication of RML(106) prions. The brains of Tg(PrP 106)Prnp/
mice infected with RML(106) prions showed spongiform degeneration,
astrocytic gliosis and extreme loss of pyramidal ceils in the CAI region of
the hippocampus. Purified prpSCl06 was protease-resistant, formed
filaments and was insoluble in non-denaturating detergents. Under conditions
that favor a-helix formation for longer PrP sequences, purified recombinant
PrPI06 derived from E.coli refolded into oligomers rich in 13-sheets
suggesting that it adopt a mctastable structure. The unique feature of
RML(106) prions offer new insights into the mechanism of prion
replication, and the small size of prpS~106 should facilitate structural
analysis. Determining the structure of PrP sc and the mechanism of prion
replication have wide-reachingimplications for understanding the plasticity
of protein conformation as well as development of effective
pharmacotherapeuties.

Etudes structurales et transg~n~tiques du m~canisme

de formation des prions
S. B. Prusiner
Institutefor Neurodegenerattvediseases, Universtty of California,
San Francisco. CA 94193, USA
Les prions provoquent des maladies neurod6g6n6ratives incluant chez
l'homme les maladies de Creutzfeld-Jacob (CJD) et chez l'animal,
l'enc6phalopathie spongiforme bovine (BSE) et la tremblante du mouton. Les
maladies ~ prion humaines peuvent se manifester par des troubles
sporadiques, g6n6tiques ou infectieux. Plus de 20 mutations diff6rentes du
g~ne PrP ont 6t6 identifi6es. Une prot6ine prion (PrP) de taille r6duite de 106
acides amin6s pr6sentant deux grandes d616tions a 6t6 exprim6e chez des souris
transg6niques (Tg) d6ficientes pour la PrP (wt) sauvage (Prnp/). Cette
prot6ine tronqu6e permet la propagation du prion. PrPl06 est compos6 des
r6sidus 89-140 et 177-231. Les prions de souche RML contenant la PrPsc de
taille normale provoquent la maladie chez les souris Tg(Prp106)Prnp : apr6s
environ 300jours, alors que l'infection, en passages suecessifs, par des prions
RML(106) contenant la prWCl06 donne la maladieapr6s seulement 66 jours
environ. Cette barri~re de transmission artificiellepour le passage des prions
RML est diminu6e par la coexpression de la MoPrP c sauvage dans des souris
Tg(PrP106)Prnp +/, qui d6veloppent la maladie en 165 jours environ. Ceci
suggSre que wt MoPrP agit cntrans pour acc616rer la r6plication des prions
RML106. Le cerveau des souris Tg(PrP106)Prnp / infect6es avec les prions
RML(106) pr6sente une d6g6ndration spongiforme, une gliose astrocytaire et
une perle importante des cellules pyramidales dans la r6gion CAI de
l'hippocampe, prpS106 rSsiste aux prot6ases, forme des filaments et est
insoluble dans des ddtergents non d6naturants. Dans des conditions favorisant
la formation d'h61ices-a pour des s6quences de PrP plus longues, la PrP106
purifi6e d6riv6e d'E. coli se replie en oligom~res riches en feuillets-b sugg6rant
que la prot6ine adopte une structure m6tastable. Cette caract6ristique unique
de RML(106) ouvre de nouvelles perspectives pour l'6tude des m6canismes
de replication du prion. Par ailleurs, la petite taille de prpSCl06 devrait faciliter
des 6tudes structurales. La d6termination de la structure de PrP~ et des
m6canismes de replication du prion peut avoir de larges implications pour
l'6tude de la plasticit6 de la conformation des prot6ines et pour le
ddveloppement d'une th6rapeutique efficace.

The IUBMB Lecture

Genetic analysis of cell cycle regulators
Mariano Barbacid
Centro Nacional de Investigaciones Oncol6gicas Carlos III
28220 Majadahonda, Madrid, Spain
Normal cell growth and differentiation requires precise control of the
mechanisms that regulate the entry, progression and exit from the cell
cycle. Most of our knowledge regarding cell cycle regulation has been
obtained from either unicellular organisms or mammalian cells in culture.
To understand the role that cell cycle regulators play in mammals, we
have targeted the pl5INK4b and pI8INK4c genes in ES cells and
generated the corresponding null mice. Mice defective for pl5lNK4b
develop normally, although they exhibit extrarnedullary hematopoiesis
and lymphoid follicular hyperplasia suggesting that pl5lNK4b plays a
role in maintaining cellular homeostasis in certain tissues. Older animals
also display a modest increase incidence of tumors. Mice lacking
p 181NK4c, albeit viable, consistently develop pituitary tumors basically
indistinguishable from those observed in Rb (+/-) and p27Kipl (-/-)
mutant mice, thus suggesting that Rb is independently controlled by
specific members of the INK and Cip families of cell cycle inhibitors.
We have also manipulated the Cdk4 locus to generate two additional
strains of mice that either do not express Cdk4 [cdk4 (neo/neo) mice] or
express a mutant Cdk4 protein (Cdk4 R24C) that cannot bind INK4
regulatory proteins. Mice devoid of Cdk4 expression are viable, but small
in size and infertile. The sterility in cdk4 (neo/neo) male mice is due to a
block in late spermatogenesis. Female sterility is due to a defect in the
formation of the corpus luteum. Cdk4 (neo/neo) mice also develop
insulin-dependent diabetes due to a dramatic reduction in beta islet
pancreatic cells. Mice expressing the mutant Cdk4 R24C protein are
viable and fertile. These mice display hyperproliferative abnormalities,
including the formation of tumors in some of the cell types affected by
the lack of Cdk4 expression including testicular Leydig cells and beta
pancreatic cells. These results illustrate the distinct roles that cell cycle
regulators play in vivo and how their mutation results in abnormal
proliferation that leads, at least in some cases, to the formation of tumors.

The EMBO Lecture

Lipid rails in membrane traffickbrg and signalling
Km Simon-,. EMBL. Meyerhofstr 1. 69117 tte~delbcLg.
Gernlan) and MPI for Molectllar Cell Biology dnd
Genetic,,. Dresden. Gernlanv
From our work on polarized sorting and dehvery of proteins and lipid, to
the cell surface in epithelial cells we have found that sphingolipids and
cholesterol form tightly packed assemblies that we call lipid rafts. These
lipid rafts form platforms that carry proteins to different destinations m
the cell. These platforms function in the delivery of proteins to the apmal
and the axonal membrane,; m epithehal cells and neurons, respect:ely.
Also fibmblasts have a raft pathway flom the trans-Golgi network to the
plasma membrane. Not only do the lipid rafts function in mcmblanc
traffic m tile cell but also as dynamic de',.ices to microcompartmenlalize
signal transductmn proces,,es, for instance, in T cell signalhng. Our
studie~ thus support a new model for membrane organisation In cell
membranes, in which dynamic assemblies of sphingohpids and
cholesterol float around m the fltud bilayer. The main functiort of
cholesterol m cells may be to dynan/ically glue together raft hpid,, and
proteins m [LlnCtion in nltracelltlldr ~.nrtltlg ;and transport as ,,',ell as in
signal transductlon

s12

Abstracts FEBS'99

The Theodor Bticher Lecture

Knowing the genes, how do plantbiochemists
proceed
M.C. Van Montagu
Department of Genetics
Flanders{ Interuniversity Institutefor Biotechnology
University of Gent
9000 Gent, Belgium

The ongoing doubling of the human population carries with it the inevitable
consequence of large scale global pollution and a dramatic irreversible loss of
natural territories. While looking at bow to stop this overpopulation, society
demands scientific and technological achievements so that the environmental
disasters could be limited, the food production secured and the industrial
production procedures less polluting.
Novelplants obtained through molecular marker assisted selection and
transgenesis have great potential.
Plant genome research helped to identify the 21.000 plant genes. Improved
methods for mRNA profiling and proteomics are bringing information on the
genes used in the different tissues and during certain developmental and stress
situations.
Metabolite profiling of solutes and volatiles can bring information on the
changes of enzymatic activities during altering growth conditions.
The improved analytical organic chemistry (GC-MS, CE-MS, HPLC-MS)
linked to mRNA profiling must make it possible to unravel the biosynthetic
pathway for plant secondary metabolites.
Engineering some key genes in related plant species can allow the
development of a less expensive medicare

Symposia

Abstracts FEBS'99

s15

1.1 New genomes

The yeast genome and multidrug resistance
A. Goffeau
Universitd de Louvain, Unit~de Biochimie Physiologique,
Place Croix du Sud 2-20, B-1348 Louvain-la-Neuve, Belgium

GENE EXPRESSION ANALYSIS OF THE BACTERIUM

MYCOPLASMA PNEUMONIAE

R. HERRMANN, H.W.H. GI3HLMANN,J.T. REGULA,

B. UEBERLE, J. WEINER III and R. FRANK
ZMBH, UniversitditHetdelberg, 1NF282. 69120 Heidelberg

In the set of the 6000 yeast proteins, called the proteome, we submitted
2~300 putative membrane proteins to binary sequence comparisons and
phylogenetic analysis. This approach identified more than 50 distinct yeast
transporter families comprising a total of 258 individual yeast transporters.
Two families, DHAI4 and DHA12, containing respectively 10 and 14 yeast
members are possibly involved in drug efflux by antiporter mechanisms.
Several of these "in silico" predictions were confirmed experimentally by
multidrug sensitivity conferred by deletions of members of these families. In
addition, X cerevisiae contains a complex pleitropic drug resistance (PDR)
network of genes controlled by the transcriptional regulators Pdrlp and
Pdr3p, which activate expression of the "ATP-binding cassette
transporters"-encoding genes PDR5, SNQ2, and YOR1 as well as other
genes. We have screened 349 toxic compounds in isogenic S cerevisiae
strains deleted of PDRS. SNQ2, or YOR1 in different combinations as well
as both PDR1 and PDR3. The screen revealed extremely promiscuous, yet
limited, and to a large extent overlapping, but distinct, drug resistance
profiles of Pdr5p, Snq2p, and Yorlp.
These transporters mediated
resistance to most currently available classes of clinically and agriculturally
important fungicides and also to many antibiotics, herbicides, and others.
Scvcral new classes of compounds were identified in the drug resistance
spectrum of these PDR5, SNQ2 and YOR1 transporters. Using genomic
microarray (collaboration with J. DeRisi and P. Brown. Stanford University)
analysis in combination with genetic and bioinlbrmatic tools, we identified
all transcriptional targets of the two major yeast multidrug resistance
regulators. Pdrlp and Pdr3p. Our analysis points out to a major role of the
transcription I5.ctors Pdrlp, in the defence of yeast cells against hydrophobic
toxic chemicals. Pdr3p has similar but partly different targets Global
resistance seems to result from parallel activations by the pdrl-3 and pdr3-7
mutations of a remarkably diversified, but largely coherent, set of
determinants most of which control multidrug transport across the plasma
membrane.

Mycobacterial genomics
S.T. Cole
Untt~ de G~n~tiqueMol~culaweBact~rienne. lnstitut Pca~teur,
28 rue du Docteur Rout. v5724 Parts Cedex 15. Fram'e

We have chosen the human pathogenic bacterium Mycoplasma pneumoniae,

the causative agent of an atypical pneumonia as a model organism to
characterize and describe a minimal self-replicating cell. The genome of N(
pneumoniae has been completely sequenced. It has a size of 816 kbp and a
coding capacity for 677 open reading frames and 39 RNA coding genes.
The sequencing data and their annotations did not provide much information
about gene expression and its regulation, but it was obvious that the number
of proteins with gene expression regulating functions was low. To
understand the mechanisms, which regulate the ordered expression of all
bacterial genes we started an analysis of all transcription (transcriptome) and
translation (protenme) products.
For the transcriptome analysis nylon membranes were prepared with
immobilized gene specific PCR products covering the complete genome.
Cross hybridization experiments with 33p labeled single stranded cDNA
from total mycoplasmal RNA provided the basis for generating transcription
profiles of M. p n e u m o n i a e cells which were grown under different
temperatures. The profiles showed significant differences concerning the
total number of transcripts and the relative number of copies from individual
genes. Since the final functional gene products are usually the proteins a
transcription analysis has to be complemented by a proteome analysis. The
essentials for a proteome analysis are the two-dimensional gel
electrophnresis for the separation of the proteins, the knowledge of the
protein sequences and mass spectrometry for characterization of the
proteins. We see in silver strained 2D-gels of protein extracts from Mi
pneumoniae about 400 proteins, so far, 100 proteins were identified by mass
spectrometry and correlated to the corresponding genes. A comparison
between transcriptome and proteome analysis revealed that a high copy
number of transcripts do not always correlate with a strong protein signal
and vice versa.

The Arabidopsis thaliana genome

and its functional analysis
M. Caboche, D. Bouchez, H. H6fle
INRA, Laboratotre de Biologte ('elhdatre. I ersatlles. France

Mycobacterium tuberculosis, the scourge of humanity, is one of the

most successful and scientifically challenging pathogens of all
time. To catalyze the conception of new prophylactic and
therapeutic interventions against tuberculosis, and to enhance our
understanding of the biology of the tubercle bacillus, the complete
genome sequence of the most widely used strain, H37Rv, has been
determined. Bioinformatic analysis led to the identification of
-.-4,000 genes in the 4.41 Mb genome sequence and provided fresh
insight into the biochemistry, physiology, genetics and
immunology of this much-feared bacterium. Genomic information
is centralized in the relational database, TubercuList, that can be
consulted via the world-wide-web
(http://www.pasteur.fr/Bio/TubereuList/).In parallel, the genome
sequence of the related leprosy bacillus, Mycobacterium leprae, is
currently entering the finishing phase. Comparisons of the two
sequences are mutually enriching and have uncovered numerous
pseudogenes and extensive genome decay in M. leprae.

Arabidopsis has been selected as a model for the analysis of the structure
and organization of plant genomes. The short reproductive cycle autogamy
and diploidy of this species led to the choice of Arabidopsis for genetic
analysis. The size of the Arabidopsis genome (130 Mb) and the relatively
low proportion of repeated sequences were also favorable to the molecular
analysis of this genome. A dense RFLP map, the coverage of the genome
with YAC and BAC clones paved the way to the complete sequencing of
this genome. From sequences data approximatively half of the genes can be
assigned a putative function. Yet the exact function of the majority of genes
remains to be discovered. Insertional mutagenesis with transposable
elements or with T-DNA is the major route to functional analysis.
Identification of knockouts in genes of potential interest can now be
achieved at a reasonable efficiency. The potential of these genomics tools for
the identification of new genes families will be illustrated.

s16

Abstracts FEBS'99

An analysis of a genetically characterised 3-megabase

region of the genome of Drosophila.
M. Ashburner (~), S. Misra (2), j. Roote m, S. Lewis ~2>
G.M. Rubin (~'~)
in Department of Genetics, Universirty of Cambridge, England," f2)Berkeley
Drosophila Genome Project, Department of Molecular & Cell Biology,
University of California. Berkeley. CA; ~3)Howard Hughes Medical
Institute, Life Sciences Annex, Unb~ersity of California, Berkeley. CA.
We have analysed a 3-rob contig from Drosophila melanogaster. This contig
covers a region of the Drosophila genome that has been subjected to a very
detailed genetic analysis. This analysis identified 73 genes, of which mutant
alleles have been recovered for 65. Analysis of the sequence predicts the
existence of 234 genes, of which 223 are protein coding (the other 11 are
tRNAs). This suggests that about one-third of Drosophila genes can be detected
by mutant phenotypes. Of the 73 known genes, 49 have at least one lethal allele,
suggesting that just under 25% of Drosophila genes are essential for viability. To
correlate the genetic and sequence maps we have used a variety of methods matches with previously sequenced genes, mapping the insertion sites of Pelements and correlating the sequence map and the breakpoints of genetically
characterized chromosome aberrations. These studies allowed 52 of the 73
known genes to be linked to the sequence.
About one-third of the protein coding genes have ESTs from the
HHMUBDGP EST project. ESTs hit genes known prior to this study more often
(71%) than they do genes newly discovered (26%), suggesting either a bias in
the sample of genes already studied or an over-prediction of known genes.
The density of genes varies greatly through the 3-mb region, but overall it is
one protein coding gene every 13-kb. In a study of about 2-mb of sequence from
the tip of the X chromosome the European Drosophila Genome Project has
estimated an average gene density of one gene in about 8-kb. Reasons for this
difference are not known, but will be discussed. About 13% of the protein
coding genes are in small tandem arrays of 2-3 genes. The sequenced region
covers 69 polytene chromosome bands, suggesting an average DNA content per
band of about 40-kb, higher than had previously been estimated.
Sixteen transposable elements have been identified in this region, a density of
one per 180-kb. A similar density is found at the X tip by the EDGP.
Some inference concerning the function or structure of gene products can be
made for 135 of the 223 protein coding genes (61%). 130 genes have
"significant" matches to protein sequences from humans or mouse (58%), 123 to
C. elegans (55%) and 96 to S. cerevisiae (43%).

Abstracts FEB S' 99

s 17

1.2 Genome analysis and evolution

The genome sequence of Rickettsia prowazekii and the
origin of mitochondria
S.G.E. Andersson and C.G. Kurland
Uppsala University. EBC, Molecular Evolunon. Uppsala. Sweden

The endosymbiotic theory suggests that mitochondria originate from a

eubacterial endosymbiont that became established in an early version of the
eukaryotic cell. Gene sequence data indicates that mitochondria are derived
from the ot-Proteobacteria, and more specifically from an ancestor of the
Rickettsia. In order to answer fundamental questions concerning the origin of
mitoehondria, it is essential that the most highly conserved mitochondrial
genomes are compared to their closest bacterial relatives. Here, we present
such an analysis.
Rickettsia prowazekii is an obligate intracellular parasite and the causative
agent of epidemic typhus. Comparative analyses of the 1.0 Mb genome of R.
prowazekii with minimally diverged mitochondrial genomes have provided
evidence for a shared evolutionary history. For example, there are striking
similarities in their bioenergetic profiles, and a close evolutionary relationship
is suggested from phylogenetic reconstructions based on the individual
components of these systems. However, the genomes o f Rickettsia and
mitochondria appear to have undergone drastic reductions in genome sizes
independently of each other. Most importantly, the identification of several
pseudogenes in the Rickettsia genome suggests.that gene deterioration is an
ongoing process in Rickettsia. Indeed, as much as one quarter of the Rickettsia
genome is noncoding DNA, which may represent mutationally destroyed
genes that have not yet been completely purged from the genome.
Comparative analyses of pseudogenes in several Rickettsia species has
allowed us to follow the step-by-step process of gene disappearance. In order
to study less diverged o~-Proteobacterial relatives of mitochondria, we are
currently sequencing the 2.0 Mb genome of Bartonella henselae, the causative
agent of cat-scratch disease. At the time of this writing, 97% of the
sequencing is done. A comparison of the genomes of Rickettsia and Bartonella
will be presented. Taken together, our data provide strong support for the
hypothesis that the bioenergetic system in mitochondria was derived from an
ancestor of ~-Proteobacteria.
Application of oligonucleotide and DNA microarrays
in genome studies
A. D. Mirzabekov
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences,
Vavilov str. 32, Moscow 117984, Russia - Argonne National Laboratory,
9700 South Cass. Ave., Argonne, IL 60439-4833, USA

Development and application of oligonucleotide and DNA microarrays is a

fast growing field. Our group has been developing the MicroAxray of Gel
Immobilized Compounds on a Chip (MAGIChiprra). The chip consists of the
array of gel-pads of the size from 10xl0x5 Ixm to 100xl00x20 ~tm and larger
chemically fixed on a glass slide. Different oligonucleotides, DNA, proteins
(such as antibodies, antigens, enzymes) or other compounds are covalently
bonded within the three-dimensional gel-pads. The gel-pads are spaced by
hydrophobic surface converting them into separate microtest tubes of
picoliters-nanoliters volume. Different specific interactions and chemical and
enzymatic reactions were carried out in parallel within microchip gel-pads.
MAGIChips have been applied to characterize the thermodynamics and
kinetics of the formation of DNA duplexes and triplexes, the effect of
modified bases on the duplex stability and on the specificity of
complementary interactions. This information allows to increase the
reliability of microchip application in hybridization analysis. Massive
collection of data on the specificity of interaction between different ligands
and nucleic acids can be obtained with oligonucleotide microchips in a short
time in comparatively simple experiments. Qualitative and quantitative
studies of mutations and of genome polymorphysm as well as the DNA
sequence analysis were performed with specific oligonucleotide microchips
or with generic microchips containing all possible 4,096 hexadeoxyoligonucleotides.
Enzymatic ligation and DNA polymerase reactions have been demonstrated
on oligonucleotide microchips for further development of PCR amplification
and single base extension analysis of DNA.
Due to their wide versatility, MAGIChips can be applied in many areas of
genome analysis. A number of other applications will be discussed, such as
identification of microorganisms and viruses, gene expression analysis,
detection of toxin and antibiotic resistence genes.

From comparative genomics to the Last Universal Common

Ancestor (LUCA)
P. Forterre
hlstitut de G~nrtique et Microbiologie, UMR CNRS C 8621,
Universit~ Paris-Sud. 91405 Orsav Cedex. France

Complete sequencing of genomes from eukarya, archaea and bacteria, together

with the recent discovery of new unique archaeal enzymes via traditional
biochemical work, have now confirmed the tripartite division of the living
world and the phenotypic coherence of archaea. However, the topology of the
universal tree of life and the nature of Last Universal Common Ancestor
(LUCA) are still matter of debate, and several scenarios compete to describe the
early history of life [1,2]. A central problem is raised by the great difference
between DNA replication mechanisms in bacteria on one side, and
eukarya/archaea on the other. I will report our recent identification of replication
origins in archaeal chromosome, which points for a close relationship between
the mechanisms of DNA replication initiation in eukarya and archaea [3]. On
the other hand, comparison between the genomes of two closely related
hyperthermophilic archaea of the genus Pyrococcus examplifies the similarity
of chromosome structure and mechanism of genome evolution in archaea and
bacteria. I will review current competing hypotheses explaining such
observations and propose a new one which involves a crucial role for ancient
viral genes in cellular evolution.
[1] Forterre, P. Current Opinion Gen. Dev., 1997, 764
[2] Forterre, P. and Philippe, H. Bioessay, in press
[3] Lopez et al., Mol. Microbiol. in press

s 18

Abstracts F E B S ' 9 9

1.3 Gene targeting, gene therapy

Gene targeting by triple-helix forming oligonucleotides
C. Giovannangeli
Laboratoire de Biophysique, Museum Nattonal d'Hlsto:re Naturelle,
CNRS UMR 8646, INSERM U201, 75005 Parts, France

Triplex-forming oligonucleotides (TFOs) and some recently

described minor groove binders (small polyamide molecules containing
pyrrole and imidazole amino acids) are at this time the most specific
ligands of the double-stranded DNA (1,2).
Gene transcription can be inhibited in a sequence-specific manner by
oligonucleotides forming triple-helical complexes with DNA . A triplex
formed with a 15-mer complementary of the HIV- 1/PPT double-stranded
DNA sequence is able to physically block the transcriptional machinery
in vitro (IC50 = 0.1/.tM), and ex vivo (IC50 - 2p.M) (3).
A quantitative method has been developped to analyze triplex formation
on genomic DNA in the nuclei of chronically infected cells (containing
HIV proviral DNA integrated in their genome): it is based on the use of
psoralen-oligonucleotide conjugate. A 15 nucleotide long TFO directed
against the PPT sequence of the HIV-1 DNA is able to specifically
recognize and to bind its 15 base pair target present in the nuclei: the PPT
sequence is accessible in chromatin within the supranucleosomal
structure (4).
A triple helix can be formed on a single-stranded RNA by clamp
oligonucleotides. A clamp oligonucleotide targeted to the polypurine tract
of HIV blocks reverse transcription of the viral RNA.

1234-

Antisense Gene Regulation by Peptide Nucleic Acid

(PNA)
P.E. Nielsen
University of Copenhagen, IMBG, Laboratory B, The Panum Institute,
Blegdamsvej 7, DK-2200 Copenhagen N, Denmark

The pseudopeptide DNA mimic PNA (peptide nucleic acid) has many of the
properties desired of an antisense reagent. It binds strongly and with high
sequense specificity to complementary RNA, it has high biostability and it
is easy to chemically synthesize and modify. Results relating to antisense
gene regulation in bacteria as well as improved methods for cellular uptake
and antisense targeting using PNA in eukaryotic cells will be presented.

Giovannangeli, In "Triplex-forming oligonucleotides" Kluwer

Academic Publishers 10 (1999)
Barre F.X.et al Nucleic Acids Res. 27 (1999) 743 :
Giovannangeli et al., J. Mol. Biol. 261 (1996) 386
Giovannangeli et al., Proc. Natl. Acad. Sci. USA 94 (1997) 79

Recent progress towards the use of lentiviral vector

in human gene therapy
D. Trono
Department of Genetics and Microbiology, University of Geneva, C.M.U.,
1 rue MicheI-Servet, CH-1211 Geneva 4, Switzerland.

Lentiviruses are unusual amongst retrovimses in their ability to infect

nondividing cells. For HIV-1, this property reflects the recognition of at least
three components of the preintegration complex by the cell nuclear transport
machinery: matrix (MA), Vpr and integrase (IN). HIVfs ability to infect
nonmitotic cells can be exploited to create retroviral vectors that allow for the in
vivo delivery, integration, and sustained long-term expression of transgenes into
non mitotic cells such as neurons, hepatoeytes, pancreatic islet cells and
hematopoietic stem cells. Several improvements have been brought to the system
as it was first described, in terms of both biosafety and performance. The Vif,
Vpr, Vpu, Env, Nef and Tat proteins, which all represent crucial virulence
factors of HIV-1, as well as the virus-derived transcriptional components were
deleted from the vector system without decreasing its efficiency to transfer genes
in vitro or in vivo. Furthermore, the vector genome was modified by addition of
the post-transcriptional regulatory element of woodchuck hepatitis virus,
resulting in a marked increase in the levels of transgene expression. The high
degree of biosafety and the remarkable efficacy of the new generation of
lentiviral vectors paves the way for their use in human gene therapy.

Self-assembling polymeric vectors for gene delivery

L.W. Seymour~, K.D. Fish&, P.R. Dash~, K. Ulbrich
"Instttute for Cancer Studies, Birmingham Universiiy UK. and
blnstitute of Macromolecular Chemistry. Czech Repubhc.

Self-assembling polymer-based vectors for gene delivery can provide a safer,

better characterised and cheaper alternative to viral delivery strategies,
avoiding the risks implicit in applying professional pathogens for therapy.
However, non-viral systems axe frequently inefficient, lacking specific
tropisms and are easily inactivated by serum. Therefore we have developed a
means of surface-modification of pre-formed cationic polymer/DNA
complexes, using multivalent hydrophilic polymers to crosslink the surface of
the complexes and introduce lateral stabilisation to prevent disruption by
serum proteins. The hydrophilic polymers can also he used to attach targeting
ligands to the surface of the complexes.
Complexes formed by self-assembly of poly(L-lysine) with DNA are allowed
to react with copolymers based on N-(2-hydroxypropyl)methacrylamide
(HPMA) bearing pendent tetrapeptide (Gly-Phe-Leu-Gly) side chains
terminated in reactive paranitrophenyl esters. The reactive esters form amide
bonds to the amino groups of pLL (but not to DNA) and liberate
paranitrophenylate anion. The resulting coated complexes typically show
diameters of 80 - 120 nm, and their surface properties can be influenced by
reaction conditions. They show significantly decreased protein binding in
serum, less non-specific association with cells, and therefore form promising
candidates for targeted delivery of DNA.
Incorporation of targeting agents onto the surface of the coated complexes
leads to enhanced association and intemalisation by cells hearing appropriate
receptors. For example, linkage of basic Fibroblast Growth Factor (bFGF) to
the surface mediates increased uptake in endothelial cells, transformed
fibroblasts and tumour cells, while Vascular Endothelial Growth Factor
(VEGF) promotes uptake into endothelial cells. Careful choice of targeting
ligand can lead to favourable intracellular distn~bution of targeted DNA,
avoiding nucleolytic degradation, and high levels of transgene expression can
result. Importantly, this receptor-mediated transfection system is operative in
the presence of serum. The concept is simple, flexible, and may find several
useful applications in vitro and in vivo.

Abstracts FEBS'99

Polycation-based DNA complexes for tumor-targeted gene

transfer
E. Wagner*'. S. Brunner*. R. Kircheis'. M.
Ogris*. S, Schtiller*, L. Wightman ~
*lnstaute of B~ochemlso'r, I ?enna Unn.ers~O, Btocentel, Dr Bohr,c:;asse 9 3,
ItemTu Boehrmger fiTge[hemt Austria. A-I I2 I l,)enna, MIISIFICt

Recently we have shown that systemic application of optimized surfaceshielded transferrin-polyethylenimine / DNA complexes into the tail vein of
Neuro2A-bear]ng mice results in preferential Eerie delivery into ~be distantly
growing subcutaneous tumors [1]. In contrast, application of standard
DN'A/Pfff complexes resulted in gene transt~r primarily to the lung. but also in
sigaifica~zt zo~:icit, g. Preger caatre~ ,~f ~ ~b.t:'sical. e~zd c x ~ i d a ~ garac~eeers e (

the transfection complexes, such as particle size. molecular weight of

polymeric carrier, stability, surface charge and modification with targeting
ligan6, was ~oun6 to slrong~iy 'mSuence m vn,o ~ N A "o'loglstfi'oufion. toY[lC'lty.
and gene transfer efficacy.
Two major mechanisms are considered to contribute for the tumor-specific
targeting as found in our model: passive targeting (by shielding the surface of
ccmple~ces from undesired interacCiotq and active tatgetitxg (65~ (ttcot-9~rati~.
off a "t~artd ~or attac'ttmeat to t'c~e ~tqaNoicate ce;:i: surfac~e re~c~eb~tc~g~ _Fnr
example, passive "targeting can be achieved by coating DNA/Tf-PEI
ccmpkes v~-ieth9otget/tg/_eng[ycol (f'~G/ el'trough, cova(et~t coug(ir~g t~ ~E[.
resulting in a reduced surface charge, plasma protein and erythrocyte binding
o f doe cqmy;~e~: ~2). PmtatL~:e,2 ctccut~fc,~a ~ct hLa,adt att,agc~ ~r.tra~a~c-zfw~ ~f
DNA complexes into distant tumor tissue which is an area of vascular
leakiness. However. active targeting may also contribute in the described
tumor model, as gene expression in the tumor is considerably higher with celtbinding ligand (transferrin) coated complexes as compared to transferrin-free
PEGylated con-tplexes.
[1] Kircheis, R.. et al. Polycation-based DNA complexes for tumor-targeted
g'~ ~l,l,e~vj f'e,,e?~,5.d. G~v,'e ']2~'N;'c@e, ~ (25, '( eqc~q~) "i'aTn~.5~.

[2] Ogris, M.. et al. PEGylated DNA/Transferrin-PEI complexes: Reduced

interaction with blood components, extended circulation in blood and potential
for systemic gene delivery. Gene T h e r a p 3 . 6 , (1999) in press.

s19

s20

Abstracts FEBS'99

2.1 Control of DNA replication

Regulation of DNA replication in bacteria
E. Boye, N. Torheim, S. Wold, K. Skarstad
Department of Cell Biology, Institute for Cancer Research,
Momebello, 0310 Oslo, Norway

Chromosomal replication in the bacterium Escherichia coli is initiated

at a unique site, oriC, and proceeds bidirectionally around the circular
chromosome. Regulation of chromosomal replication occurs
predominantly at the initiation step. Regulatory mechanisms must
make sure that i) initiation occurs at the appropriate cell physiology,
i.e. there must be a coupling mechanism to general cell growth, and
that ii) initiation occurs only once per cell doubling time for each origin
of replication. The DnaA protein binds to several recognition
sequences in oriC and is required to perform the first steps in initiation.
Also, since a higher DnaA concentration results in initiation at a lower
cell mass, DnaA is a positive regulatory factor for initiation. The SeqA
protein negatively affects initiation and, in particular, prevents two
successive initiations at one and the same origin. We have investigated
the molecular function of SeqA in initiation and its interaction with
other proteins. SeqA binds hemimethylated DNA and is known to
sequester oriC in a hemimethylated form. It also binds specifically to
fully methylated oriC and inhibits formation of an initiation complex
("prepriming complex") in vitro. Once formed, the prepriming complex
is inhibited by SeqA. Evidence will be presented that SeqA may
interact on many levels to regulate chromosomal initiation in E. coll.

Replication in normal and neoplastic cells.

R.A. Laskey, G.H. Williams, K. Stoeber, T. Krude,
A.D. Mills.
Wellcome/CRC Institute, Tennis Court Road, Cambridge CB2 1QR, UK

We have studied the control of DNA replication using a cell-free system

derived from cultured mammalian cells. Nuclei from G1 3T3 mouse fibroblasts,
synchronised by release from contact inhibition, initiate DNA replication in
extracts of S phase HeLa cells. Competence of nuclei to initiate replication arises
several hours after release from GO at a time that coincides with Cdc6 synthesis
and MCM binding to chromatin. Addition of recombinant Cdc6 protein advances
the onset of competence to replicate by several hours and increases the
proportion of nuclei that initiate replication in vitro, indicating that Cdc6 is one of
the limiting components for initiation of replication.
We have exploited the absence of Cdc6 protein and the changes in MCM
proteins during entry and exit from quiescence or differentiation to test the value
of these proteins as markers for neoplastic and pre-neoplastic cells in clinical
samples. We have focused initially on detection of abnormal cells in cervical
smears and biopsies. Antibodies against Cdc6 or MCM proteins show
exceptional promise in detecting abnormal cells in these and other clinical
samples, raising the possibility of improved early diagnosis of cancer.
We have developed a DELFLA immunoassay for detecting Cdc6 or MCM
proteins in body fluids such as urine and shown that this can be used to detect
bladder cancer non-invasively.

Replication of Chromosomes
B. Stillman
Cold Spring Harbor Laboratory
Cold Spring Harbor, NY USA

During the cell division cycle in eukaryotic cells, chromosomes replicate

and separate once during the S phase of the cell cycle. There are two
principal steps in the control of the cell division cycle. These occur at
transitions from G1 to S phase and from G2 to M phase. However, the
assembly and disassembly of protein complexes that affect these processes
occurs throughout the entire cell division cycle. For the initiation of DNA
replication a key protein is the Origin Recognition Complex (ORC). ORC
recognizes origins of DNA replication and acts as a landing pad for a series
of protein-protein interactions that result in the assembly of a number of
higher order complexes that control initiation. The loading of proteins such
as the Cdc6p and Mini-Chromosome Maintenance (MCM) proteins is
required for formation of a pre-Replication Complex (pre-RC) and later,
other proteins are loaded onto the pre-RC to form a pre-initiation complex.
These latter proteins include the Cdc45p, a protein that interacts with the
MCM proteins. In addition to these and many other proteins required for
the initiation of DNA replication, two protein kinase systems, including the
S phase-cyclin CDKs and the Cdc7/Dbf4 protein kinases are both required
to affect initiation of DNA replication. We have characterized the steps
involved in kinase activation of the initiation of DNA replication, including
the cell cycle regulation of these protein kinases. Initiation of DNA
replication is regulated at every single origin of DNA replication and the
frequency of initiation is controlled by the levels of the Cdc6 protein. We
have begun to understand the cell cycle dynamics of these events in human
cells, including the identification of the Origin Recognition Complex, Cdc6
and other proteins required for the initiation of DNA replication. These
and other studies will be discussed.

Dynamic organization of the genome for the

regulation of DNA replication
during embryonic development
M. M~chali, J.M. Lemaitre, S. Menut, Y. Vassetzky, Maiorano
lnstitut de G~n~tique Humaine, CNRS, Genome Dynamics
and Development, 34396 Montpellier, France

The organization of the eukaryotic chromosomes in the nucleus is

becoming a major issue to understand how chromatin domains are regulated
for their replication and transcription. Two aspects of the dynamic of this
organisation will be presented that could be essential to the developmental
programs. The first concerns the organisation of the chromosomes for cell
division and the adaptation of the nucleus to the rapid cell cycles occurring
during early development in Xenopus, when chromosomes behave as
structurally and functionally independent units of replication [ 1].
The localization of origins of DNA replication constitutes a second
aspect of this plasticity. We observed that during Xenopus development,
origins become specifically localized only after the mid-blastula transition
when transcription resumes in the embryo [2]. We report that the formation
of competent transcription complex on reporter genes promotes site-specific
initiation of DNA replication, although transcription per se is not necessary.
This phenomenon might be essential to developmental programs, when the
control of DNA replication could be more integrated to the control of gene
expression than in unicellular eukaryotes.
[1] Lemaitre et al (1998) Dynamics of the Genome during Early Xenopus
laevis Development : Karyomeres as Independent Units of Replication. J.
CellBiol, 142, 1159-1166.
[2] Hyrien et al (1995) Transition in specification of embryonic metazoan
DNA replication origins. Science, 270, 994-997.

Abstracts FEBS'99

s21

2.2 DNA recombination, repair and plasticity

DNA double-strand break repair
by homologous recombination
R. Kanaar

DNA double-strand break repair by non-homologous end

joining and responses to spontaneous DNA damage

Cell Biology and Genetics, Erasmus University. P.O. Box 1738,

3000 DR Rotterdam, The Netherlands

ICRF, Clare Hall Laboratories, South Mimms. Herts EN6 3LD. U.K.

Error-free repair of ionizing radiation (IR)-induced DNA double-strand breaks by

homologous recombination requires the RAD52 group proteins, including Rad51
and Rad54, in the yeast Saccharomyces cerevisiae. A key step in recombination,
formation of a joint molecule between the damaged DNA and the homologous
repair template, is mediated by Rad51 and stimulated by Rad54. The biological
importance of the RAD52 pathway for genome stabllity is underscored by its
conservation from fungi to humans..
We have analyzed the phenotype associated with disruption of the mouse
RAD54 (mRAD54) gene. mRAD54 knockout embryonic stem cells are sensitive to
ionizing radiation, mitomycin C and methyl metbanesulfonate, but not to UV light.
In addition, gene targeting experiments demonstrate that the frequency of
homologous recombination in mRAD54 knockout cells is reduced compared to
wild-type cells. These results imply that, besides DNA end-joining mediated by
DNA-dependent protein kinase, homologous recombination contributes to repair of
DNA double-strand breaks in mammalian cells
lmmunofluorescence experiments show that the mRad54 protein forms nuclear
focl upon treatment of the cells with ionizing radiation. Interestingly, upon
irradiation mRad54 partly colocalizes with mRad51. Immunopreclptation
experiments Indicate that the two proteins form a stable complex, but only upon
induction of DNA lesions that require mRad54 for their repair.
Rad54 belongs to the SWI2/SNF2 protein family whose members modulate
protein-DNA interactions in an ATP-dnven manner. We have purified the human
Rad54 (hRad54) protein and show that it has ATPase activity that is absolutely
dependent on double-strand DNA. Results of a topological assay suggest that
bRad54 can unwind double-stranded DNA at the cxpense of ATP hydrolysts.
Unwinding of the homologous repair template could promote the formation or
stabilization of hRad51-medlated joint molecules. Rad54 appears to bc reqmred
downstream of other RAD52 group protein~, such as Rad52 that assist Rod51 in
interacting with the broken DNA

Control

of recombination

in Saccharomyces

cerevisiae.
F. Fabre, E. Coic and C. Soustelle
C E.A, Fontenay-aux-RosesFrance

Among the different mechanisms that can repair double-stranded

breaks in Saccharomyces cerevisiae, homologous recombination by
strand invasion of an identical duplex is preponderant.
The proteins required for successful recombinational repair between
homologous chromosomes are Rad51, Rad52, Rad54, Rad55 and
Rad57. These proteins interact together and possibly form a
recombinosome. Many other genes are involved but their function is
not necessary.
Rad51 is the functional homologue of the bacterial RecA protein. It
forms filaments on single- and double-stranded DNA and catalyses
strand exchange between a linear duplex and an homologous singlestranded circle, a reaction largely dependent on the presence of the
single-strand binding proteins RPA. Addition in the assay of Rad52 or
Rad54 or Rad55 and Rad57 stimulates or accelerates strand exchange.
Homologous recombination is also negatively controlled by systems
that presumably reverse recombination structures. One known
mechanism is the inhibition of recombination when the DNA are
similar but not identical in sequence. Mismatch repair proteins are
largely responsible for this inhibition. Another mechanism, which we
study, consists in reversion by the Srs2 helicase of recombination
structures that are not or slowly maturate. We have evidence that Srs2
acts in a late stage of recombination, after the steps catalyzed by the
"RAD51 group" of genes. Furthermore, the srs2 mutation is a
suppressor of leaky alleles of Rad51 which confer a partial sensitivity
to ionizing radiations. It has no effect on a null tad51 mutation. Thus
in presence of leaky alleles of Rad51, Srs2 likely reverses
recombination structures and in its absence repair is completed thanks
to the leakiness of the system. This activity would account for the
hyper-recombination phenotype of srs2 cells, as well as for their
radiation sensitivity.

T. Lindahl

A distinct eukaryotic DNA ligase, different from the enzymes that

account for joining during replication and excision-repair processes,
is required for joining of double-strand breaks. This DNA ligase IV
is nonessential in yeast, but knockout mice deficient in the function
die during late embryonic development. The lethal phenotype is
associated with massive apoptosis in the central nervous system but
not in other organs, revealing an unexpected requirement for the
joining of DNA double-strand breaks during early neural
development [1 ]. Mammalian DNA ligases active in repair and
recombination interact with scaffolding proteins through a specific
fold for protein interactions, the BRCT domain. The threedimensional structure of one such domain has been solved [2], and
site-specific mutagenesis experiments provide further insights into
these interactions. Endogenous damage to DNA may be caused by
active oxygen species, and the repair pathway for such damage has
been reconstituted with purified human proteins [3]. The properties
of gene knockout mice deficient in various functions required for
correction of spontaneous DNA damage and the fidelity of the repair
process will be described. A human counterpart to the dnaQ/mutD
editing enzyme ofE. coli has been identified, cloned and expressed,
and this human factor should improve the accuracy of DNA
replication, repair and/or recombination.
[1]
D.E. Barnes et al., Curr. Biol. 8, t395-1398; 1998
[2]
X. Z h a n g e t a l . , E M B O J , 17,6404-6411; 1998
[3]
A. Klungland et al., Molecular Cell 3, 33-42; 1999

Role of the yeast Rad5 protein for channeling repair of

DNA double strand breaks into homologous recombination
F. Eckardt-Schupp, C. Klaus, G Drexler, B. Fellerhoff,
S. Fichter, M. Klechle, M Kistler, A.A Friedl, F. Ahne
GSF-Institute of Radiobiology,85758 Nenhcrbetg, Germany.
Genomic stability of cells is severely endangered if DNA double strand breaks
(DSB) or gaps are not repaired Several genetically and mechanistically
distinct pathways of DSB repair have evolved in eukaryotic cells. In yeast,
homologous recombination (HR) mediating error-free DSB repair is of major
importance whereas yKu-dependent non-homologous end-joining 0NltEJ) is a
minor "back-up" pathway which is more error-prone than HR NHEJ in yeast
has an increased potential for mutations by loss of base pairs of DNA ends to
be joined [1] and by joining "false ends" leading to chromosomal
translocations [2] In mammalian cells, NItEJ is the prevailing mechanism, and
HR is of minor importance In order to investigate the regulatory basis for the
differential use of HR versus NHEJ, we have established a plasmid-based
system in yeast which allows to analyse whether an enzyme-mediated deletion
(169 bp) is correctly repaired by HR using the chromosomal homologous
sequence or by NHEJ which - as opposed to HR - does not restore the
biological function of the plasmid (uracil proficiency). We have shown that the
Rad5 protein is required for channeling gap repair of the plasmid into HR
(97 %) RAD5 belongs to the SNF2 family of helicase-like ATPases, members
of which are involved in DNA damage processing, transcriptional regulation
and maintenance of chromosome stability, rod5 mutants are "mammalian celllike" as the majority of the enzyme-induced gaps (75 to 80 %) are joined by
NHEJ although rod5 mutants are HR-profient for chromosomal markers and
the chromosomal homologous sequences are present [I]. Absence of the Rad5
protein has no effect on the efficiency of repair. Deletion of the yKU70
(HDF1) gene in the rod5 mutant abolishes NHEJ: gap repair occurs
exclusively by HR (95 %) and with high efficiency.These results let us propose
a regulatory role for Rad5p in favour of HR as a prevailent pathway for DSB
repair in yeast. To test this hypothesis, we study the regulation of RAD5 gene
expression and possible functions of RadSp to elucidate the molecular basis of
the putative channeling function, and we analyse whether the role of Rad5p is
specific for gaps in plasmids with nucleosomal structure or accounts as well
for DSB and gaps in chromosomes.
[1] Ahne, F et al, Nucl Acid Res 25 (1997) 743
[2] Friedl, A A. et al., Genetics 148 (1998) 975
The financial support given by the European Commission is gratefully acknowledged.

s22

Abstracts FEBS'99

Meiotic double-strand breaks and recombination

in yeast and mammalian DNA
G. Simchen, D. Zenvirth, S. Klein, A. Arbel
Department of Genetics, The Hebrew University, Jerusalem 91904, Israel

Yeast chromosomes, as well as artificial chromosomes (YACs) carrying

human-derived DNA, are subject to meiosis-induced double-strand breakage.
Each chromosome and YAC has a characteristic set of preferred meiotic
double-strand break sites, which reflect their recombination behaviour[1, 2].
For YACs from the human XY pseudoautosomal region, levels of doublestrand breakage during yeast meiosis correlate with levels of recombination
during human meiosis. Our data strongly support the notion that meiotic
recombination events are initiated as DSBs in yeast as well as in human
meiosis.
Repair of DSBs in mitotic yeast cells usually occurs by recombination
with the sister chromatid, whereas in meiosis the non-sister is preferred as a
template. How are such preferences carried out? We developed a novel
method to identify genes that are required for repair by the sister chromatid,
using haploid strains that embark on meiosis and genome-wide DSBs[3]. We
show that the recombinational repair gene RAD54 is required primarily for
sister-chromatid based repair, whereas TID1, a yeast RAD54 homologue, and
the meiotic gene DMC1, are dispensable for this type of repair. The latter are
required for recombination with the non-sister, in meiosis. Under some
circumstances the sister-chromatid repair pathway, which involves RAD54,
and the non-sister repair pathway, which involves DMC1, can substitute for
one another[3]. Deletion of RAD54 in yeast results in a similar phenotype to
that found in mammalian cells, namely impaired DNA repair and reduced
recombination during mitotic growth, with no apparent effect on meiosis. The
principal role of RAD54 in sister-chromatid based repair may also be shared by
mammalian and yeast cells.
[1] Klein, S., et al., Chromosoma, 105 (1996): 205.
[2] Klein, S., et ai., Nature Genetics 13 (1996): 481.
[3] Arbel, A., et al., E M B O ~ 18 (1999): 2648.

Abstracts FEBS'99

s23

3.1 Cell cycle

Protein kinases regulating centrosome function.
E.A. Nigg, A.M. Fry, T. Mayor, and P. Meraldi
Department of Molecular Biology, Sciences lI, University of Geneva,
30 Quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland

Centrosomes are the principal microtubule-organizing centers of animal

cells. They duplicate once per cell cycle and contribute to determine the
biolarity of the mitotic spindle. This bipolarity in turn is important for the
error-free segregation of sister chromatids during mitosis, and
chromosomal imbalances (aneuploidies) have long been recognized as an
important factor in tumorigenesis. Our laboratory is studying two human
protein kinases, the NIMA-related kinase Nek2, and the polo-like kinase
Plkl, both &which function in relation to the centrosome cycle (albeit not
exclusively). We found that overexpression of active Nek2 in mammalian
cells causes premature centrosome splitting, suggesting a possible role for
Nek2 in the induction of centrosome separation at the onset of mitosis
[1,2], while antibody-mediated abrogation of Plkl function prevents proper
maturation of centrosomes and leads to either G2 arrest or the formation of
monopolar spindles, depending on cell type [3]. In order to better
understand how centrosome duplication is coordinated with DNA
replication, we have recently begun to study the mechanisms that regulate
centrosome duplication during the cell cycle. Using a novel centrosome
duplication assay in mammalian somatic cells, we found that centrosome
duplication requires both transcription factors of the E2F family and
Cdk2/cyelin A activity.

Centrosomes and cells: one becomes two

David M. Glover
Cancer Research Campaign Cell Cycle Genetics Group,
University of Dundee,DDl4HN and Department of Genetics.
University of Cambridge, CB2 3EH

The spindle poles play a crucial rote in directing the segregation of

chromosomes, and in setting up the structure that will divide the cell into two at
cytokinesis. Several of the molecules that mediate these two processes localise to
the centrosomes or polar MTOCs during M-phase. We have taken a genetic
approach to understand the roles of both structural and regulatory components of
the centrosome, an organelle that itself has to duplicate. Several regulatory
protein kinases associate with the centrosome during mitosis. Of these the pololike kinases are thought to play multiple roles during mitotic progression by
regulating Cdc25, centrosome function to form the bipolar spindle, activation of
the anaphase promoting complex, and the early events of cytokinesis. Their subcellular localisation is consistent with each of such roles. A search for polo
kinase substrates has identified the product of the abnormal spindle (asp) gene,
an asymmetrically localised component of the centrosome during mitosis. The
Asp protein is required to focus the poles of the mitotic spindle in vivo.
Removing Asp protein function from Drosophila embryo extracts either by
mutation or immunodepletion, resulted in loss of their ability to restore
microtubule organising centre (MTOC) activity to salt stripped centrosome
preparations. This was corrected by addition of purified Asp protein. Thus Asp
appears to hold together the microtubule nucleating ~/-tubulin ring complexes to
organise the mitotic centrosome, an activity that is in part regulated by polo
kinase.

[1] Fry, A.M., Meraldi, P., and Nigg, E.A. (1998) EMBO J., 17, 470-481.
[2] Fry, A.M., Mayor, T., Meraldi, P., Stierhof, Y.-D., Tanaka, K., and
Nigg, E.A. (1998) J. Cell. Biol., 141, 1563-1574.
[3] Lane, H.A., and Nigg, E.A. (1996) J. Cell Biol., 135, 1701-1713.

Blocking C-terminal tyrosine of -tubulin inhibits Mphase entry in starfish and xenopus oocytes and eggs.
Soplue Vee*, Laurence Lafanech~re*, Didier Job* and Andr~ ptcard*.
: DBMS CE4 l 7, rue des martyrs F 38054 Grenoble cedar 9
*: laboratoire A t ago, BP 44, F-66651 Banvuls sur mer cedex

A dynamical equilibrium between a specific carboxypeptidase and a

Tubulin-Tyrosine Ligase (TTL) regulates the presence of a tyrosine residue
at the C-terminal end of ot-tubulin. The microinjection of antibodies specific
for TTL in fertilized starfish eggs induces cell cycle arrest in G2 of the first
mitotic cell cycle. More stfickingly, microinjection of antibodies specific for
the tyrosylated C terminus (YL1/2 and 20C6 ) inhibits H1 kinase reactivation, after meiosis I or at the G2-M transition in the first mitotic cell
cycle, according to the time of microinjection, even in the complete absence
of polymerized microtubules (201ag/ml Nocodazole). While YLI/2
microinjection does not inhibit hormone-induced meiosis reihitiation in
starfish, it counteracts the effects of progesterone on xenopus oocytes.
These observations can be accounted for by a strong inhibition of the ERK2
MAP kinase activation, and of cyclin B synthesis in both starfish and
xenopus oocytes. This work supports the view that abundant proteins like
tubulin or eventually chaperones, usually considered as "house keeping
proteins", play an essential role in the very close regulation of the master
regulators of the cell cycle.

Yeast Cdc20 and Hctl, conserved regulators of

APC-mediated proteolysis
W. Seufell
b~slttlt/e o] ]ndttslrla[ (JeJ~ellc;% l~'ml'e~ ~itl' of .'~'lull~art, ,4lhmmdrmg 31
D- 70569 Stuttgart, GermanF

Progression through the eukaryotic cell division cycle depends on stagespecific proteolysis of regulatory proteins by the ubiquitin-proteasome
system. During mitosis and GI a large ubiquitin ligase termed anaphase
promoting complex (APC) mediates destruction of anaphase inhibitors as
well as mitotic cyclths and thereby promotes chromosome separation and
exit from mitosis. Substrates ubiquitinated by the APC carry a short motif
called destruction box. Studies in budding yeast showed that the APC
cooperates with two related WD40-repeat proteins, Cdc20 and Hct 1/Cdh 1,
which are critical for the stage-specific selection of substrates by this
pathway 111,2]. Cdc20 is needed for proteolysis of Pdsl at anaphase, while
Hct 1 mediates Clb2 cyclin degradation in telophase and G1. Homologs of
Cdc20 and Hctl have been identified in other organisms including lmmans.
Recent results help to explain how these regulators ofproteolysis work [3].
Hctl and Cdc20 were both found to bind to the APC. Cdc20-APC binding
and Cdc20-mediated proteolysis were unaffected by cyclin-dependent
kinase (Cdk) activity. In contrast, binding of Hctl to the APC and Hctlmediated proteolysis were inhibited through phosphorylation of Hctl by
various cyclin-Cdkl complexes. This difference in sensitivity to Cdkl
allows Cdc20 to be active at anaphase and restricts Hctl activity to late
mitosis and G1. The exact mechanism how these WD40-proteins activate
the ubiquitin ligase activity of APC in a substrate-specific fashion is the
subject of current studies.
[1] Schwab el al. Cell 90 (1997) 683
[2] Visintin et al. Science 278 (1997) 460
[3] Zachariae et al. Science 282 (1998) 1721

s24

Abstracts FEBS'99

Sister chromatid cohesion in yeast

A. Toth,R. Closk, F. Uhlmann, M. Galova,

A. Schleifer, K. Nasmyth
Research Inst. Molecular Pathology, Dr Bohr Gasse, 1030 Vienna

Sister chromatid cohesion is crucial for chromosome segregation

during mitosis. Loss of cohesion very possibly triggers sister
separation at the metaphase to anaphase transition. This process
depends on the destruction of anaphase inhibitory proteins like
Pdslp (Cut2p), which is thought to liberate a sister separating
protein Esplp (Cutlp). By looking for mutants that separate sister
centromeres in the presence of Pdslp, this and a previous study
have identified 6 proteins essential for establishing or
maintaining sister chromatide cohesion. Four of these proteins,
Scclp, Scc3p, Smclp and Smc3p, are subunits of a ~ Cohesin complex that binds chromosomes from late G1 until the onset of
anaphase. The fifth protein, Scc2p, is not a stoichiometric Cohesin
subunit but it is required
for Cohesin's association with
chromosomes. The sixth protein, Ecolp is not a Cohesin subunit.
It is necessary for the establishment of cohesion during DNA
replication but not for its maintenance during G2 and M phases.

Abstracts FEBS'99

s25

3.2 Cell cycle and cancer

GI/S control and its deregulation in cancer
J. Bartek, C. Lukas, C. Svrensan, E. Santoni-Rugiu,
J. Bartkova, J. Lukas
Institute of Cancer Biology, Danish Cancer Society, Copenhagen,

Denmark

Cancer is increasingly viewed as a cell cycle disease, a notion supported by

recent accumulation of data on the molecular basis of the cell cycle
machinery and its defects commonly found in turnout cells. Strikingly, the
cell cycle phase targeted most frequently in multistep oncogenesis is the
control of G1/S transition. This period includes the late-G1 commitment to
replicate the genome and complete the cycle (the restriction point control),
and the initiation of DNA replication, events regulated by the so-called "RB
pathway". While the key components of the RB pathway qualify as protooncoganes or tumour suppressors and their aberrations may provide direct
proliferative advantage to cancer cells, defects in the so-called checkpoint
mechanisms that monitor and help ensure the error-free execution of the cell
cycle transitions, act more indirectly yet affect both tumour progression and
response to anti-cancer therapy. Examples of both the oncogenic defects in
the G I/S-controlling machinery, and the ways proto-oncogenic events may
activate checkpoint responses, will be presented. In addition, evidence in
favour of existence of a potential parallel pathway, independent on, and
likely cooperating with the classical pl 6-cyclin D/CDK-pRB-E2F axis (the
RB pathway) to govern timely S-phase entry, will be presented. Finally, the
proposed candidacy of the RB pathway for the molecular mechanism
underlying the late-G1 restriction point switch will be critically considered,
and emerging data on novel functions of the 1t13 pathway in coordination of
the cell cycle events from late G1 until mitosis will be summarized. These
new discoveries have significant implications for our understanding of the
mammalian cell cycle control and its subversion in tumour cells, with
emerging applications in oncological diagnostics, turnout prognosis, and
attempts to device new strategies to treat cancer.

CDKN2A GENE PRODUCTS IN CELL CYCLE CONTROL

AND TUMOR SUPPRESSION. Gordon Peters. Imperial
Cancer ResearchFund, 44 Lincoln's Inn Fields, London, WC2A
3PX, U.K

The CDKN2A locus on human chromosome 9p21 encodes two

structurally unrelated proteins that both function in tumour
suppression. The product of the alpha transcript, pl6 DqK4a,induces a
G1 cell cycle arrest by inhibiting phosphorylation of the
retinoblastoma protein (pRb) by the cyclin-dependent kinases,
CDK4 and CDK6. In contrast, the product of the beta transcript,
p14 AR~, activates a p53 response resulting in elevated levels of
MDM2 and p21 crP~ and a cell cycle arrest in both G1 and G2/M.
Activation of p53 by ARF appears to be a result of a direct
interaction between ARF and MDM2 which inhibits the MDM2mediated degradation of p53. Conversely, p53 negatively regulates
ARF expression and there is an inverse, albeit imperfect correlation
between ARF expression and p53 status in human tumour cell lines.
Ectopic expression of ARF in human diploid fibroblasts results in
phenotypic characteristics typical of replicative senescence and these
effects are relieved by coexpression of the HPV16 E6 protein, which
targets p53 for degradation. We are currently investigating the
possible role for ARF in senescence using human diploid fibroblasts
from an individual who is homozygous for a deletion in exon 2 of
the 1NK4a-ARF locus. These cells are "functionally null for pl6 ~K4a
but express a fusion protein in which the amino terminal region of
ARF is fused to the carboxy terminus of pl6n~K4L
In these fibroblasts and in a p53 negative T-cell line, ARF
levels increase as cells progress from quiescence into cycle with
maximal expression at S-phase. This would be consistent with
regulation by the E2F family of transcription factors and the
sequence of the ARF promoter region reveals a potential E2F
binding site upstream of the presumed start of transcription.
Promoter-reporter assays have confirmed that E2F-1 can induce
transcription from the ARF promoter. These findings imply that
ARF provides a link between the pRb and p53 pathways and can
explain why oncogenic agents, such as HPV E7, adenovirus E1A
and Ras, that functionally intersect with pRb, also activate p53.
They also suggest a model in which the inactivation of pRb that
occurs as cells leave quiescence and initiate DNA synthesis results in
the induction of ARF and the priming of the p53-MDM2 pathway.

p53 - upstream regulators and downstream effectors

M. Oren ~, A. Damalas ~, A. Elkeles', T. Gottlieb ~, T. JuvenGershon", R. Maya", T. Unger', J. Zhurinsky ~, E. Moallem b, R.
Vogt-Sionovb, M. Berger b, D. Shkedy ~, R. Khosravi ~, A. BenZe'ev ', B. Geiger", Y. Shilo~ and Y. Haupt~
"Department of Molecular Cell Btology, the Weizanann Institute of Sctence,
Rehovot 76100, Israel; t'Lautenberg Center for General and Tumor
hnmunology, The Hebrew Untversttv Hadassah Medical School.
Jerusalem 91120. Israel: 'Department of Haman Genettcs and Molecular
Medtcme, Sackler
School of Medicine, Tel Avtv Universtty, Ramat Avtv 69978, Israel.

The p53 tumor suppressor gene is a major target for genetic alterations in
human cancer. The p53 protein is a sequence-specific transcnptional aettvator.
It is activated m response to a variety of stress signals, including genomic
damage and deregulated oncogenes, On the other hand, p53 ~s subject to
effective inactivation by the Mdm2 protein, itself a product of a p53-regulated
gene. The binding of Mdm2 to p53 results m the rapid proteolyttc degradation
of p53, serving to keep p53 activity very low m the absence of stress. In
response to genotoxtc stress, the cells accumulate increased amounts of p53.
This is largely due to an escape of p53 from Mdm2-directed proteolysis. This
escape is achieved through covalent modifications of both p53 and Mdm2. We
report that phosphorylation of p53 on serine 20, occurring in response to DNA
damage, greatly interferes with the ability of Mdm2 to bind p53 and cause its
degradation. The ATM kinase can also phosphorylate p53 in response to DNA
damage. In addition, however, ATM can also promote the phosphorylation of
Mdm2. This modification also contributes to p53 stabtlization, apparently
through Mdm2 inactwation.
Mutational events that occur early during colorectal carcinogenesis lead to
nuclear accumulatmn of activated [3-catenin. At a later stage of colorectal
tumor progression, p53 mutations occur at a high frequency. We demonstrate
that excess deregulated [3-catenin triggers the accumulation of transcriptionally
active p53, by interfering with both Mdm2-dependent and Mdm2-mdependent
p53 degradation. This p53 activatmn presumably serves as a safeguard
mechanism against cancer development, and may explain the selective pressure
for subsequent p53 inactivation.
When p53 becomes activated m response to stress, it stimulates transcription of
an array of specific target genes. We report that one of the p53 targets, at least
in certain cell types, is the c-fos ~ene. Thus, p53 may s~t at the cross-roads
Anti-tumoral responses mediated by the INK4a-ARF locus.
M. Serrano, M. Barradas, F. Bringold, C. Pantoja, I. Palmero.
Department of lmmlmolog9, and Oncolog3, National Center of
Biotechnolog3. Madmd, Spare

Normal, healthy, cells possess mechanisms that protect them from becoming
neoplasically transformed by the simple action of oncogenes. Another
characteristic of normal cells is their inability to grow indefinitely in culture:
indeed, upon accumulation of a certain number of doublings, normal cells
enter into a permanent arrest known as senescence.
The 1 N K 4 a - A R F l o c u s encodes two tumor suppressor, pl6 I~'K4~ and
p19 ARF, that regulate twv different tumor suppressor pathways: pl6 INK4a
activates Rb by inhibiting the CDK4 and CDK6 kinases; and p19 ARF
activates p53 by inhibiting the destabilizing effects of MDM2. The cellcycle inhibitor p21 mediates some of the effects of p53 in arresting
proliferation.
Expression of oncogenic Ras in primary cells is initially mitogenic
but, after a period of a few days, it triggers an anti-proLiferative response
mediated by both p l6 INK4aand p19 ARF [1,2]. We regard this response as an
anti-tumoral mechanism. Indeed, rodent cells genetically deficient in either
p 16jNK4aor p 19Aa~ a r e efficiently transformed by oncogenic Ras.
We have compared senescence in fibroblasts derived from mice
deficient in pl61>''4a, p53 or p21 [3]. We have found that fibroblasts
derived from p2 l-deficient mice senesce in a manner indistinguishable from
wild-type cells, whereas pl6- or p53-deficient cells are immortal.
Moreover, p2 l-deficient cells, like wt cells, are refractory' to transformation
by oncogenic Ras, which is in contrast to p53-deficient cells. We conclude
that the p l6tskaa/Rb and p19ARV/p53 pathways are essential for senescence
and for resistance to transformation, but p21 is dispensable for both
processes. These data correlate well with the tumor susceptibility of the
corresponding knock-out mice.
[1] Serrano et al.. Celt 88, 593 (1997).
[2] Palmero et at., Nature 395, 125 (1998).
[3] Pantoja et al., Oncogene, in press (1999).

s26

Abstracts FEBS'99

3.3 Programmed cell death

CD95(APO-1/FAS)-MEDIATED APOPTOSIS: SIGNALING
AND DISEASES
Krammer. P.H. Tumorimmunology Program, German Cancer
Research Center, Heidelberg, Germany
CD95, a member of the tumor necrosis factor (TNF) receptor superfamily
induces apoptosis upon receptor oligomerization. The receptor and its ligand
are important for apoptosis of peripheral T cells, for downregulation of an
immune response and most likely, at least in part, also for peripheral T cell
tolerance. In Aids, apoptosis mediated by this system might contribute to the
depletion of T helper lymphocytes. Likewise, in diseases in which liver cells
are destroyed the CD95 system might play a major role.
In a search to identify intracellular signalling molecules coupling to
oligomerized CD95 several cytotoxicity-dependent APO-1-associated 12roteins
(CAP) were immunoprecipitated from the apoptosis-sensitive human leukemic
T cell line HUT 78 and the lymphoblastoid B cell line SKW6.4. CAP1-3 and
CAP4 instantly detectable after crosslinking of CD95 were only associated
with aggregated and not with monomeric CD95. CAP1 and CAP2 were
identified as phosphorylated (MORT1/FADD). CAP4 was a new molecule,
designated FLICE (for FADD like ICE), and showed homology to the death
effector domain of FADD and to ICE-like proteases (FLICE is now called
caspase 8). Thus, FLICE was identified as the most CD95 receptor proximal
protease which starts the cascade of protease reactions important for CD95mediated apoptosis. Association of CAP 1~ with CD95 was not observed with
C-terminally truncated non-signalling CD95. CAP1 and 2 did also not
associate with an CD95 cytoplasmic tail carrying the lprCg amino acid
replacement. Moreover, no CD95/CAP association was found in the CD95 +,
anti-CD95 resistant pre B cell line Boe. CAPI-4 form a death-inducing
signalling complex (DISC) with the CD95 receptor and are, thus, the first
CD95 associating proteins of a signalling cascade mediating apoptosis.The
function of the DISC is discussed in detail, particularly with respect to its role
in sensitivity and resistance to apoptosis.
The CD95 death system plays a role in destruction of liver tissue. In
hepatitis cytotoxic T lymphocytes might use the CD95 system to kill infected
hepatocytes. In M. Wilson copper overload leads to upregulation of the CD95
ligand that may finally contribute to acute liver failure. In HCC from patients
treated with chemotherapeutic drugs the CD95 receptor and ligand are
upregulated and may contribute to apoptosis of the tumor or, dependent on the
drug sensitivity of the tumor, to the status of the tumor as an
immunoprivileged site.

Bcl-2 family members and their interaction with mitochondria

J.-C. Martinou,S. Desagher,A. Osen-Sand,A. Nichols.R. Eskes,
S. Montessuit,S. Lauper.B. Antonsson.
Semno PharmaceuticalResearchInstitute,Plan-les-Ouates.
Geneva, Switzerland

The Bcl-2 family members play a major role in the control of various
types of apoptosis. Bax, a pro-apoptotic member of the family, is
responsible for the mitochondrial cytochrome c effiux which activates
a cascade of caspases through binding to Apaf-I and easpase 9. We
will show that in the early phases of apoptosis, Bax undergoes a
conformational change induced by Bid, another pro-apoptotic member
of the Bcl-2 family. This change in conformation leads to Bax
insertion in mitochondrial membranes and possibly to channel
formation. The mechanisms by which Bax triggers the release of
cytochrome c from mitochondria is unclear. We will show that the
Bax-induced cytochrome c release occurs independently of the
opening of the permeability transition pore (PTP) as it is not blocked
by the PTP inhibitor cyclosporin A.

Lipid signaling in Fas-indueed apoptosis

Roberto Testi
Deptartment of Experimental Medicine and Biological
Sciences, University of Rome "Tor Vergata", Italy.

Understanding the molecular mechanisms which regulate the

expression and function of surface death receptors might prove useful in
designing therapeutic strategies for a variety of diseases. We are
currently focusing on the characterization of the molecular pathway
which originates from the surface death receptor Fas and leads to cell
death by apoptosis. We have defined the role of a series of lipid and
glycolipid intracellular mediators, such as ceramide and GD3
ganglioside, which are generated upon Fas receptor clustering in cells
undergoing apoptosis. Specifically we found evidence for a role of GD3
ganglioside in recruiting the mitochondria to the cell death program by
causing mitochondrial inner membrane permeability transition, rapture
of the outer membrane and subsequent release of apoptogenic factors
from mitochondria. Interfering with this pathway largely prevents cell
death and bears promises as a potential approach for delaying tissue
destruction in selected conditions.

Abstracts FEBS'99

s27

4.1 Structural motifs, dynamics and function in proteins

Motifs, folds and functions
C. Orengo, J.M. Thornton
Biochemistry and Molecular Biology Department,
University College London, Gower Street, London WCIE6BT, UK

As more gene sequences are determined, the demand for better methods to
predict protein function from sequence grows. Currently the only reliable
approach is to predict function by recognising sequence and/or structural
homology to a related protein, whose function is known. Recognising close
relatives is easy using standard algorithms, but recognition of more distant
homologues becomes more difficult as sequences and structures diverge.
Recent methods have improved recognition by using family information and
iterative scanning to build up a 'sequence profile'. Our interest lies in the
relationship between protein sequence, structure and function. An overview
will be presented of our current knowledge of the universe of protein
structures, and how sequences, structures and functions evolve. In particular,
we will consider the relationship between fold and function using our
classification scheme of protein structures called CATH [1 ]. Every structure
includes much information on detailed interactions, involved in biological
function, yet very little of this information is used in genome analysis. We
have therefore developed some software tools which seek to improve the
annotation of sequences by the structural and functional information,
especially regarding inter- and intra-molecular interactions. This resource is
available on the web at http://www.biochem.ucl.ac.uk/bsm. Various methods
to extract and classify functional information from structural work will also
be discussed [3].
Reference~
[1] Orengo, C.A. et al. CATH - A Hierarchic Classification of Protein
Domain Structures. Structure, 5, 1093-1108 (1997).
[2] Milbum, D., R. A. Laskowski and J. M. Thornton. SAS: Sequences
Annotated by Structure: a Tool to Facilitate the Use of Structural
Information in Sequence Analysis. Protein Engineering, 11,855-859.
[3] Wallace, A.C., Borkakoti, N. & Thornton, J.M., (1997). TESS: A
Geometric Hashing Algorithm for Deriving 3D Coordinate Templates for
Searching Structural Databases. Application to Enzyme Active Sites. Protein
Science, 6, 2308-2323.
Structure and activation by dimerisation
of the integral membrane enzyme OMPLA,
outer membrane phospholipase A
B.W. Dijkstra, A. Snijder
Laboratory of Biophysical Chemistry, Groningen University,
Nijenborgh 4, 9747 AG Groningen, the Netherlands

Outer membrane phospholipase A (OMPLA) is an integral membrane enzyme

located in the outer membrane of Gram-negative bacteria. Its structure has
been determined by X-ray crystallography. Monomeric OMPLA consists of a
12-stranded anti-parallel [3-barrel with a convex and a fiat side and with
approximate dimensions of 16x30x45 A 3. The 12 amphipathic [~-strands
traverse the membrane in an up-and-down fashion forming a hydrophobic
outer surface.
The active site contains Ser144 and His142, as established from chemical
modification and site-directed mutagenesis experiments. It is located on the
exterior of the ~-barrel, positioned just outside the outer leaflet ring of
aromatic residues. Besides the serine and histidine, the active site contains an
asparagine residue (Asn156). Its carboxamide side chain is at hydrogen
bonding distance from the NSI atom of His142, probably fixing the
orientation of the His142 side chain.
The activity of the enzyme is regulated by reversible dimerisation with the
dimer being the active form. Covalent inhibition of OMPLA results in
increased dimer stability which allowed for the crystallisation of the dimer.
The monomers associate in a parallel fashion to form a homo-dimer such that
the two active sites are located at the outer leaflet side of the membrane.
Surprisingly little structural rearrangements occur (r.m.s. difference between
monomer and dimer is 0.4 A for 257 Co~ atoms). The key interaction
stabilising the dimer seems to be a double hydrogen bond formed between the
side chains of glutamine 94 from one monomer and its counterpart in the other
monomer, nearly in the middle of the membrane-embedded part.
Dimerisation results in productive active sites, with two substrate binding
pockets created at the interface between the two monomers.

Folding mechanisms of proteins with simple folds and

complex topologies
S.E. Radford
School of Btochemistry and Molecular Bmlogy, Umverstty ol'Leeds.
Leeds LS2 9JT

Our understanding of how a protein folds has increased enormously in recent

years, principally through the combination of novel protein engineering
experiments, ingenious experimental methods and rapid developments in
theoretical approaches. Views of folding ranging from rapid simple twostate transitions to more complex mechanisms involving parallel pathways
and partially folded states have emerged. An important question that arises is
whether current views of folding are representative of the wide variety of
native protein structures found in the structure database or whether important
new clues about folding might emerge from investigations of the folding of
different protein folds. To address some of these issues, we have initiated
studies of the folding of the Greek key protein, pseudoazurin, which has a
complex double wound Greek key fold. Our recent results on the folding of
this protein and the insights learned will be described.
In contrast with our studies on the folding of complex 13-sheet proteins, we
have also recently initiated a study of the family, the bacterial immunity
proteins, which have a very simple four helix fold. We have analysed the
folding of two of the family members, Im7 and Ira9. Our data show that
these proteins fold very rapidly, but despite being 60% identical, appear to
fold with mechanisms of different complexity. These data will be reviewed
and the implications of the results obtained thus far for the folding of helical
proteins discussed.

Structural insights on the pivotal role of the LBD

in signal transduction by nuclear receptors
J.-P. Renaud
CNRS UPR9004 Btologie Structurale, IGBMC, 67404 lUkirch, France

Nuclear receptors form the largest family of transcription regulators

in higher organisms, controlling numerous physiological events in
development, homeostasis, and cellular life. They activate in general the
transcription of a set of specific genes in response to cognate ligands,
usually small lipophilic molecules. Upon binding, the ligand triggers a
conformational switch in the ligand-binding domain (LBD) which
abrogates the interaction with a corepressor and in turn promotes the
recruitment of a coactivator. Coactivators then facilitate the assembly of the
pre-initiation complex including general transcription factors and RNA
polymerase II, which results in enhanced transcription rates. This liganddependent transcriptional activation has been recently related to chromatin
remodelling, which has lead to a coherent picture of the role of nuclear
receptors: they are locks for chromatin access - ligands being the keys.
The first crystallographic studies on nuclear receptor LBDs had
suggested the existence of a conserved fold within the superfamily [1],
which has been confirmed by subsequent structures [2], and had brought
first evidence for the ligand-dependent conformational switch, enlighting
the crucial role of the "activation helix" H12 found at the LBD C-terminus.
The conformational switch between the apo and holo forms of the LBD has
also been documented by dynamic simulations.
The mode of action of agonists and antagonists has now been
detailed. Agonists bind to and remodel LBDs, yielding the transcriptionally
active conformation [3]. On the other hand, antagonists bind to LBDs but
fail to produce the LBD surface to which coactivators bind. Recent
crystallographic studies have brought some insights on the coactivator
interaction surface and on how some antagonists prevent coactivator
binding by relocating H 12, thus destroying the interaction surface [2],
[1] Wurtz J.M. et al., Nature Struct. Biol., 3 (1996), 87.
[2] Moras D. et al., Curr. Op. Cell Biol., 10 (1998), 384,
[3] Klaholz B. et al., Nature Struct. Biol., 3 (1998), 199.

s28

Abstracts FEBS'99

Further structural insights into the regulation

of the Src kinases
J. K u r i y a n
The Rockefeller University and
Howard Hughes Medical Institute
1230 York Avenue, New York, NY 10021-6399
The Src family of tyrosine kinases are a closely related group of signaling
proteins that are characterized by the presence within them of two peptide recognition
modules known as the Src homology domains SH2 and SH3. The SH2 and SH3 domains
serve a dual function in regulating the biological and chemical activity of Src kinases. In
the active form of Src kinases these domains are responsible for targeting the proteins to
specific sites containing phosphotyrosine residues or Pro-X-X-Pro motifs, which bind the
SH2 and SH3 domains, respectively. In the absence of incoming signals the SH2 and
SH3 domains cooperate to inactivate the Src kinases by turning inwards and binding to
elements within the parent protein.
A breakthrough in our understanding of the structural basis for the inhibition of
catalytic activity by the SH2 and SH3 domains came with the determination of the first
structures of the Src family kinases, human c-Src and human Hck, in the auto-inhibited
form. While these first structures 0,2), along with a subsequent report of the structure of
chicken c-Src (3), have clarified much of the structural mechanism that underpins the
regulation of the Src kinases, one piece of the puzzle is still missing. In each of the
structures the central portion of the activation segment, which includes Tyr 416~ was not
modeled. We now report the crystal structure of the Src-family tyrosine kinase Hek
eomplexed with the inhibitor PPl, determined at a resolution of 2.0 A. In this structure
the entire activation segment is modeled reIiably. Most interestingly, Tyr 416 is
positioned within the catalytic center of the kinase domain and, together with the flanking
regions of the activation segment, it blocks the binding of peptide substrates. The
structure thus provides an explanation for why the phosphorylation of Tyr 416 is perhaps
the most crucial element of the switch to the activated state of the protein. In addition,
consideration of the structure of PPl bound to the active site reveals a means for
modifying this compound to build in specificity for the inactive forms of Src-family
tyrosine kinases.
(1) Sicberi, F., Moarefi, 1., and Kuriyan, J. (1997). Crystal structure of the Src family
tyrosine kinase Hck. Nature 385, 602-609.
(2) X u, W., Harrison, S., and Eck, M. 0997). Three-dimensional structure of the tyrosine
kinase c-Src. Nature 385, 595-602.
(3) Williams, J., Weijland, A., Gonfloni, S., Thompson, A., Courtneidge, S., SupertiFurga, G., and Wierenga, R. (1997). The 2.35 A crystal structure of the inactivated form
of chicken Src: a dynamic model with multiple regulatory interactions. Journal of
Molecular Biology 274, 757-775.

Abstracts FEBS'99

s29

4.2 Macromolecular recognition and assembly

Biomolecular Cages for Protein Folding and
for Protein Degradation.
Robert Huber
Max-Planck-lnstitutfur Biochemie, D-82152 Martinsrted
Recent crystal
structural studies of the Thermosome, an archaeal
homologue of the eukaryotic chaperonin
CCT (I), of the yeast 20S
proteasome (2), and of the E. coli HslV protein (3) provide detailed views of
the subunit structures and arrangements of these large homo- or heterooligomeric complexes. The fold of the thermosomal ct- and 13- subunits
resembles that of the E.coli GroEL monomer and the proteasomal and the
HslV subunits resemble the Ntn hydrolases. The particles are cyclic
symmetric, pseudo-D8 in the hexadecameric thermosome, pseudo-D7 in the
(0t,13) tetradecameric proteasome, and D6 in the duodecameric HslV. By
these architectures hollow particles with large internal cavities are generated
in which the protein substrates are processed. All three complexes observed
are closed in their respective crystal forms and the cavities are quite
inaccessible from the outside such that substantial rearrangement of
segments, domains, or subunits is required for macromolecular substrate
entry and binding. Detailed structural information for any of these
hypothetical open forms is still lacking. Binding of ATP and its analogues
induces small local changes and large domain rotations in the thermosome
possibly representing intermediates between closed and open forms in the
catalytic cycle, but leaves the overall conformation closed in all crystal
forms studies. Very detailed structural information is available for
proteolysis by the proteasome through small substrate binding studies and
mutagenesis experiments explaining the specificity, the processivity, and
the preferred length distribution of its peptide products.
(1) Crystal structureof the Thermosome,the archaeal chaperoninand homologueof CCT.
L. Ditzel, J. L6we, D. Stock, K.-O. Stetter, H. Huber, R. Huber, S. Steinbacher. (1998) Cell
9.3_,125-138.
(2) Structureof the 20S proteasomefrom yeast at 2.4,~ resolution.
M. Groll, L. Ditzel, J. Lbwe, D. Stock, M. Bochtler, H.-D. Bartunik, R. Huber. (1997)
Nature 386, 463-47l.
(3) Crystalstructure of the heat shock locus V (HslV fromE. coll.
M. Bochtler,L. Ditzel, M. Groll, R. Huber
(1997) Proc. Natl. Acad.Sci.USA94, 6070-6074.

TROSY - a new NMR approach for studies of

intermolecular recognition in macromoleeular assemblies
K. Wiithrich, K. Pervushin, R. Rick, M. Salzmann, G. Wider
lnstitutfiir Molekularbiologieund Biophysik, ETH H6nggerberg,
CH-8093 Ziirich, Switzerland
A new NMR experiment, TROSY (transverse relaxation-optimized spectroscopy), promises to greatly expand the molecular size limit for NMR studies
in solution. TROSY makes use of the fact that cancellation of transverse relaxation effects can be achieved for one of the four multiplet components observed for 15N-tH moieties, and TROSY exclusively observes this narrow
component [ 1]. Theory predicts that the extent of this cancellation effect is dependent on the polarizing magnetic field Bo: at 1H NMR frequencies in the
range 900-1000 MHz it may be nearly complete, but TROSY yields significantly narrower spectral linewidths and improved sensitivity for observation
of 15N-1H groups already at 750 MHz, as was recently verified using experiments with a protein of molecular weight 110000. In TROSY-type experiments, transverse relaxation-optimization is applied during evolution periods
and data acquisition. The more recently introduced CRINEPT scheme now
makes further use of cross relaxation effects for improved efficiency of magnetization transfers in very large molecules [2]. NMR experiments based on
TROSY and CRINEPT, and using suitably isotope-labeled systems can now
yield informative data on proteins or nucleic acids in particles with molecular
weights of several hundred thousand. Potentially attractive applications are
studies with isotope-labeled membrane proteins solubilized in unlabeled micelles or lipid vesicles, labeled proteins attached to unlabeled nucleic acid
fragments, or vice versa, homooligomeric proteins, and segmentally labeled
large proteins or nucleic acids, i.e., quite generally systems for which the
NMR spectra are not overly complex in spite of the large size.
[1] Pervushin, K. et al., Proc. Natl. Acad. Sci. USA, 94, (1997), 12366. [2]
Riek, R. et al., Proc. Natl. Acad. Sci. USA, (1999), in press.

Structure and Assembly of Modular Proteins

lain D. Campbell
Departmentof Biochemistry, Universityof Oxford,
South Parks Road, Oxford, OX] 3QU, UK

Many proteins are constructed from a series of domains or modules structural scaffolds that have been favoured in evolution[i,2]. In most
cases modules can be recognised relatively easily at the sequence level and
examples of many different module structures are now known. This
information has been derived, in most cases, using a "dissection"
approach, where identified regions of a protein are produced by
recombinant expression and their structures determined by NMR or X-ray
crystallography. Modularity appears to provide biological systems with
several advantages including: a convenient way of presenting appropriate
ligand binding sites in the right position; a way of producing regulatory
effects by spatial rearrangement of modules and a way of forming
appropriate signal-transducing modular assemblies. Recent studies in this
laboratory, of a wide range of modules derived from proteins of the cell
surface, the extracellular matrix and intracellular signalling will be used to
illustrate some common features of module structure and assembly.
Special emphasis will be placed on sites on modules that interact with
other molecules and on relative flexibility between modules.
[1] Campbell I.D. (1998) The modular architecture of leukocyte cellsurface receptors. Immunological Reviews163, 11-18
[2]. Campbell 1D & Downing AK (1998) NMR of modular proteinsNature
Struct. Biol. 5, 496-499

Function and structure of 2D

membrane protein crystals
H. Stahlberg, B.Heymann, D.J. Mi,iller, L. Hasler,
D. Fotiadis, S. Scheuring, S.A. MUller & A. Engel
M.E.Miiller-lnstitute, Klingelbergstrasse 70, CH-4056 Basel.
One third of all sequenced genes code membrane proteins. In contrast, only
12 membrane proteins have been solved to atomic resolution compared to
4000 soluble proteins. To assess structure and function of a membrane
protein it is reconstituted into a two-dimensional (2D) crystal in the presence
of lipids. Molecular symmetry and stability of the solubilized protein promote
2D crystallization, which is driven by hydrophobic interactions during
detergent removal. Crystallographic packing depends on the nature of the
lipid, lipid-to-protein ratio, temperature, counterions, ionic strength and pH.
Stable proteins allow the use of octyl type detergents that are suitable for
dialysis driven crystallization. Fragile proteins require dodecyl type detergents
that are removed by adsorption to biobeads or diluted below their critical
micellar concentration.
Since the protein is reconstituted into a bilayer, its function can be assessed.
Examples to be discussed are the water flux in aquaporin crystals (1) and the
closure of a bacterial porin (2). The atomic force microscope is the tool of
choice to determine the surface topography of membrane proteins under
physiological conditions, as well as their dynamics. Their three-dimensional
structure, however, is elucidated by electron crystallography of frozen
hydrated 2D crystals. An example is the emerging structure of AQPI, the
water channel of the human erythrocyte (3).
(1) T. Walz, B. L. Smith, M. L. Zeidel, A. Engel, P. Agre, J Biol Chem
269, 1583-1586 (1994; (2) D. MUller, A. Engel, J. Mol. Biol. 285, 13471351 (1999); (3) T. Walz, et al., Nature 387, 624-7 (1997).

s30

Abstracts FEBS'99

5.1 RNA splicing and trafficking

Structure and function
of major and minor spliceosomal UsnRNPs
R. Ltihrmann, lnstitutftirMolekularbiologieundTumorforschung,

Negative regulation of splice sites recognition by RSF1,

a pre-mRNA splicing repressor
J. Tazi~, E. Labourier~, E. Allemanda, H-M Bourbon b
"1 GM. UMR5535/CNRS, F34293Montpelher. France
I'CBD. UMR5547/CNRS, F31062 Toulouse, France

Philipps-Universitiit Marburg, Emil-Mannkopff-Straj3e 2, D-35037

Marburg (Germany)

The major snRNPs Ul, U2, U4/U6 and U5 are trans-acting factors
required for pre-mRNA splicing. These particles, together with additional
factors, assemble in an ordered fashion onto the pre-mRNA to form a
functional splicing complex, the spliceosome, in which catalysis occurs.
We are analysing the structure of snRNPs and their role in spliceosome
assembly and splicing, with particular emphasis on the snRNP protein
moiety. With more than 60 distinct proteins identified, snRNPs purified
from HeLa cells exhibit a surprisingly complex protein composition This
is particularly true of the 25S [U4/U6.U5] tri-snRNP which, in addition to
the Sm core polypeptides, contains 24 particle-specific proteins not present
in U1 or U2 snRNPs, cDNA cloning of the tri-snRNP proteins revealed a
striking diversity of sequence motifs including novel RNA-binding motifs,
RNA helicase and GTPase domains as well as a novel cyclophllin. Several
of the proteins are implicated in the regulation of both RNA and protein
conformational changes which occur in the tri-snRNP during the splicing
process We have also carried out a comprehensive characterization of the
proteins of purified [U4AJ6.U5] tri-snRNPs from the yeast S. cerevisiae,
using mass spectrometry (in collaboration with G. Neubauer and M.
Mann) Most of the tri-snRNP proteins are evolutionarily highly conserved
between yeast and human The results of biochemical and genetic
experiments investigating the role of individual tri-snRNP proteins m
splicing will be presented Finally, we have started to investigate the
protein composition of the minor snRNPs UI 1, UI2, U 4 and U6,~ from
HeLa cells These particles, together with U5 snRNP, form the so-called
minor spliceosome which catalyses the splicing of a rare class of introns
with unique splice sites. Interestingly, in addition to proteins which are
uniquely present in the minor snRNPs, a large number of proteins are
shared by the major and minor snRNPs. Our data shed light on the
evolutionary relationship between the minor and major spliceosome.

Specific recognition of splice sites within metazoan mRNA precursors

is a stage of possible regulation by alternative splicing. Such regulated
splicing can lead, from a single array of coding sequences, to the
production of different proteins or alternatively, to a lack of protein
expression by introduction of a premature stop codon. Splice sites can
be activated by exonic enhancer sequences (ESE) which are bound by
splicing factors of the SR protein family. These factors which contain
one or two RNA binding domain and arginine (R) serine (S) repeats
(RS domain), are required for early recognition of splice sites during
assembly of the ribonucleoprotein structure "spliceosome" where
splicing take place. The SR proteins are known to stabilize the binding
of constitutive splicing factors like the U1 small nuclear
ribonucleoprotein particle (U1 snRNP) to the 5' splice site and U2
auxiliary factor (U2AF) to the 3' splice site. More recently, we have
characterized a novel splicing regulator (RSF1), identified from
Drosophila, that functionally antagonizes SR proteins. Like SR
proteins, RSF1 shows a modular organization with an amino(N)terminal RNA binding domain while its earboxyl(C)-terminal part is
enriched in glycine (G), arginine (R) and serine (S) residues (GRS
domain). RSF1 binds ESE but inactivates splice sites selection. RSF1
induces a dose-sensitive inhibition of splicing for several reporter premRNAs, an inhibition that occurs at the level of early splicing
complexes formation. RSFI interacts, through its GRS domain, with
the RS domain of the SR protein SF2/ASF and prevents the latter
from cooperating with U1 snRNP in binding pre-mRNA.
Furthermore, overproduction of RSF1 in the fly rescues several
developmental defects caused by overexpression of the splicing
activator SR protein B52/SRp55. Therefore, RSF1 may correspond to
the prototypical member of a novel family of general splicing
repressors that selectively antagonize the effect of SR proteins on 5'splice-site recognition.
Analysis of RNA export routes in yeast

Structure and biosynthesis of snoRNPs

I. Bozzoni, T. Villa, A. F. E. Caffarelli
Department of Genetics and Molecular Biology,
University of Rome "La Sapienza". 1 - 00185 Rome. Italy.

The eukaryotic nucleoli contain a large number (more than 100) of small
RNAs (snoRNAs) that have been classified into two major classes based on
their: i) function ii) primary sequence and structural conserved elements and iii)
protein factors with which they interact. Besides a few snoRNAs which are
involved in rRNA processing, most of the members of the two classes
participate in the two major post-transcriptional modifications of rRNA: 2'-0methylation (C/D box snoRNAs) and pseudouridilation (H/ACA box snoRNAs).
Another interesting feature is that most of the members this new family of RNAs
are encoded in introns of protein coding genes and are produced not by
independent transcription but, instead, by processing of the pre-mRNA in which
they reside. Thus, the function, structure and biosynthesis of these RNPs are
very new and interesting topics to be studied. We have extensively studied the
structure and biosynthesis of U 16 snoRNA in X.laevis and that of U I8 snoRNA
in S.cerevisiae. Both snoRNAs belong to the CJD box family. UI6 snoRNA is
released from its intron precursor by endonucleolytic cleavages through a
pathway which is alternative to splicing. The conserved C and D box elements,
which have been shown to be indispensable both for processing and for stability
of the snoRNA, play their role by binding to specific protein factors. In vivo UV
cross-linking experiments have shown that three proteins specifically interact
with U16 snoRNA: p40 (fibrillarin) and p68 which require intact boxes C and D
and p62 which requires a region comprised between the two boxes. These
factors are likely to represent common general factors for the whole class of C/D
box snoRNAs. The yeast intron-encoded UI8 snoRNA is processed mainly
through exonucleolytic digestion of the host intron, but also through a splicingindependent pathway relying on endonucleolytic digestion of the unspliced host
pre-mRNA. Inspection of the host intron revealed the presence of two long and
complementary sequences surrounding the UI8 coding region, potentially
forming an intramolecular stem. Mutational analysis of these sequences showed
that the external stem is necessary for accumulation of UI8 snoRNA from both
processing pathways. From the comparison of the yeast and vertebrates studies a
general model for the intron-encoded snoRNA biosynthesis can be derived.

E.C. Hurt

Biochemie-Zentrum Heidelberg (BZH), Universit?itHeidelberg,

Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany
The nuclear pore complex (NPC) is a 120 MDa structure in the nuclear
membrane that mediates bidirectional nucleocytoplasmic transport. Through
the combination of genetic approaches and biochemical assays, we identified a
vast number of yeast nuclear pore proteins (collectively termed nucleoporins or
Nups) and transport factors specific for different cargo molecules passing
through the nuclear pores. By genetically exploiting mutants of the Nup84p
complex, we found the nuclear import receptor for hnRNP-like proteins
(Mtrl0p/Kapllp),

and export factors for mRNA (Mex67p, Mtr2p).

By

genetically exploiting the Nsp 1p complex, a nuclear export receptor for tRNA
(called Los lp) could be characterized. To identify components involved in the
nuclear export of ribosomes, an in vivo assay was developed exploiting a GFPtagged version of ribosomal protein L25. After its import into the nucleolus,
L25-GFP assembles with 60S ribosomal subunits which are subsequently
exported into the cytoplasm. In wild-type cells, GFP-labeled ribosomes are
only detected by fluorescence in the cytoplasm. However, thermosensitive
rnal-I (Ran-GAP), prp20-1 (Ran-GEF) and nucleoporin nup49 and nspl
mutants are impaired in ribosomal export as revealed by nuclear accumulation
of L25-GFP. Nuclear export of ribosomes thus requires the nuclear/cytoplasmic
Ran-cycle and distinct nucleoporins.

Abstracts FEBS'99

A novel RNA binding protein component

of the U snRNA export complex
M. Ohno, A. Segref, A. Bachi, M. Wilm,
M. Fornerod, I,W. Mattaj
European Molecular Biology Laboratory,
Meyerhofstrasse 1. D-69117 Heidelberg, Germany.
In metazoans, splicesomal U snRNAs are initially exported from the nucleus
after transcription, and the cytoplasmic phase is essential for U snRNP
assembly. The monomethylated cap structure of U snRNAs is an essential
signal for the export process. Two factors are already implicated in U snRNA
export, the leucine-rich nuclear export signal receptor CRMl/exportin 1
(Xpol) and the nuclear cap binding complex (CBC). We hypothesised that
Xpol interacts (directly or indirectly) with CBC in a RanGTP-dependent
manner while CBC binds to U snRNA via the cap structure. However, a
mixture of U1 snRNA, CBC, RanGTP and Xpol fails to form a complex in
vitro.
We have identified a novel 55 kd RNA binding protein (p55) that appears to
bridge the interaction between Xpol and CBC. Formation of the complex
containing Xpol, p55 and CBC is strictly dependent on the presence of
RanGTP, and capped U I snRNA stimulates complex formation, suggesting
strongly that this complex represents the U snRNA export complex. Xenopus
oocyte microinjection experiments indicate that p55 moves from the nucleus to
the cytoplasm together with U snRNA via the Xpol pathway and recycles
back to the nucleus via the classical NLS pathway. Moreover, co-injection of
recombinant p55 stimulates U snRNA export. These results indicate that p55
is a novel component of the U snRNA export complex. Interestingly,
interaction with Xpol, and therefore U snRNA export complex assembly, is
controlled by modification of p55.

s31

s32

Abstracts FEBS'99

5.2 RNA editing and other epigenetic modifications

Mechanism and evolution of RNA editing in
mitochondria of kinetoplastids
R. Benne, D. Blom, J. van den Burg, A de Haan,
C.K.D. Breek, D. Speijer, P. Sloof
Dep. of Biochemistry/AMC, Amsterdam, The Netherlands

In kinetoplastid protozoa, mitochondrial (mt) mRNAs are posttranscriptionally altered by insertion and deletion of uridylate
residues, the information being provided by guide (g) RNAs. This
remarkable process thus generates transcript sequences that are
different from those encoded by the mt genome and is one of the
many forms of RNA editing that are currently known. U
insertion/deletion editing is widespread in the kinetoplastid lineage,
suggestive of an ancient evolutionary origin. Current popular
mechanisms envisage a series of consecutive 'cut-and-paste'
reactions, during which pre-edited mRNAs are cleaved at one editing
site by endonucleases, followed by terminal uridylate transferasemediated U-addition to the 3' end of the 5' cleavage product in the
case of insertion or exonuclease-mediated U-removal in the case of
deletion. In the final step, the two halves of the RNA are joined by
an RNA ligase. In order to shed further light on the mechanism of
RNA editing, we have identified and characterized a number of mt
proteins that have affinity for the 3' oligo (U) extension of gRNAs in
UV cross-linking assays. Most of our recent work has been focused
on two gRNA-binding proteins of approximately 30 kDa from
mitochondria of the insect trypanosomatid Crithidiafasciculata. One
of these proteins, uBP30.1, turned out to be the C fasciculata
homologue of a gRNA binding protein of 21 kDa (gBP21) from
Trypanosoma brucei, a protein that has recently been found in
association with active RNA editing complexes ('editosomes'). The
other protein, uBP30.2, has no homology to known proteins. Both
proteins are basic with pI's around 9.7, bind to gRNAs with high
affinity provided the gRNAs are equipped with a U-tail and are
present in large macromolecular complex(es) that also contain
gRNAs and mRNAs, but no rRNAs. We conclude that we have
identified two components of the C. fasciculata editosomal
machinery that interact with the U-tail of gRNAs via novel RNAbinding motifs.
Guide snoRNAs for the modification of rRNA nucleotides
J.P Bachellerie, J. Cavaille and L H Q u
Laboratotre de Biologte ivlol~culatre Eucaryote du (~N R.,S~. Untverslte
Paul-Sabatwr, 31062 Toulouse Cedex, France

Eukaryotic ribosomal RNAs contain two prevalent types ofnucleotide

modifications, methylations on the ribose and pseudouridines, each present at
about 50-100 sites per ribosome. Both modifications occur on the primary
transcript, exclusively within strongly conserved domains of the mature rRNA
sequences.
Over the last few years, more than one hundred novel small nucleolar RNAs
(snoRNAs) have been identified, which are intronic in vertebrates but mostly
non-intronic in yeast, and belong to only two distinct families. The first family
is termed box C/D antisense snoRNAs, because of the presence of two short
sequence motifs and of a 10-20 nt long complementarity to rRNA The second
family is called PdACA snoRNAs, because of an ACA trinucleotide located 3
nt from the 3' end, and of a related H motif in the hinge region linking the two
long stem structures shared by all these snoRNAs. These two families have
been shown to guide the ribose methylations and the pseudouridylations,
respectively, of eukaryotic rRNAs, in both cases through formation of a
specific RNA duplex at the modification site [1, 2].
We will review our present knowledge about how those novel RNAs guide the
nucleotide modifications and will also report further investigations aimed at
using tailored guide snoRNAs as tools for targeting nucleotide modifications
onto non-ribosomal RNAs, in order to alter gene expression in a highly
selective way.
An intriguing aspect of the two families of guide snoRNAs is the diversity and
peculiarities of their genomic organization and mode of expression among
eukaryotes. However, despite this diversity, some common features can now be
seen, reflecting a coordination of their biosynthesis with that of components of
the translational machinery. This is apparent not only at the transcriptional
level, but also at the RNA processing level. Thus, in yeast the processing of
both box C/D antisense snoRNAs and H/ACA snoRNAs involves enzymes
which also process pre-ribosomal RNA, i.e. exonucleases Rat l and Xrn 1 and
endonuclease RNase 1II [3].
References
1. Bachellerie, J.P, and Cavaillr, J. 1997. TrendsBiochem. 22:257-261.
2. Ganot, P., etal. 1997. Ce//89:799-809.
3 Qu, L H . , e t a l . 1999. Mol. Cell. Biol. 19'1144-1158.

Pre-mRNA editing and tRNA modification by adenosine

to inosine conversion is catalyzed by related enzymes
W. Keller, J. Wolf, A. Gerber
Biozentrum. Umversity of Basel, CH-4056 Basel, Switzerland

Eukaryotic adenosine deaminases that act on RNA (ADARs) comprise a

family of enzymes that convert adenosine (A) to inosine (I) in RNA. The
mammalian ADARI and ADAR2 proteins convert A to I in extended dsRNA
and are implicated in the site-specific editing of diverse synaptic receptor premRNAs in the brain [1]. Recently, we reported the identification of an RNAspecific adenosine deaminase from Saccharomyces cerevisiae (termed
Tadlp/scADAT1) that selectively converts A at position 37 to inosine in
eukaryotic tRNA-AIa. Tadlp has significant sequence similarity to the Cterminal part of the mammalian editing enzymes [2]. These findings suggest an
evolutionary link between pre-mRNA editing and tRNA modification.
We have now identified two additional yeast proteins (termed Tad2p and
Tad3p) that selectively convert adenosine to inosine at the wobble position
(position 34) of the anticodon in tRNAs. Both genes are essential and
temperature sensitive mutants were obtained. Extracts prepared from these
mutants fail to deaminate A to I in tRNA. Tad3p copurifies with a tagged
version of Tad2p complementing a TAD2A strain. Pure fractions containing
Tad2p and Tad3p selectively convert A to I at position 34 in several tRNAs.
Interestingly, both proteins contain a deaminase domain that is more related to
the catalytic domain found in cytidine and deoxycitydylate deaminases than
to that of ADARs. Therefore, Tad2p and Tad3p belong to a new family of
adenosine deaminases that act on RNA.
In addition, we have cloned E. coli and human TAD2 homologs. The
partially purified K coli ADAT2 selectively converts A to I at the wobble
position of E. coil tRNA-Arg2.
[1] Maas, S., Melcher, T. and Seeburg, P.H. (1997) Curr. Opin. Cell Biol. 9,
343.
[2] Gerber, A., Grosjean, H., Melcher, T. and Keller, W. (1998) EMBO J. 17,
4780.

Transfer RNA modification: Influence on translational

frameshifting and metabolism
G.R.Bj6rk., J.M.B.Durand, T.G.Hagervall, R.Leipuviene,
H.K.Ltmdgren, K.Nilsson, C.Peng, Q.Qian and J.Urbonavirius
Department of Mwrobiology Ume~ University, Umegt,Sweden

According to the dual error model for frameshiffing [1], a pause

occurs induced by a shortage of an aa-tRNA or by a slow entry
of it into the A-site. During this pause, a near-cognate tRNA is
accepted (first error). Following a normal three-nucleotide
translocation this near-cognate tRNA slip into the +1 or -1
frame, since its anticodon-codon interaction in the P-site is
aberrant (second error). Instead of a near-cognate tRNA, a
modification deficient or otherwise altered tRNA may also be
prone to frameshift in the P-site. According to this new model,
modification deficient tRNA may influence reading frame
maintenance at two steps during the translation cycle: 1) at the
aa-tRNA selection step if modification deficiency reduces this
rate and thereby causing a near-cognate tRNA to be accepted
instead at the A-site. Following a three-nucleotide translocation
of the near-cognate tRNA, it may shift frame while residing in
the P-site. Of course, if the undermodified tRNA is inducing a
long pause and if no near-cognate tRNA can compete, the wild
type tRNA already in the P-site may shift frame. 2) following the
translocation step when the hypomodified tRNA resides in the
P-site the frequency of frameshifi may increase provided that it
is accepted at a reasonable rate to the A-site. tRNA modification
as a regulatory device will be discussed and evidence that tRNA
modification regulates central [2] and thiamine metabolism as
well as the virulence of a bacterium [3] will be reviewed.
[1] Qian, Q. et al., Mol. Cell, 1, (1998) 471.
[2] Person, B. C. et al., J.Bacteriol., 180, (1998) 1344.
[3] Durand, J. M. B. et al., J. Bacteriol., 179, (1997) 5777.

Abstracts FEBS'99

s33

5.3 Architecture, design and evolution of RNA

Inhibition of gene expression with the
hammerhead ribozyme

RNA Folding and C a t a l y s i s

David M.J. Lilley

B. Bramlage, E. Luzi and F. Eckstein

CRC Nuclew A cid Slrlt('lltlV Research Grolq~, l)epttflment of Btochemisto~

Untversity qf l)umlee. I)mulee 1)1)1 4HN, UK. dmjhllev@ba~Ldumlee.ac.uk

The small nucleolytic ribozymes carry eta site-specific cndonuclcolytic

transestefification reactions, whereby the oxygen atom of the 2"-hydroxyl attacks
the 3'-plmsphorus via an SN2 mechanism. The reaction proceeds via a transition
state in which the attacking 2" oxygen atom, the 3' phosphorous atom and the
leaving 5' oxygen atom are colincar, and a naaitn"part of the rate enhancement is
likely to come from conformational facilitation of the tra,icctory into the in-line
transition state. Thus folding and catalysis are tightly linked in the mechanism of
fibozyme cleavage.

Max-Planck-lnstitut fiir experimentelle MedJzin.

Hermann Rein Str 3, D-37075, Gottingen, Germany

The luciferase gene, in two different constructs, has been selected as target for
testing the hammerhead ribozyme for the inhibition of gene expression in cell
culture. One is a cell line which stably expresses Iuciferase under control of
the HIV- l promoter where the ribozyme was directed against a position in the
LTR. In the other construct the ribozyme was directed against the luciferase
gene under control of a tetracycline-regulated promoter. Ribozyme-susceptible
sites were identified by incubation of transcripts with ribozymes with
randomised binding arms. Transfection was done with cationic liposomes.

The hammerhead and hairpin ribozymcs tllldCl'gO well-dcfiricd ion-irtduced

folding events that are essential to generate the catalytically competent species.
The hammerhead ribozymc undergoes two separate folding events induced by the
sequential binding of two magnesium ions. The first is the formation of a coaxial
belic',.d stacking that forms the scaffold on which the structure is built, while the
second is the formation of the catalytic core itself. The two structural metal ions
bind with apparent association constants tit I(},/R)Oand 1,1)0() M -1 respectively.

Ribozymes with inverted binding arms served as control and inactive

ribozymes with mutations in the core served to determine any antisense effect.
Also ribozymes with chemical modifications at the U-position and at the
termini were used. The degree of inhibition was around 50% for both gene
constructs with little difference for the catalytically inactivated ribozymes,
indicating a considerable antisense effect for inhibition. Also, a comparison
between chenfically modified and non-modified ribozymes showed similar

The natural l'onn of the hairpin ribozymc is a four-way helical junction, two arms
of which calvy Rn'mally-unpaimd loops. Physical association between these loops
is induced by the cooperative binding of two magnesium ions. This is required for
catalytic activity, and changes in the coufomaation of the helical junction lead to
reduction in cleavage activity.

degrees of inhibition. Thus, as observed by others, the cationic liposomes

offer sufficient protection against nucleases.
Cellular uptake was monitored by fluorescence microscopy using 5"fluorescein-labeled

ribozymes.

Chemically modified

ribozymes

were

predominantly present in the nucleus whereas the non-modified were

G S Bas~.i, A I I I Murclfic, F Wahcr, R M ('long and 1) M .I IAllcy (1997) Ion-reduced

distributed between cytoplasm and nucleus. As both types of ribozymes were

fokling o| the JiilflllnCl'llcHd I'it'Ki/ylnC, il I]tIOIC~.CCIK'CI'C>,OII~LIIC(2CIICr[2y[rHll~fcf MtJdy EMBO

equally active in inhibiting gene expression, it is assumed that the ribozymes

J. 1 6, 7481-7489.
A.I.I]. Murchie. J.B. Thomson, 1.. Vv'~tllcraild DM.J. I,dlcy (1998) Folding el the hairpifJ

in the cytoplasm are responsible for this activity. Cholesterol-modified

ribozyine ill its n~lulral olifornllilioti ~lChicvc~ close plly~ic:ll proxilnil)' of the loops

Molecular Cell l. 873-g81.

Catalysis of splicing reactions by group II intron

ribozymes.
Main Jacquier
URA 1300 CNRS, D6pt. des Biotechnologies, Institut Pasteur
25-28 rue du Dr. Roux, F-75724 PARIS cedex 15, FRANCE
Group I/introns are ribozymes able to catalyze their own excision. They use
the same splicing pathway as nuclear pre-mRNA introns. In particular, the first
splicing step consists in a transesterification reaction initiated by the nucleophilic
attack of the 5' splice junction by the 2' hydroxyl of an adenosine internal to the
intron. It generates a 2'-5' branched structure responsible for the lariat form of
the excised intron. This branching reaction, characteristic of this splicing mode,
is reversible. Wild type introns are however poorly competent for this reverse
branching reaction because a conformational rearrangement, occurring after the
first splicing step, switches the intron structure from a step one specific
conformation to a step two specific conformation. Only the first step specific
structure is competent for the reverse branching reaction. In contrast, mutant
lariat introns, in which a tertiary interaction specifically involved in the
stabilization of the step two specific structure has been disrupted, remain
predominantly in the step one specific structure and are able to perform reverse
branching very efficiently. Here, we present the use of these mutants and of the
reverse branching reaction to study both, RNA folding and catalysis of the
branching reaction in group II introns.
First, we used the reverse branching reaction to set up an in vitro selection
scheme. This approach was use to select 5'-exons optimized for their specific
interaction with the intron. We found that accessibility of 5'-exert sequences
involved in Watson-Crick interactions with the intron was limiting the folding
efficiency of the usual model precursor transcripts. Precursor molecules
harboring the selected 5'-exons splice faster and more homogeneously than any
of the previously analyzed precursor transcripts.
Second, the use of the reverse branching reaction allowed us to analyze for
the first time the catalytic rate of the f r s t transesterification reaction under
conditions where chemistry appears rate limiting. A detailed analysis of the metal
ion requirement for catalysis under these conditions showed that, unexpectedly,
substitution of magnesium ions by manganese ions accelerates the catalytic rate
of the first transesterification reaction by two order of magnitude. Furthermore,
we could show that this spectacular rate enhancement is due in part to the fact
that at least one metal ion binding site important for catalysis exhibits a
considerably higher affinity for Mn 2ions than for Mg 2ions.

ribozymes, although catalytically active in vitro, could not inhibit gene

expression when applied without carrier. They were contained mainly in the
cellluar membrane.
RNA recognition and splicing of p r e - t R N A
G.P.Tocchini-Valentini, P.Fruscoloni, M.I.Baldi, E.Mattoccia
ln.~titute o[ Cell Bioh~gy, C N R., "Campu~ A, Buzzatl-Traverso",
Via E. Ramarmi, 32, 00016 Monterotomh~ Scah~ (RM), Italy

Accuracy in tRNA splicing is essential for the formation of fanctional

tRNAs, and hence also for gene expression, in Bacteria, Archaea and
Eukaryotes. In both Archaea and Eukaryotes, the specificity ["or
recognition of the pre-tRNA resides in the endonucleases. The enzymes
remove the intron by making two independent endonucleolytic cleavages.
The Archaeal enzyme acts without making any reference to the mature
domain (mature-domain independent mode, MDI) and recognizes local
structures at the intron-exon boundaries. The Eukaryal enzyme normally
acts in a mature-domain dependent mode (MDD) [l]. We have previously
shown, however, that the Eukaryal endonuclease retains the ability to
operate in the MDI mode [2].
We constructed three series of variant substrates that can be cleaved by the
Eukaryal endonuclease either in the MDD or MDI modes or in both. The
substrates were utilized both in vitro and in vivo.
The results suggest an evolutionary pathway for cleavage site recognition.
[1] E. Di Nicola Negri et al., Cell 89, (1997) 859.
[2] S. Fabbri et al., Science 280, (1998) 284.

s34

Abstracts FEBS'99

Creation and Evolution of New Ribozymes

D. Barrel, P. Unrau, E. Schaltes, W. Johnston
Whitehead Institutefor Biomedical Research and Departmentof
Biology, MIT, 9 Cambridge Center, CambridgeMA, 02142

We have been isolating new RNA catalysts (ribozymes) from large pools
of random RNA sequences in an effort to better understand the basic
properties of RNA as a catalyst and to see whether these properties are
compatible with the RNA world scenario. The hypothesis that certain RNA
molecules may be able to catalyze RNA replication is central to the RNA
world scenario. In support of this idea, we have generated an RNA that
synthesizes short segments of RNA using the same reaction as that employed
by protein enzymes that catalyze RNA polymerization. This ribozyme is
serving as a starting point in efforts to evolve ribozymes capable of more
extensive and accurate polymerization. Another challenge for the RNA world
hypothesis is the source of nucleotide substrates needed for RNA
polymerization. Such nucleotides must have been synthesized from simpler
precursors. We have isolated RNA molecules that catalyze the synthesis of a
pyrimidine nucleotide at their 3" terminus. Optimization of these ribozymes to
the point where they synthesize non-tethered nucleotides would further
support the idea of an RNA world that included nucleotide synthesis and other
metabolic pathways mediated by ribozymes.
Other experiments are exploring the distribution of catalysts in RNA
sequence space. Examination of natural ribozyme isolates shows that the
same ribozyme motif can be represented by very different sequences.
Because the different sequences have descended from a common ancestor,
there are likely to be neutral paths in sequence space that connect these
different sequences, allowing evolutionary drift from one sequence to the
other without loss of function. The set of all possible neutral paths for a
particular ribozyme is thought of as a neutral network. We have evidence that
neutral networks for completely different ribozylne motifs can intersect. That
is, we have generated an RNA sequence that can fold into two different
ribozyme motifs. Because the molecule sometimes folds into one motif and
sometimes folds into the other motif, the same sequence catalyzes two
different reactions. The fact that networks for functional RNAs intersect
makes RNA an attractive biopolymer substrate for the birth of new function
early in the evolution of life. In principle, only one ribozyme needed to
emerge from arbitrary sequences at the beginning of the RNA world; all other
ribozymes could have descended from this founding ribozyme. A related
implication is that RNAs in modem biology with no structural or functional
similarities may have common ancestry. Furthermore, our results show that
evolutionary divergence can precede duplication, which differs from the
canonical view of divergence following duplication.

Abstracts FEBS'99

s35

6.1 Structure and function of chromatin

Structure and Function of Chromatin
T. J. Richmond, K. Luger, A. W. M~ider and D. F. Sargent
blstitut far Molekularbiologie und Biophysik, ETH Ziirich
Ztirich, Switzerland

The nucleosome, the elemental repeating unit of chromatin, is responsible for

the two most fundamental levels of DNA organization within the eukaryotic cell
nucleus. The nucleosome core (206 kDa) consists of an octameric protein
assembly comprising two copies of each of the four core histones, H2A, H2B,
H3, and H4, and a roughly equal mass of DNA. Compared to the nucleosome,
the core lacks only linker DNA and associated histone H 1.
The crystal structure of the nucleosome core particle has now been refined to
2.0 ~, resolution. The improved quality of the X-ray structure permits the reliabk
location of nearly 1000 water molecules and ions in the electron density map
(Rer,e = 21.4% ). These additional atoms make important contributions to the
histone-histone and histone-DNA interactions.
The histone protein chains are divided into three types of structures: I) rigid
alpha-helical domains named the histone-fold, 2) histone-fold extensions which
interact with each other and the histone-folds, and 3) flexible 'histone tails'. The
histone-fold domains form the H3-H4 and H2A-H2B heterodimers that are
responsible for organizing 121 bp of the 147 bp DNA in its 1.65 turns of lefthanded superhelix. The extensions just prior to the H3 histone-folds bind the
first and last 13 bp of the superbelix. The flexible tails of the histones reach out
between and around the DNA gyres to contact neighboring particles. Only about
one-third of these flexible histone tails is observed in the electron density map,
implying that these regions are meant to make inter-nucleosomal interactions
which perhaps facilitate the formation of nucleosome higher-order structure and
its dynamic alteration. One such possible interaction is suggested by the crystal
packing.
Hydrogen bonds between the protein mainchain amide and DNA phosphate
groups appear to rigorously specify the DNA conformation, most likely making
the DNA structure at each of the 14 histone-DNA binding sites independent of
sequence contributions. An approximately equal number of sidechain-DNA
hydrogen bonds are both direct and water mediated. A twist distortion seen in
one half of the DNA superhelix suggests a mechanism for chromatin
remodeling.

Chromatin Remodeling In Vivo

W. H6rz, P.D.Gregory, M.Mttnsterk6tter, G.Ertinger
lnstttutfur Physiologtsche Chemte, UniversitditMiinchen,
Schillerstr 44, 80336 Miinchen, German3,

We are investigating the mechanism of chromatin remodeling that is characteristic of many regulated promoters during gene activation and are focusing on the P H 0 5 promoter from yeast [1]. In the repressed state, the
P H 0 5 promoter is organized in an array of positioned nucleosomes that is
only interrupted by a short hypersensitive site. Upon activating the gene by
phosphate starvation, two nucleosomes upstream and two downstream of
the hypersensitive site are disrupted, and the transcription factor Pho4 binds
to two UAS elements.
The transcription factor Pho4 is strictly required for the chromatin
transition, and we can show that not only the DNA binding domain but also
the activation domain is needed. Heterologous activation domains, for example from the glucocorticoid receptor, are also capable of triggering the
chromatin transition when fused to the Pho4 DNA binding domain.
When we bring the P H 0 5 promoter under galactose control by replacing either or both UAS elements by Gal4 binding sites we find that one
high afflmty Gal4 binding site is sufficient for the chromatin transition and
that again four nucleosomes are remodeled. The Gal4 DNA binding domain
can brad to a nucleosomal site in vivo, generating a triple complex between
bistones, DNA and factor. This binding results in a local chromatin perlurbation which is very different, however, from the four nucleosome transition
usually seen.
The historic acetyl transferase Gcn5 nmkes only a minor contribution
to P H 0 5 activation and chromatin opening and is largely dispensable. Hawever, under submaximally inducing conditions it is required for activation.
In its absence, a novel chromatin pattern at the promoter is observed consisting of randomized nucleosomes.
We have extended our studies to another structural gene of the PHO
family, the P H 0 8 gene which encodes a weakly expressed alkaline phosph,ttas~.. The contribution of the SWi/SNF remodeling machine and of Gcn5
at the P H 0 8 and the P H 0 5 promoter are strikingly different, and the role of
these activities will be discussed.
[1] Svaren J, Hrrz W: Tramcription Factors vs nack'o:somes: Rcgulation ofthc P H 0 5 promoter in yeast. Trends Btochem.Sci. 1997, 22:93-97.

CHRAC, an energy-dependent nucleosome mobiliser

Peter B. Beaker, Edgar Bonte, Davide Corona, Cedric Clapier,
Simona Ferrari and Gernot Lfingst
EMBL, 69012 Heidelberg, Germany.
The CHRomatin Accessibility Complex (CHRAC) has been purified from a
Drosophila embryo extract due to its ability to increase the accessibility of
chromatin towards DNA binding proteins in a global way. CHRAC is able to
convert the energy of ATP hydrolysis to catalyse several different reactions
involving nucleosomal substrates: (1) their remodeling, facilitating the inter-action
of a diverse range of DNA binding regulators with nucleosomal sites with
functional consequences in cell-free systems; (2) their repositioning with respect to
a ehromatin boundary and (3) their even spacing during nucleosome assembly
under physiological conditions. In order to account for these diverse effects we
suggest that CHRAC functions by a general "mobilisation" of nucleosomes.
Supporting this model we have recently demonstrated CHRAC-induced
movements of single nucleosomes.
The 670 kDa CHRomatin Accessibility Complex consists of five subunits. A 130
kDa subunit was identified as ISWI, a nucleosome-stimulated ATPase that is also
part of other chromatin remodeling complexes. The 160 kDa subunit proved to be
an active dimer of topoisomerase II. CHRAC therefore combines enzymes
involved in modulating DNA topology as well as nucleosome structure in a
complex molecular machinery with multiple enzymatic functions and several DNA
binding motifs.
Understanding the mechanism by which CHRAC establishes a dynamic chromatin
environment means defining the contributions of each individual subunit to the
remodeling reactions. Surprisingly, we discovered that the recombinant ISWI
ATPase itself, removed from the context of a large remodeling assembly, was able
to recognise nucleosome structure and to trigger bona fide energy-dependent
nucleosome remodeling and positioning reactions. The finding that the enzyme is
able to remodel chromatin by itself is at odds with the current paradigm, according
to which nucleosome remodeling requires the functional interaction of two or more
subunits physically associated in a remodeling complex. Recombinant ISWI is
also able to trigger nucleosome movements, Interestingly, the directionality of
nucleosome movements differs depending on whether ISWI alone or in the context
of CHRAC induces mobility. Thus the ISWI associated subunits most likely
regulate and modulate the activity of ISWI and further may define the
physiological context in which the remodeling reaction occurs.

Histone deacetylation and gene activity:

Mechanismeof repression
JianahengWu, Noriyuki Suka, Andrew Carmen and
MichaelGrunstein

Histone deacetylation is a key mechanism by which genes

may be repressed in eukaryotic cells. The yeast Saccharomyces
cerevisiae contains five histone deacetylases (HDAI, RPD3,
HOSI, HOS2 and HOS3) and our laboratory is pursuing the
different functions of these various enzymes. Of particular
interest are their targets and the effects of these enzymes on
the genes they regulate.

The histone deacetylase RPD3 interacts with the adaptor

protein SIN3 and the DNA binding factor UME6 to repress genes
adjacent to UME6 binding sites. To determine how repression
occurs, we have analyzed histone H4 and H3 acetylation in vivo
at genes adjacent to UME6 binding sites. This was done by
using antibodies against individual histone acetylation sites
in order to immunoprecipitate chromatin fragments. We have
identified all H3 and H4 sites deacetylated at the promoter
of the UME6 regulated gene, IN01. The structure and
specificity of other histone deacetylases in yeast will also
be discussed.

s36

Abstracts FEBS'99

CHROMATIN-INDUCED SILENCING AND THE

MECHANISM OF CELLULAR MEMORY

R. PARO, A. BREILING,I. CHEN, G. RANK and G. CAVALLI,

ZMBH, Universityof Heidelberg, 1NF282, D-69120Heidelberg

In Drosophila the Polycomb group (PcG) and trithorax group (trxG)

genes are necessary for a stable and heritable maintenance throughout
development of, respectively, repressed and active expression patterns of
many important regulatory genes. PcG components interact in multimeric
complexes and are bound to specific chromosomal elements termed PREs.
PcG have been proposed to act by packaging repressed chromosomal
domains into a heterocbromatin-like structure, perhaps via stable
interaction with the chromatin fiber. In collaboration with E. Bonte and P.
Becker (EMBL) we have obtained evidence that the Polycomb (PC)
protein, a major constituent of the PcG, depicts a specific in vitro binding
affinity for reconstituted nucleosomes. The main nucleosome binding
domain coincides with a region in the C-terminal part of PC previously
described as the repression domain. Our results suggest that PC interacts
with the N-terminal tails of the core histones and the nucleosomal DNA.
Components of both PcG and trxG exert their regulatory function
through chromosomal maintenance elements like Fab-7. We have set-up a
transgenic reporter system to analyze how F a b - 7 can sustain gene
expression patterns during development. A reporter gene like lacZ or white
flanked by F a b - 7 shows normally a reduced expression, indicating a
constitutive silencing ability of Fab-7. However, by utilizing an GAL4inducible lacZthe Fab-7 can be switched from the default silenced state to
an activated state with a single pulse of GAL4 early in embryogenesis.
This activated state can be maintained through development even after
GAL4 levels have decayed. Interestingly, activated F a b - 7 is also
meiotically stable and can maintain activity on neighboring reporter genes
into the next generations. With specific antibodies against different forms
of acetylated histones we found that mitotically stable activated F a b - 7
constructs are marked by hyperacetylated H4 but not by hyperacetylated
H3. Conversely, we found that transient gene activation of a Fab-7
reporter by GAL4 only results in a short and moderate H4
hyperacetylation. Thus, histone acetylation appears to play an important
part in the epigenetic marking of memory elements like Fab-7.

Abstracts FEBS'99

s37

6.2 Molecular recognition in transcription: the machinery

Mechanisms of transcription complex assembly
in eukaryotes and in Archaea
S.P. Jackson, S.A. Qureshi and S.D. Bell
Wellcome/CRCInstitute, Tennis Court Road, Cambridge, UK

The Archaea are a diverse range of organisms that constitute a defined

domain of life distinct from Bacteria and Eucarva. However, in recent
years it has become apparent that the transcription machinery of archaeal
organisms is fundamentally homologous to the core components of the
eucaryal RNA polymerase (RNAP) II apparatus. Archaea possess
orthologues of the TATA-box binding protein (TBP) and TFIIB (termed
TFB in Archaea). In addition, the archaeal RNAP has similar subunit
sequence and complexity to the eucaryal RNAPs. We have established a
defined basal transcription system for the hyperthermophilic archaeon
Sulfolobus acidocaldarius using recombinant TBP and TFB and highly
purified RNAP. This system has been employed to identify sequence
determinants of promoter strength. Remarkably, regions upstream of the
TATA-box were found to be important for full promoter activity. This
element, the BRE, is contacted by a helix-turn-helix DNA-binding motif in
TFB. The BRE is found in many archaeal promoters and plays a key role
in establishing the unique orientation of transcription.
Analysis of the complete sequence of archaeal genomes reveals the
presence of bacterial-like operons, some of which contain homologues of
bacterial-like transcriptional regulatory molecules. It is of considerable
interest to determine how these bacterial-like regulators impinge on
eucaryal-like basal machinery. We have characterised one such repressor
homologue and found that it specifically negatively regulates expression of
its own operon. It binds co-operatively to three operator sequences
overlapping the transcription initiation site. Repressor binding does not
affect binding of TBp and TFB to the TATA-box and BRE but does block
polymerase recruitment. Because of the relatedness of the archaeal and
eucaryal basal transcription machinery it is expected that further studies of
the archaeal transcription system will shed light on the evolution of the
eucaryal RNAP II machinery.

TAFu-mediated transcription in Drosophila

F. Sauer, An_h-DungPham & S. Mtlller
ZMBH, lm Neuenheimer Feld 282, 69120 Heidelberg, Germany

In eukaryots, enhancer-bound transcription factors are thought to contact

components of the general RNA polymerase II transcription machinery to
activate transcription. This machinery is a multi-protein complex comprised of
basal transcription factors (TFIIA, B, D, E, F, H, I), RNA polymerase II and
associated cofactors (1). One component targeted by activators is the basal
transcription factor 'ITIID, which is comprised of the TATA-box-binding
protein (TBP) and at least eight different TAFus. Different classes of
activation domains (i.e. glutamine-, proline-rich) interact with distinct TAFlts
and these interactions mediate transcriptional activation in vitro (2). However,
little is known about the role and function of TAFs in vivo. We have
combined powerful Drosophila genetics and in vitro biochemistry to
investigate the functional importance of TAFus for transcriptional regulation
in a metazoan model system Drosophila (3,4).
In Drosophila, the maternal morphogen Dorsal initiates the formation of
embryonic mesoderm by activating the zygotic expression of two regulatory
genes, twist and snail. Twist encodes a basic helix-loop-helix transcription
factor (Twist), which cooperates with Dorsal in order to activate its own
expression and snail expression (5). In vitro analyses indicate that specific
TAFn-subunits within the basal transcription factor TFIID mediate
transcriptional activation by Dorsal and Twist in vitro and in the Drosophila
embryo. Dorsal interacts with TAFH110 and TAFn60, Twist with TAFu110.
These TAFas serve as coactivators in order to mediate simple and synergystic
transcriptional activation by Dorsal and Twist in vitro. In Drosophila,
mutations in the corresponding TAFn-coactivators alleviate activation of twi
and sna expression by Dorsal or Twist. Gene dosage assays indicate that a
synergystic interplay of Dorsal and Twist with TAFn 110 activates mesodermdetermining gene expression, and hence initiates mesoderm formation, in
Drosophila. These results provide evidence that TAFns may play an essential
role for the molecular events leading to activation of gene expression in
metazoans (3,4).
(1) Roeder, R., Trends Biochem. Sci. 21 (1996) 327;
(2) Saner, F. & Tjian, R.,. Curt. Opin. Gen. Dev. 7 (1997) 176;
(3) Sauer, F., et al., Cell 87 (1996) 1271;
(4) Pham A.-D. et al., MOD, in press.
(5) Rush, J. and Levine, M., Curr. Opin. Genet. Dev. 6 (1996) 416.

Protein-protein interactions
within the RNA polymerase HI transcription machinery
A. Sentenac, E. Deprez, H. Dumay, M.L. Fen-i, C. Conesa,
O. Lefebvre, C. Marck and M. Werner
S.B.G.A~, CEA/Saclay, 91191 Gif-sur-YvetteCedex, France

A collection of 26 polypeptides participates in transcription complex

formation on the small class III genes in yeast, amounting to a total mass of
- 1500 kDa. A cascade of protein-DNA and protein-protein interactions
lead to the recruitment of RNA polymerase III. Two auxiliary multiprotein
complexes, TFIIIC and TFIIIB, direct the recruitment of Pol III to its
promoters. TFIIIC, a flexible, multifunetional factor plays the role of
enhancer binding proteins (via B block interaction), of proximal element
binding factor (through A block binding), of an antirepressor (relieving
chromatin repression) and of an activator (by recruiting TFIIIB). All six
subunits of yTFIIIC have been cloned and found to be essential for cell
viability. TFIIIB, the general initiation factor for class III genes, comprises
TBP, TFIIIB70/BRFI, and B"/TFIIIB90. The assembly of TFIIIB on the
DNA involves complex interactions between at least two subunits of
TFIIIC, x 131 and x60, and TFIIIB components, including TBP.
The mechanism of recruitment of PolIII by the TFIIIBoTFIIIC*DNA
complex also appears to be a sophisticated pathway. At least two specific
subunits of PolIII interact with TFIIIB. The PolIII subunit C34 plays a
critical role in transcription initiation. C34.TFIIIB70 interaction is
important both for PoUII recruitment and for open complex formation.
Another subunit of PollII, C17, interacts with the N-terminal part of
TFIIIB70 and its role is not redundant with that of C34. Our preliminary
results even suggest additional interactions between two other subunits of
PolIII and x131, the major TFIIIB-assembling subunit of TFIIIC. These
multiple interactions suggest that RNA polymerase is precisely guided to
the initiation site by the preinitiation complex and brought into an initiation
competent configuration. Evidence in favor of a PolIII holoenzyme will be
presented.
Chedin et al., In : Mechanisms o f Transcription, Cold Spring Harbor Symp.
Quant. Biol. Vol 63, (1998) 381.

Dissecting heavy metal stress response in mammals

W. Schaffner, O. Georgiev, A. Auf der Maur,
B. Zhang, P. Lichtlen
Institutfitr Molekularbiologieder UniversitMZitrich, CH-8057 ZLirich

The heavy metal-responsive transcriptional activator MTF-1 regulates the

basal and heavy metal induced expression of metallothioneins. Embryos lacking
MTF-I die in utero at around day 14 of gestation from liver decay, and
embryonic cells cultivated from earlier stages are sensitive to cadmium and
hydrogen peroxide. Concordant with this notion, transcription of metallothionein
I and II genes is very low, and transcription of the heavy-chain subunit of the
gamma glutamylcysteine synthetase, a key enzyme for glutathione biosynthesis,
is reduced. The expression of a number of other genes is also affected in such
MTF-1 null mutant embryos. The available data suggest that MTF-1 improves
the ability of cells to cope with heavy metal load and oxidative stress. To further
dissect the molecular aspects of heavy metal induction, we have established an in
vitro transcription system which depends on MRE sequences in the promoter and
responds to the presence of MTF-1, zinc and other heavy metals. We have also
cloned MTF-1 from other species, and find that MTF-1 from the pufferfish Fugu
can restore heavy metal response in mouse MTF-I null mutant cells.
Westin, G. & Schaffner, W. (1988) A zinc-responsive factor interacts with a
metal-regulated enhancer element (MRE) of the mouse metallothionein-I gene.
EMBO J., 7, 3763-3770.
Heuchel, R., Radtke, F., Georgiev, O., Stark, G., Aguet, M. and Schaffner,
W. (1994) The transcription factor MTF-1 is essential for basal and heavy metalinduced metallothionein gene expression. EMBO J, 13, 2870-2875.
Giines, C., Heuchel, R., Georgiev, O., MUller, K.-H., Lichtlen, P.,
Bltithmann, H., Marino, S., Aguzzi, A. and Schaffner, W. (1998) Embryonic
lethality and liver degeneration in mice lacking the metal-responsive
transcriptional activator MTF-I. EMBO J, 17, 2846-2854
Auf der Maur, A., Belser, T., Elgar, G., Georgiev, O. and Schaffner, W.
(1999) Characterization of the Transcription Factor MTF-I from the Japanese
Pufferfish (Fugu rubripes) reveals Evolutionary Conservation of Heavy Metal
Stress Response. Biol. Chem. 380, 175-185

s38

Abstracts FEBS'99

6.3 Gene regulations by nutrients and hormones

Peroxisome function in S a c c h a r o m y c e s cerevisiae

studied by SAGE
H.F. Tabak, MJ.A. Groot Koerkamp, A.J. Kal
Department of Btochemistry, Academic Medical Centre,
Metbergdreef l S, 1105 AZ Amsterdam, The Netherlands

We have studied peroxisome biogenesis and function in S a c c h a r o m y c e s

cerevisiae g r o w n on the fatty acid oleate, a condition that results in
peroxisome proliferation, using genome-wide transcript analysis by
SAGE.
A major fraction (>20%) of the 15,000 mRNA molecules in oleategrown cells comprised transcripts, which were derived from only 2% of
the total number of -6300 genes. Most of these mRNAs code for
enzymes or for other proteins participating in metabolism (e.g.
metabolite transporters). This was exemplified by the huge increase of
mRNAs encoding the peroxisomal [3-oxidation enzymes required for
degradation of fatty acids. The data provide evidence for the existence of
redox shuttles across organellar membranes that involve peroxisomal,
cytoplasmic and mitochondrial enzymes. We also analysed the mRNA
profile of a mutant strain with deletions of the P I P 2 and O A F 1 genes,
encoding transcription factors required for induction of genes encoding
peroxisomal proteins. Induction of genes under the immediate control of
these factors was abolished; other genes were upregulated in response to
the changed metabolism imposed by the genetic impairment.

Molecular genetic analysis of glucocorticoid-dependent gene expression

Holger M. Reichardt.Jan Tucker'mann,FrancoisTronche.ChrlstophKellendonk,

AntonBauer.HartmutBeug,Peter Angeland GuenlherSchuetz

German Cancer Research Center, Im Neuenhetmer Feld 280. 69120 Heidelberg.

Germany. D~vtston of Molecular Biology of the ('ell I

Glucocorticoids are involved in the regulation of numerous physiological

processes. Most of these effects are mediated by the glucocorticoid
receptor (GR) via activation and repression ofgene expression. Activation
requires DNA-binding of the receptor while repression is mainly mediated
by cross-talk with other transcription factors. To analyse GR function in
vivo several mo*use mutations were generated by gene targeting.
In order to separate the different modes of action, we developed mice
carrying a DNA-binding-defective GR. In contrast to mutants with a
disrupted glucocorticoid receptor gene these mutants termed GRdim are
viable, demonstrating that DNA-binding of the GR is not essential for
survival. Transactivating functions of the GR are absent in GRdim mice as
demonstrated by transfection studies, bandshift-experiments and loss of in
vivo-induction of gluconeogenic enzymes. However, transrepression by
the GR via cross-talk with AP-I is unaltered. Studies performed with
these mice revealed that glucocorticoid-dependent thymoeyte apoptosis
and proliferation of erythroblasts are abolished whereas the control of the
hypothalamo-pituitary-adrenal axis is only partially affected. In contrast,
repression of cytokine expression in thymocytes and macrophages is
unaffected. Additional experiments on the in vivo role of the DNA-binding
activity by GR in skin pathology, stress erythropoiesis and
immunosuppression will be discussed.
To adress the question of tissue-specific functions of the glucocorticoid
receptor mice carrying somatic mutations in the GR gene were generated
using the Cre/loxP system. Thereby it could be shown that selective
absence of GR in the nervous system leads to deregulation of the
hypothalamo-pituitary-adrenal axis as well as to behavioural deficits.
Parallels between these mutants and patients suffering from Cushing's
syndrom will be discussed.

Gene regulation by glucose

PPAR-alpha mediates an adaptive response to fasting

S. Vaulont, A. Kahn

W. Wahlia, J. Seydouxb, J. M. Peters c, F. J. Gonzalez c,

B. Desvergnea, S. Kerstena

1CGM, I N S E R M U:129,
24 Rue du Fg. Saint-Jacques, 75014 Paris - FRANCE

Glucose is an important regulator of gene transcription in all types of

organisms. In vertebrates, it especially regulates transcription of metabolic
genes in the liver and fat tissue, activating genes encoding enzymes and
regulators of the glycolytic and lipogenic pathways.
Working with the L-type pyruvate kinase gene we have found that in
hepatocytes glucose-dependant gene regulation requires :
- Presence of the GLUT2 glucose transporter, necessary to allow for an
effective depletion in glucose 6-phosphate (G-6P) under gluconeogenk
conditions.
- Phosphorylation of glucose to G-6P assured either by insulin-dependent
glucokinase or by another hexokinase isoform.
- Most likely, entry of G-6P in the pentose phosphate pathway.
- Modulation of a kinase/phosphatase cascade, in particular inhibition of the
5'AMP-activated protein kinase.
- Signalling through a glucose response complex assembled onto a glucoseresponse element (G1RE) located in regulatory regions of glucose-responsive
genes.
- The activators USFs belong to the complex, and are required for a normal
gene activation by glucose, as evidenced from the phenotype of knock-out
mice deficient in USF. The study of USF-defective knock-out mice suggests
that USF could be involved in nutritional activation of a whole class of genes
regulated by glucose, and not by insulin itself. In particular, lipogenic genes are
abnormally responsive to diet in USF-/- mice.
- The transactivation potential of USF would be modulated by a glucose
sensor system implying the COUP-TFII transcription inhibitor.
- The main role of insulin in the glucose response of genes like the L-PK gene
is to induce the glucokinase gene.
- Glucagon, through cyclic AMP, inhibits L-PK gene transcription mainly
through activation of PKA. The PKA catalytic subunit could act by
phosphorylating member(s) of the glucose-response complex, or of
contiguous transcription factors, e.g. HNF4.

"institute of Ammal Biology, Universay of Lausanne, CH-IOI5

Lausannne," ~Department of Physiology, University of Geneva Medtcal
School, CH-1200 Geneva, 'National Cancer Institute, NIH, Bethesda MD

Fatty acids and their metabolites are very potent signallingmolecules

during development and in adulthood. Release of large amounts of
fatty acids from the adipose tissue, followed by their oxidation in the
liver is induced by deprivation of food. Since the nuclear receptor
PPAR-alpha plays a role in regulating mitochondrial and peroxisomal
fatty acid oxidation it is likely to be involved in the transcriptional
response to fasting. To investigate this possibility, PPAR-alpha null
mice were subjected to a high fat diet or to fasting and their response
compared to wild-type mice. PPAR-alpha null mice chronically fed a
high fat diet showed a massive accumulation of lipid in the liver. This
phenotype was also observed in fasted PPAR-alpha null mice,
which, in addition displayed severe hypoglycemia and hypothermia
and showed a plasma metabolite profile that suggests a dramatic
impairment of fatty acid metabolism. These data demonstrate that
PPAR-alpha is part of a complex network of signalling systems in
the liver, which alters transcription to channel fuel molecules into the
appropriate metabolic pathways.

Abstracts FEBS'99

s39

Retinoid signaling
D. Metzger and P. Chambon
IGBMC, CNRS/INSERM/ULP/CoIII~ge de France,
BP 163, 67404 lllkirch Cedex, C.U. de Strasbourg, France.

Retinoids, the active metabolites of vitamin A, regulate complex gene networks

involved in morphogenesis, organogenesis, growth, cellular differentiation and
homeostasis. Two families of receptors that belong to the nuclear receptor
superfamily, the retinoic acid receptors (RARs) and the retinoid X receptors
(RXRs), are implicated in the transduction of the retinoid signal. RARs are
activated by all-trans retinoic acid (tRA) and its 9-cis isomer (9cRA), while
RXRs are activated by 9cRA only. The various RAR (RARct, [5 and T) and
RXR (RXRo~, I~ and T) isotypes are encoded by different genes, while their
isoforms (which differ in their N-terminal regions) are generated by differential
promoter usage and alternative splicing. The multiple RAR and RXR isotypes
and isoforms are conserved in vertebrate evolution, and display distinct spatiotemporal expression patterns in developing embryos and adult tissues
suggesting that each receptor performs some unique functions. As RXR/RAR
heterodimers bind much more efficiently to RA response elements than their
respective homodimers in vitro, it was suggested that these heterodimers
represent the functional units transducing the retinoid signal in vivo [ 1].
To better understand RAR and RXR functions, a panel of F9 murine
embryonal carcinoma cell lines has been generated which are null for the
expression of either RARe, RAR T or RXRct, and RARct/RXRct or
RART/RXRot combinations [2, and refs therein]. The F9 cell line is a well
established cell autonomous model system for the study of retinoic acidinduced differentiation, antiproliferation and apoptosis. Mutant mouse lines
were also established for each receptor by homologous-mediated gene targeting
[3 and refs therein]. The analysis of single and compound mutant F9 lines and
mice revealed that RXR/RAR heterodimers are the functional units mediating
the retinoid signal in vivo, and that RXR/RAR heterodimers can exert both
specific and redundant functions.
To investigate the physiological role in defined target tissues of the receptors
that mediate signals with highly pleiotropic effects, and/or whose gene
knockouts are lethal in utero or during the perinatal period, somatic
mutagenesis is required. A method to create spatio-temporally controlled sitespecific somatic mutations will be presented.
[1] Chambon, P. Semin. Cell Biol. 5, (1994) 115. Chambon, P. FASEB. J.
10, (1996) 1187. Mangelsdorf, D. et al., Cell, 83, (1995) 835.
[2] Chiba, H. et al., Molecular and Cellular Biology, 17, (1997) 3013.
[3] Kastner, P. et al., Cell, 83, (1995) 859. Kastner, P. et al., Development
124, (1997) 313.

s40

Abstracts FEBS'99

6.4 Molecular recognition in transcription: tissue specificity

The specification of skeletal muscle cells
M.E. Buckingham, U. Borello +, G. Cossu +, P. Daubas, J.
Hadchouel, M. Primig, D. Rocancourt & S. Tajbakhsh
URA 1947 CNRS. hl~titut Pasteur, 25 rue Dr. Roux. 75015 Parts.. France
+ Um~el~i[~ of Rome, Via A. S~opa 14, 00161 Rotor, Itah"
Most skeletal muscle cells are derived from somites, segments of paraxial
mesoderm which form along the anterior/posterior axis of the vertebrate
embryo (1). Cells in the somite are initially multipotent and signals from
surrounding tissues specify their fate. Members of the Wnt famdy and Sonic
hedgehog are implicated in the activation of M3f5 and MyoD (2). In the
absence of the earliest myogenic determination factor Myf5 prior to the
activation of MyoD, cells in the mouse embryo which have activated the gene
migrate aberrantly and may adopt other somitic cell fates depending on the
local environment (3). A second factor Pax3 which is required for longer
range cell migration acts together with Myf5, upstream of MyoD in the
genetic hierarchy which regulates skeletal myogenesis (4). The correct
positioning of myogenic progenitor cells in the embryo is therefore a key
event prior to the activation of other members of the MyoD family such as
myogenin which are required for muscle cell differentiation.
Unexpectedly Myj5 is transcribed in distinct regions of the central nervous
system, marking certain early axonal tracts. There is no apparent neuronal
perturbation in Myf5 null mice and in fact the protein is not detectable in the
brain. The same signalling molecules are present in regions adjacent to cells
which transcribe M3f5 in both the somite and the central nervous system. We
suggest that this phenomenon of inappropriate activation of key regulatory
genes by the same signalling molecules deployed at different sites in the
embryo, necessitating post-transcriptional regulation, may be more
widespread, particularly in the developing brain. Interestingly the repression
of Myf5 function which operates in vivo is lifted in vitro; neurons which
transcribe M)J5 can convert to myogenesis in culture (5).

Differentiation of the exocrine pancreas

Peter Wellauer
Swiss Institutefor ExperimentalCancerResearch (ISREC)
Chemin des Boveresses 155, CH-1066 Epalinges, Switzerland
We have generated a mouse bearing a null allele of the gene encoding bHLH
protein p48, the cell-specific DNA-binding subunit of transcription factor PTF 1,
a heterotrimeric protein complex containing three distinct bHLH proteins that
directs the expression of genes in the exocrine pancreas. The null mutation,
which establishes a lethal condition shortly after birth, leads to a complete
absence of exocrine pancreatic tissue and its specific products even though the
dorsal and ventral pancreas Anlagen are initially formed in the mutant embryo.
p48 is thus required for differentiation and/or proliferation of the exocrine cell
lineage. It is the only developmental regulator so far known to be required
exclusively for committing cells to an exocrine fate. The hormone secreting cells
of all four endocrine lineages are present in the mesentery that normally harbors
the pancreatic organ until El6. Towards the end of embryonic life, cells
expressing endocrine functions are no longer detected at their original location
but are now found to colonize the spleen where they persist in a functional state
until postnatal death of the organism occurs. These findings suggest that the
presence of the exocrine pancreas is required for the correct spatial assembly of
the endocrine pancreas and that, in its absence, endocrine cells are directed by
default to the spleen, a site which in some reptiles harbors part of this particular
cellular compartment.

(1) Buckingham, M. et al_ hi: Cell Lineage and Fate Determination. S.A.
Moody Ed., Academic Press, Chapter 41, (1998) 617; (2) Tajbakhsb, S. et
al., Development, 125, (1998) 4155; (3) Tajbakhsh, S. et al., Nature, 384,
(1996) 266; (4) Tajbakhsh, S. et al., Cell, 89, (1997) 127; (5) Tajbakhsh,
S.et al., Neuron, 13, (1994) 813.

Transcription factors in thyroid development, diseases and evolution

R Di Lauro
Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Napoli, haly

Morphogenesis of the thyroid gland begins with the formation of an

endodermal bud in the posterior region of the pharyngeal floor (thyroid
anlage). The thyroid bud migrates dorso-caudally to reach the anterior wall of
the trachea, where it will merge with other ceil types derived from the
brachial pouches However, the hormone-producing thyroid follicular cells
(TFCs) of the adult thyroid are largely, if not exclusively, derived from the
thyroid anlage Only after completion of migration the thyroid follicular cells
undergo functional differentiation and begin to express cell-type specific
genes such as thyroglobulin, thyroperoxidase and the thyroid stimulating
hormone receptor Subsequent to the onset of differentiation, the TFC
population expand to reach a size compatible with sufficient hormone
production
Three transcription factors, thyroid transcription factor I (TTF-1), thyroid
transcription factor 2 (TTF-2) and Pax8 have been implicated in the onset of
functional differentiation However, functional inactivation of these genes m
mouse model showed that they have an important role at stages of
organogenesis that precedes differentiation. In agreement with these finding
in mouse models, mutations in the genes encoding either TTF-2 or Pax8 have
been shown to be responsible for some cases of thyroid dysgenesis, a rather
frequent human condition presenting alteration in thyroid organogenesis
It has been proposed that the most primitive example of thyroid function is
the tunicate endostyle, a pharyngeal organ containing cells capable of
concentrating iodine The TTF-I encoding gene from the tunicate Ciona
intestinalis has been isolated and shown to be expressed in the endostyle,
suggesting that at least some of the mechanisms responsible for endostyle and
thyroid organogenesis are in common and thus providing genetic support to
the hypothesis of the tunicate endostyle as precursor in evolution of
mammalian thyroid

Essential role of Pax5 in B-lineage commitment

S. L. Nutt a, B. Heavey a, A. Rolink b and M. Busshngera
aResearch Institute of Molecular Pathology, Vienna, Austria
aBasel Institute.for Immunology, Basel, Switzerland

The Pax5 gene codes for the transcription factor BSAP which is expressed
throughout B-cell development except in terminally differentiated plasma cells.
Gene inactwation in the mouse revealed that Pax5 is essential for the
progression of B-cell development beyond an early pro-B cell stage m the bone
marrow (1,2). The early pro-B cells present in Pa,r5 (4-) bone marrow can be
cultured in vitro on stromal cells in the presence of IL-7 and express a large
number of B-lymphoid-specific genes, suggesting that they correspond to
comrmtted B-lymphocytes (3).
We now present evidence that the Pax5 (4-) pro-B cells are in fact noncomrrntted hematopoietic progenitor ceils which, upon appropriate st~mulatmn,
are able to differentiate along many diffferent hematopoietic lineages except
along the B-ceU pathway. Upon IL-7 withdrawal, these pro-B cells can
uniformly differentiate in vitro to phagocytic macrophages in the presence of MCSF (instead of IL-7), to bone-resorbing osteoclasts in the presence of
TRANCE, to antigen-presenting dendritic cells in the presence of GM-CSF, to
natural killer cells in the presence of IL-2 and to granulocytes in the presence of
G-CSF. In addition, the Pax5 (-/-) pro-B cells can efficiently reconstitute T-cell
development in vivo, as they participate in the formation of a normal thymus
upon injection into RAG2-deficient truce lacking mature T-lymphocytes.
Moreover, Pax5 (-/-) pro-B cells can in vivo rescue the osteoclast detect of cfos-deficient mice. Pax5 (4-) pro-B cells have therefore many characteristics of
a stem cell. Retrovirus-mediated expression of Pax5 m these cells restricts,
however, their developmental potential to the B-lymphoid lineage, indicating
that Pax5 is the critical B-lineage determination gene.
Recently we have demonstrated that the Pax5 gene is monoallelically
expressed at the onset of B-cell development (4). The selection of one of the
two Pax5 alleles for transcription is independent of the parental origin and thus
random. In summary, these data indicate that the transcription initiation of Pax5
is a stochastic process resulting in B-lineage comrmtment.
[1] Urb~nek et al. (1994). Cell 79, 901-912. [2] Nutt et al. (1997). Genes Dev.
11, 476-491. [3] Nutt et al. (1998). EMBO J. 17: 2319-2333. [4] Nutt et al.
(1999). Nature Genet., in press.

Abstracts FEBS' 99

s41

7.1 The translational machinery

Initiation of translation in the yeast Saccharomycescerevisiae
M. Altmann, C. Berset, P. Danaie, D. Dominguez, S. Helliwell &
H. Trachsel
Institute of Biochemistry and Molecular Biology, Umversityof Bern,
Buhlstrasse 28, P.O. Box, CH-3000 Bern 9, Switzerland

The yeast Saccharomyces cerevisiae is a widely used model system to study

the mechanism and regulation of eukaryotic translation initiation. This multistep biochemical pathway is catalyzed by a large number of translation
initiation factors (elF) which cooperate to place ribosomes at the initiator AUG
codon of an mRNA. Regulation of translation initiation is mediated by extraand intracellular signals and executed mainly through initiation factor
modifications. Changes in translational activity are important in many
physiological situations including the response to amino acid deprivation and
entry into S-phase.
The first step in translation initiation is the recognition of the 5' cap structure
of the mRNA by the cap-binding protein eIF4E. This protein recruits the large
scaffold factor elF4G to the mRNA. The interaction between elF4E and eIF4G
is regulated by a small protein, p20, which competes with elF4G for binding to
eIF4E, p20 carries an RNA-binding site and this site is required for inhibition
of translation by p20. In a complex with elF4E, elF4G most likely contacts the
mRNA at several sites since we were able to identify three domains in eIF4G
which have RNA-binding activity. In the next step, the mRNA-protein
complex then binds the 40S preinitiation complex through interaction between
elF4G and elF3 bound to the 40S ribosome. This step requires the RNA
helicase elF4A. We were recently able to demonstrate that yeast eIF4A
interacts directly with a central domain in yeast eIF4G. Bound eIF4A most
likely unwinds RNA secondary structure in the 5' cap proximal region of the
mRNA in a reaction which is facilitated by elF4B/Tif3. Finally, the 40S
preinitiation complex moves in the 5' to 3' direction on the mRNA to the first
AUG codon where it joins the 60S ribosomal subunit to form an 80S ribosome
competent to translate the open reading frame of the mRNA.

Translation termination in yeast and its regulation by a

prion-like protein
M.F.Tuite, S.Eaglestone, P.Mugnier, F.M.Graves and B.S.Cox
Dept Biosciences, Universityof Kent, Canterbury, Kent CT2 7NJ, UK

Our understanding of the events that take place when a ribosome encounters
a translation termination codon has been greatly facilitated by genetic
screens for yeast mutants that alter the efficiency of a SUQ5, a weak ochre
suppressor tRNA set. This screen has identified the two essential components
of the yeast release factor: eRF1 (encoded by the SUP45 gene) and eRF3
(encoded by the SUP35 gene) [1]. eRF1 is an RNA-binding protein and
deletion analysis of its C-terminus has identified a region important for its
interaction with both eRF3 and the ribosome [2] although binding of the two
eRF components to the ribosome appear to be independent events. The Cterminus ofeRF1 also contains one or more sites that can be phosphorylated
in vitro by a ribosome-associated kinase raisingthe possibility that
translation termination may be regulated through phosphorylation of eRF 1.
A second novel mode of regulation of translation termination in yeast is
mediated by the unusual prion-like behaviour of eRF3. In certain strains
(designated [PS/+]) eRF3 is found primarily as a high mol. wt. amyloid-like
aggregate which is established and maintained via a mechanism analogous
to the establishment of the prion form of the mammalian PrP protein. In
[PS1+] strains there are only very low levels of soluble eRF3 that can form a
functional release factor with eRF1 and this defect in termination can be
detected by enhancement of the efficiency of the SUQ5 suppressor. In
ethanol-stressed yeast cells there is a transient and partial resolubilisation of
the eRF3 polypeptide from the priori-like form thereby restoring efficient
translation termination under conditions of stress [3]. We have also
identified conditions under which the prion form of eRF3 can be stably
eliminated from yeast cells. The importance of the prion-like behaviour of
eRF3 in the regulation of translation termination in this simple eukaryote
will be discussed.
References: [1] Stansfield, I et al., EMBO J. 14 (1995) 4365; [2]
Euwilaichitr, L. et al., Mol. Microbiol. (1999) inpress; [3] Eaglestone, S. et
al., EMBO J. (1999)

Relation of structure and function at the elongation step

B.F.C. Clark, P. Nissen, M. Kjeldgaard, S. Thirup & J. Nyborg

Institute of Molecular and Structural Biology
Aarhus University, 8000 Aarhus C, Denmark
The elongation step of protein synthesis is one of the most well understood
biological processes in structural terms. We and others have solved
structures for all of the cycle steps of elongation factor, EF-Tu, off the
ribosome, mRNA is decoded by aminoacyl-tRNA which is brought to the
ribosome in the form of a ternary complex with elongation factor Tu and
GTP. Our group has in particular determined the structure of the ternary
complex consisting of yeast Phe-tRNA, T. aquaticus EF-Tu and the GTP
analogue GDPNP. The ternary complex has an extended shape of
approximately 115,~ in the longest dimension. The EF-Tu:GDPNP
component binds exclusively to the acceptor arm of the Phe-tRNA, involving
all three domains of the protein. The 3'-CCA end of the Phe-tRNA binds in a
cleft formed by domains 1 and 2. In the GDP conformation of EF-Tu, a large
conformational change takes place, destroying the binding site, so that the
factor is no longer capable of binding tRNA. We can now precisely
characterise why all aminoacyl-tRNAs are recognised by one protein and
have identified a new RNA binding motif.
The structural similarity of the ternary complex with the published structure
of elongation factor G in the GDP or empty form has led us to propose the
concept of structural macromolecular mimicry of protein and RNA. It is
proposed that release factors RFI and RF3 together would also have
structural similarity with the ternary complex at the ribosomal A-site.
We now consider that the ternary complex is a step in molecular evolution
from the RNA to the protein worlds.

The Three-Dimensional Structure o f B a c t e r i a l Ribosomal RNA at 13 A Resolution

R. Brimacombe
M~ix-P1a~-Ir~b~f~rl~lel~ulazeG~net.~.
Ihrz~cca~e 73, 14195 Be~,Lrl, G e ~ .

Recent advances in the c r y o - e l e c t r o n microscopy o f E. c o l i

ribosomes have led to computerized r e c o n s t r u c t i o n s at eve--rhz-~er resolution. A reconstruction of 70S ribosomes carrying tRNA
at the P site and an EF-Tu/tRNA ternary complex at the "pre-A
site" (I> has now been refined to 13A in the laboratories of W.
Wintermeyer (Witten, FRG) and M. van Heel (Imperial College,
London>. At this resolution, the bound ligands are directly visible, end many fine structural elements can be seen which correspond to individual helices of the rRNA molecules.
Previously, models for the 3D folding of the E. coli rRNA
molecules have been constructed on the basis of the large body
of biochemical data (from cross-linking, foot-printing and similar studies> that has been accumulated over the years. Now,
these models can be combined with the EM density maps to derive
the 3D structure of the rRNA in situ within the ribosome (2).
With the help of a number of ~ w - - ~ c h e m i c a l data sets, including cross-links between the rRNA and mRNA, tRNA or the growing
peptide chain, or within and between the 5S, 16S and 23S molecules themselves (3) (collected in collaboration with the laboratoy of O. Dontsova, Moscow), we have fitted all three rRNA molecules to the 33 A reconstruction just mentioned. The structure
produced satisfies both the EM density data end the great majority of the biochemical facts. Furthermore, the X-ray structures
of tRNA, EF-Tu and a number of individual ribosomal proteins (in
collaboration with S. White, Memphis USA, V. Ramakrishnan, Salt
Lake City USA, I. Tanaka, 5apporo dapan, and M. Gorlech, dena
FRG) have been satisfactorily fitted to the rRNA models in the
70S ribosome. The results are giving us an increasingly accurate
and detailed picture of the structure of the ribosome and of its
interactions with functional ligands.
(1) H. Stark et el, Nature, 389, (1997) 403.
(2) F. Mueller & R. Brimacombe, d. Mol. Biol., 271, (1997) 524.
(3) P. Sergiev et el, Nucl. Acids Res. 26, (1998) 2519.

s42

Abstracts FEBS'99

7.2 Translational control and molecular mimicry

Mimicry between mRNA and tRNA recognition
by E. coli threonyl-tRNA synthetase

A network of regulation by RNA-binding proteins in

the C. elegans germline M. Wickans, B. Kraemer, C.

M. Springer a , J. Caillet a, T. Nogueira a, D. Moras c, B.

Ehresmann b, C. Ehresmann b & P. R o m b y b.
a IBPC, Paris, b IBMC, Strasbourg, IGBMC, lllkirch, France

Luitjens, S. Crinanden and J. Kimble.* Dept Blochem, U WisMadison, 53706 USA and * H.H.M.I.

Many cases of molecular mimicry can be found within the translational

apparatus. Mimicry of EF-G by the EF-Tu:PhetRNA:GTP complex, and
tRNA and m R N A by bacterial 10Sa RNA, which is involved in protein
degradation, are just two examples. Another is the regulation of the ribosomal
protein operons at the translational level in E. coli, where it has been
proposed that control ribosomal proteins bind to their mRNAs at sites that
share structural features with rRNA. A similar type of mimicry exists in the
control of the expression of the threonyl-tRNA synthetase (ThrRS). ThrRS is
involved in aminoacylation of tRNA TM isoacceptors, but has also been
shown to bind to its own mRNA and inhibit translation. The site of the
rrLRNA responsible for the control, called the translational operator, contains
two hairpins, each sharing features with the anticodon domain of tRNA TM.
Both carry sequences that are identical to the anticodon of tRNA TM, the two
last bases of which are essential for recognition by the synthetase.
The proof that these two domains in the mRNA are recognised
similarly to tRNA TM, comes from experiments performed on both the mRNA
and the synthetase. We have shown that autocontrol by ThrRS is affected by
changes in nucleotides of the m R N A corresponding to positions in the
tRNA TM anticodon known to be essential for aminoacylation. The tRNA-like
nature of the operator has also been verified using tRNA identity rules. For
instance, changing the Thr anticodon-like sequence in one of the hairpins on
the mRNA to the anticodon of tRNA Met, an essential identity element for
recognition by methionyl-tRNA synthetase (MetRS), switches the regulatory
specificity from ThrRS to MetRS.
The residues of the protein responsible for recognition of the tRNA TM
anticodon have been identified with the recent determination of the structure
o f T h r R S complexed with its tRNA TM. Mutations in some of these residues
cause defects in both aminoacylation and control, indicating that both
processes depend on common sites of the protein.
Although m R N A and tRNA recognition by the synthetase have many
common features, catalysis is not required for regulation since mutants of
ThrRS inactivated by changes in the catalic site are still able to perform
control. Therefore, regulation relies on the capacity of the synthetase to
specifically recognise a tRNA-like structure on its own mRNA but not on the
catalytic act itself.

Translational control of cell m e t a b o l i s m ,

differentiation and development
M.W. Hemze
European Molecular Biology Laboratory, 69117 Heidelberg, Germany

The translation of cellular m R N A s is determined by the 5' cap structure,

the 3' poly-A tail and specific regulatory sequences usually contained
within the 5' or 3' untranslated regions of the m R N A s [1]. Specific
regulatory elements have been identified in both the 5' untranslated
regions (Iron-responsive elements, sex-lethal binding sites) and in the 3'
untranslated regtons (Differentiation Control element, DICE) of specific
translationally regulated mRNAs. Their specific binding proteins (iron
regulatory proteins, IRPs; sex lethal, SXL, hnRNP K, hnRNP E l ) have
been identified and cloned, and their mechanism of regulation studied in
vivo and in vitro. Results will be discussed with regard to the regulation of
translation initiation during cell differentiation, in metabolic control and in
early development [2,3].
Furthermore, the role of general translation initiation factors involved in
the recruitment of the 43S translation initiation complex will be discussed,
also in the light of the m e c h a n i s m by which translational regulatory
proteins can act.
[1] Sachs, A.B. et al., Cell, 89, (1997) 831.
[2] Gebauer, F. et al., RNA, 4, (1998) 142.
[3] Muckenthaler, M. et al., Cell, 2, (1998) 383.

Development of the C. elegans germline requires coordination of

several biological functions. The decision between mitosis and
meiosis, between spermatogenesis and oogenesis, and the execution
of proper gametogenesis, are all critical events. We will discuss recent
results that suggest that families of regulatory mRNA-binding
proteins are vital in these decisions. FBF, a relative of the Drosophila
Pumilio protein, is a critical player, as are several of the proteins with
which it interacts. These interacting proteins include relatives of
known translational regulators in the embryos of other species, such
as Nanos and CPEB. Relatives in other species of these proteins, and
of FBF, regulate specific m R N A s by binding to their YUTRs. We
suggest that a network of interacting and conserved proteins
coordinates development in the germline by controlling specific
mRNAs.

Localization-dependent translation of oskar mRNA

N. Gunkel, T. Yano, S. Castagneni, F. Gebauer, M. Hentze
and A. Ephrussi
European Molecular Biology Laboratory, Meyerhofstrasse 1.
69117 Heidelberg, Germany
The establishment of antero-posterior and dorso-vantral polarity of the Drosophila
embryo relies on the correct localization of maternally provided determinants in the
oocyte, during oogenesis. One such determinant is oskar, which induces formation of the
posterior pole plasm, the germ plasm of Drosophila, at the posterior pole of the egg 1.
The pole plasm contains the determinants of both the germline and of the abdomen, and
is therefore crucial to proper development of the embryo. During oogenesis, oskar is
Iocahzed as an RNA to the posterior pole of the oocyte, oskar mutants and mutants in
which oskar RNA remains unlocalized in the oocyte develop into embryos lacking an
abdomen and a germline. Conversely, deliberate mislocalization of oskar to the anterior
pole of the oocyte causes pole plasm assembly at the anterior and the resulting embryos
develop an ectopic abdomen and functional germ cells in the place of the head. Hence,
oskar is both necessary and sufficient to induce pole plasm assemby. Furthermore,
correct localization of oskar activity to the posterior pole is imperauve, to ensure proper
establishment of antero-posterlor polarity.
Prior to its localizauon to the posterior pole via its 3' untranslated region (3'UTR), oskar
RNA is translationally repressed by Bruno protein which binds to several regions within
the 3' UTR2 We have found that, when the mRNA reaches the posterior pole, its
translation is derepressed by an active process that requires a specific element in the 5'
region of oskar mRNA 3. This novel type of element is a translational derepressor
element, whose functional interaction with the previously identified repressor region in
the oskar 3'UTR is required for activation of oskar mRNA translation at the postenor
pole. The derepressor element only functions at the posterior pole, suggesting that a
locally restricted interaction between trans-actmg factors and the derepressor element may
be the link between mRNA localization and translational activation. We also observe the
specific interaction of two proteins with the oskar mRNA 5' region;one of these also
recognizes the 3' repressor element. Ongoing expenmants are aimed at understanding the
mechanisms by which translational repression and derepression ofoskarare effected, and
at obtaining mutants in the genes encoding the RNA-bmding proteins that we have
identified and testing their involvement in localization-dependanttranslatmn.
To begin to understand the molecular mechamsms by which oskar translation is
regulated - how repression of oskar by Bruno is effected, and ultimately, how thzs
repression is allevtated - we are reconstituting oskar translational regulation in vitro.
Drosophila embryo extracts sustain efficient cap and poly(A)-dependent translation of a
variety of mRNAs (Gebauer, Corona, Becker and Hentze, personnal communication). We
are making use of these and ovarian extracts, of recombinant Brunn and other RNAbinding proteins suspected to play a role in oskar translation, to reproduce and
characterize regulation of oskar translation in vitro.

Abstracts FEBS'99

s43

7.3 tRNA identity and aminoacyl-tRNA synthetases

From the RNA world to the protein theatre:
tRNAs and their synthetases
P. Schimmel
The Skaggs Institute for Chemical Biology, The Scripps Research Institute,
BCC-379, 10550 North Torrey Pines Road, La Jolla, Califorma 92037 USA

Aminoacyl tRNA synthetases are thought to have arisen early, as essential

components of the genetic code. Many contemporary tRNA synthetases
aminoacylate small RNA oligonucleotides that reconstruct the acceptor
ends of their cognate transfer RNAs. A variety ofoligonucleotide substrates
for aminoacylation have been reported. These include RNA duplexes,
conventional hairpin helices, RNA tetraloop hairpins, and linear and
circular RNA pseudoknots. These substrates may be molecular fossils of an
RNA world where aminoacylation was catalyzed by ribozymes. The
relationship between the sequences/structures in these oligonucleotides and
the specificity of aminoacylation constitutes an operational RNA code for
amino acids. The operational RNA code may have led to the genetic code
and the development of a two-domain tRNA structure. One domain contains
the acceptor helix and amino acid attachment site, while the other harbors
the anticodon triplet. The two-domain organization of the tRNA structure is
recapitulated in the tRNA synthetases, suggesting that the two partners may
have co-evolved at least in part. Contemporary synthetases use tRNA not
only as a substrate but also as a cofactor to achieve highly accurate RNAfine structure discrimination of amino acids through editing reactions This
RNA-dependent fine structure discrimination may reflect an early history
where ribonucleoprotein complexes were intermediates in evolution
between ribozymes and contemporary protein synthetases. The evolution of
synthetases as proteins has gone further than just aminoacylation. Most
recently, a link between protein synthesis and signal transduction has been
forged. This link is made through a specific tRNA synthetase that can be
split into two distinct cytokines. Thus, understanding the long evolutionary
history oftRNAs and their synthetases is essential for clarifying the
development of the genetic code and the connections between protein
synthesis and other parts of biology.

Structures and substrate recognitions of class I

aminoaeyi-tRNA synthetases
S. Yokoyama
Department of Biophysics and Biochemistry, Graduate School of Science,
University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, and
Cellular Signaling Laboratory, The Institute of Physical and Chemical
Research (RIKEN), Mikaduki-cho. Sayo-gun, Hyogo 679-5143, Japan

The class I arninoacyl-tRNA synthetases are characterized by the

catalytic Rossmann-fold domain. We have determined the crystal structures
of four class-I aminoacyl-tRNA synthetases, isoleucyl-tRNA synthetase
(IleRS) [1], methionyt-tRNA synthetase (MetRS) [2], glutamyl-tRNA
synthetase (GluRS) [3], and arginyl-tRNA synthetase (ArgRS), from
Thermus thermophilus HB8. Here, we describe functional structures of these
class I aminoacyl-tRNA synthetases which are active in the monomeric form.
All these class I aminoacyl-tRNA synthetases, as well as the Escherichia coli
glutaminyl-tRNA synthetase, share the [~-a-a-[~-a junction domain, which
has the KMSKS motif in a long loop and connects the Rossmann-fold domain
and the anticodon-binding domain. This structural unit may contact with the
inner side of the L-shaped tRNA molecule, and therefore precisely locate the
anticodon trinucleotides for the strict recognition.
The crystal structure 72 thermophilus GluRS complexed with
tRNA Glu (an in vitro transcript) has also been determined. The tRNAbinding mode of GluRS is different from that in the tRNA-GlnRS complex,
with respect to the following points. First, GluRS recognizes tRNAGlu
without disruption of the first base pair of the acceptor stem. Second,
anticodon bases are recognized without disruption of their stacking
interactions. Third, the major identity determinants on the minor groove of
the D-stem helix are recognized by GluRS. Only three aminoacyl-tRNA
synthetases, GluRS, GInRS, and ArgRS, require cognate tRNAs for the
aminoacyl-adenylate formation.
Mechanisms of the tRNA-dependent
reaction will be discussed on the basis of the structures.
[1] O. Nureki et al., Science, 280 (1998) 578-582.
[2] I. Sugiura et al., submitted.
[3] O. Nureki et al., Science, 267 (1995) 1958-1965.

How class II aminoaeyl-tRNA synthetases work

S. Cusack
EMBL Grenoble Outstation, c/o ILL, BP 156, 38042 Grenoble, France

Similarities and differences in the modes of recognition of tRNAs

and amino acids by their cognate aminoacyl-tRNA synthetases will be
discussed with reference to crystal structures of substrate complexes of
five Thermus thermophilus class II synthetases: seryl-, lysyl-, pro|yl-,
histidyl- and asparaginyl-tRNA synthetases.
The native ProRSTT structure has been determined at 2.4,3,
resolution and with proline bound, at 2.9A resolution. Preliminary data on
the complex of ProRSTT with cognate tRNA have been published at 3.5A
resolution [3]. New results on a ternary complex of ProRSTT with tRNA p'
and a prolyl-adenylate analogue at 2.85A resolution will be presented. In
addition a new structure of HisRSTT has been determined at 2.4A
resolution without any substrate bound whereas previously the structure
was known with histidine and histidyl-adenylate bound [1]. The structure
of AsnRS with an analogue of asparginyl-adenylate shows how
discrimination between aspartic acid and asparagine is achieved [2].
These structures shed further light on (a) specific amino acid
recognition (b) conformational changes associated with amino acid binding
(c) the mechanism of amino acid activation (d) the mode of anti-codon
recognition by the anti-codon binding domain in class Ila synthetases (e.g.
ProRS) compared to class lib synthetases (e.g. LysRS).
[1] A. Aberg, A. Yaremchuk, M. Tukalo, B. Rasmussen and S. Cusack
Biochemistry. 36, 3084-3094 (1997).
[2] Berthet-Colominas, C., Seignovert, L., H~rtlein, M., Grotli, M.,
Cusack, S. and Leberman, R. E M B O J . 17, 2947-2960 (1998).
[3] Cusack, S., Yaremchuk, A, Krikliviy, I. and Tukalo, M. Structure 6,
101-108 (1998).

Threonyl-tRNA synthetase, a paradigm for class II

enzymes
D. Moras
lxtboratoire de Biologie Structurale. IGBMC, I rue Laurent Fries - 67404
lllkirch Cedex- France

E. coli threonyl-tRNA synthetase (ThrRS) is a class I! enzyme which

represses the translation of its own mRNA. The crystal structure of the
complex between tRNAT M and ThrRS, reveals structural features showing
novel strategies for providing specificity in tRNA selection. These include
an amino terminal domain containing a new protein fold that makes minor
groove contacts with the tRNA acceptor stem. The anticodon loop is
characterized by a large deformation and an unique interaction between two
adjacent anticodon bases accounting for their prominant role in tRNA
identity and translational control. Despite the presence of all class II
conserved residues necessary for aminoacylation, the active site exhibits a
catalytic zinc ion. never observed so far in aminoacyl-tRNA synthetases,
that is shown to be essential in vivo. This finding open new perpectives from
the point of view of catalysis and evolution.

s44

Abstracts FEBS'99

8.1 Protein degradation

The yeast anaphase promoting complex
W. Zachariae, M. Shirayama, M. Galova, K. Nasmyth
Institute of Molecular Patholgy, Dr Bohr-Gasse 7, 1030 Vienna, Austria

Control of NF-~cB activity by IgB molecules

,Main Isr~l, ShojiYamaoka,Gilles Counois,ChristineBessia, Simon
WhitesideandRobertWeft
Unit# de Biologic Mol~culaire de I'Expression G#nique-URA 1773
CNRS, lnstitut Pasteur, 25 rue du Dr Roux, 75015 Paris, France

The anaphase promoting complex (APC), also known as the cyclosome, is a

large particle which mediates ubiquitination of cell cycle regulators which are
subsequently degraded by the proteasome. We shall present studies on the
subunit composition and the regulation of this ubiquitin-protein ligase which
has emerged as a key component of the mechanism used by all eukaryotic
cells to regulate progression through the cell cycle. The yeast APC contains
at least 12 different subunits most of which have homologues in human
cells. Subunits include three tetratricopeptide-repeat proteins, the RINGdomain protein Apel 1, and the cullin-related subunit Apc2. Apc2 shows
similarity to the yeast cullin Cdc53, a subunit of the ubiquitin-protein ligase
SCF which is essential for the initiation of DNA replication. APC-dependent
degradation of the anaphase inhibitor Pds I occurs shortly before anaphase
and is required for sister chromatid separation. Degradation of B-type
cyclins during anaphase is important for cytokinesis and a new round of
DNA replication. Ubiquitination mediated by the APC depends on two
WD40 proteins, called Cdc20 and Cdh 1, which bind to the core particle in
substoichiometric amounts. Their activities are rate limiting, substratespecific and cell cycle-regulated. Degradation of Pdsl depends on Cdc20
whereas that of the mitotic cyclin Clb2 requires Cdh 1. Cdc20 is not only an
activator but also a substrate of the APC. Cdc20 levels are low in GI and
accumulate during G2 and M-phases. Cdhl is present throughout the cell
cycle but its binding to the APC is, unlike that of Cdc20, inhibited by cyclindependent kinases (CDKs). Therefore, Cdhl is only active from late
anaphase until late G1. The mutual inhibition between the APC and CDKs
explains how cells suppress mitotic CDK activity during GI and then
establish a period with elevated kinase activity from S-phase until late
anaphase. The reciprocal regulation of the two WD40 activators helps to
ensure that cytokinesis and chromosome re-duplication occur only after
sister chromatids have been separated. The order of events is further
enforced by the fact that activation of APC-Cdhl requires prior degradation
of Pdsl by APC-Cdc20 and that APC-Cdhl but not APC-Cdc20 depends
on the polo-like kinase Cdc5.

Macromolecular assemblies designed for

controlled proteolysis
Wolfgang Baumeister
Max-Planck-Institut fuer Biochemie
D-82152 Martinsried b. Mtinchen, Germany
Intracellular protein degradation must be subject to spatial and temporal
control to prevent the destruction of proteins not destined for
degradation. A basic stratagem in controlling protein degradation is
compartmentalization, that is the confinement of the proteolytic action
to sites that can only be accessed by proteins displaying some sort of
degradation signal. Several unrelated proteases have converged toward
a common architecture in which proteolytic subunits self-assemble to
form barrel-shaped complexes. These enclose inner cavities, which are
several nanometers in diameter and harbour the active sites. Access to
those inner compartments is restricted to unfolded polypeptides which
can pass narrow channels guarding the entrance. The target proteins
thus require interaction with a machinery capable of binding and
presenting them in an unfolded form to the proteolytic core complex. In
eukaryotic proteasomes these tasks are performed by the 19S
regulatory complex which binds to either one or both ends of the
barrel-shaped 20S core complex. Jointly they form the 26S proteasome
a 2.5 MDa molecular machine built of approx. 35 different subunits.

The NF-~B family of transcription factors is critically

involved in the i m m u n e and inflammatory responses.
Homo- or heterodimeric complexes of members of the NFKB family are usually retained in the cytoplasm of nonstimulated ceils by inhibitory molecules collectively
referred to as I~B's. Activation of the NF-B signaling
cascade (through TNF, LPS, ILl or multiple other stimuli)
results in phosphorylation of the IKB's, followed by their
degradation through the ubiquitin-proteasome pathway.
Recently a major progress has been made in the field as the
kinases responsible for IKB p h o s p h o r y l a t i o n have been
identified. These kinases are part of a multisubunit high
molecular weight complex w h o s e other c o m p o n e n t s
remain unidentified. We describe here the identification by
genetic complementation of a core component of the
kinase complex.

Abstracts FEBS'99

s45

8.2 Structure and function of chaperones

Highly efficient disaggregation and refolding of a
wide range of protein aggregates by a multichaperone machinery of clpb and dnak
P. Goloubinoff ~and B. Bukau 2
ISilberman Institute of Life Sciences, The Hebrew University of Jerusalem,
91904 Jerusalem, Israel 2lnstitutfuer Biochemie und Molekularbiologie,
University of Freiburg, Herman-Herder-Str 7, D-79104 Freiburg, Germany.

Molecular chaperones are highly efficient at preventing protein aggregation

during stress and promoting the correct refolding of stress-denatured proteins.
However, most chaperones remain poorly effective at refolding previously
aggregated proteins. CIpB from E. coli belong to the Hspl00 chaperone family,
which, in the case of yeast Hspl04, has been implicated in prion propagation,
cellular thermotolerance and in the rescue of minor fractions of aggregating
luciferase and b-galactosidase in vitro (Glover and Lindquist 1998, Cell, 94, 7382). We show here that previously aggregated proteins were efficiently
disaggregated and solubilized by sub-stoichiometric amounts of a specific
combination of CIpB and DnaK/DnaJ/GrpE chaperones (KJE). CIpB alone, or in
the presence of other E. coli chaperones such as HscA/B, HtpG, GroELS and
IbpB could not substitute the specific ClpB+KJE-mediated disaggregation and
refolding reaction. In addition to model substrates, such as MDH, a-glucosidase
and luciferase, more than 100 different aggregated proteins from E. coli were
efficiently re-solubilized.
CIpB did not interact with native proteins, nor prevented protein aggregation,
but, rather, interacted directly with previously aggregated proteins. CIpB was
more hydrophobic and displayed a higher affinity for aggregated proteins in the
presence of ATP, or without nucleotides, then in the presence ADP. ClpB alone
with ATP increased the hydrophobicity and the size of inactive MDH aggregates.
In contrast, CIpB+KJE with ATP dramatically decreased the hydrophobic
exposure and b-sheets content of protein aggregates. Order-of-addition
experiments suggest a mechanism where: 1) CIpB initially binds exposed
hydrophobic regions in large protein aggregates, 2) ATP-binding induces
changes in aggregate-bound CIpB that transiently exposes new hydrophobic sites
in the aggregate, to which 3) DnaJ and DnaK bind and thus displace the
equilibrium towards dissociation. 4) KJE actively dissolve small aggregate and
mediate refolding of solubilized polypeptides into native proteins.
This novel efficient mechanism, whereby sub-stoichiometric amounts of a bichaperone machinery can catalytically mediate the solubilization and refolding of
a large array of aggregated proteins in the cell, is a major addition to the
molecular arsenal of the organism to cope with stress damage and possibly with
amyloid and prion diseases.

Role of HspT0 chaperones in protein folding

in the cytosol of bacteria
A. Mogk, P. Goloubinoff, T. Tomoyasu, H. Langen*,
D. R6der, B. Bukau
Institute of Biochemistry and Molecular Biology, Umversity of Freiburg,
D-79104 Freiburg; *Hoffmann-La Roche AG, CH-4002 Basel

The DnaK (Hsp70) chaperone machinery constitutes a protein repair system of

E. coil that prevents aggregation and assists refolding of misfolded proteins

and that is essential for cell survival under stress conditions such as heat shock
to 42C [ 1]. In addition, this system facilitates the degradation of shortlived
and misfolded proteins by proteases. While the protein repair function of the
DnaK system is well established for model substrates, the nature of the
misfolded protein substrates which accumulate in E. coli under heat shock
conditions is largely unknown and was analyzed in this study.
When soluble total extracts ofE. coli cells were subjected to severe heat shock
treatment a significant fraction of proteins aggregated. Among several
exogeneously added chaperones of the E. coli cytosol tested for their ability to
prevent protein aggregation in these extracts, only the DnaK system was
found to be highly effective. This finding correlates well with the essentiality
of this system in vivo at 42C. To identify the heat labile protein substrates of
DnaK, we analyzed the cellular proteins which aggregated in a DnaKdependent fashion in both, cell extracts and in vivo (in AdnaK52 nutants),
using 2-D gel electrophoresis and mass spectroscopy. More than 50 different
proteins aggregated in dnaK null mutants upon shift to 42C, and a similar
protein pattern was found in heat-shocked cell extracts. These protein
substrates exert a multitude of cell functions and show no specificity towards
their pI value. However, large and oligomeric proteins were more abundant
while small proteins (< 25 kD) were largely excluded, showing that
multidomain and oligomeric proteins are particularly heat labile and require the
chaperone activity of the DnaK system. For one natural substrate identified in
our analysis, the MetE protein, we verified with purified components that the
DnaK system acts directly to prevent its aggregation. Together, these results
provide for the first time information on the nature of the heat labile proteins in
E. coli that require the chaperone function of the DnaK system.
In a second approach we investigated the ability of the DnaK system to
cooperate with other chaperones oftbe E. coli cytosol in the disaggregation of
aggregated proteins. Data will be presented which document such activity.
[1] Hesterkamp and Bukau, EMBO J. 17, (1998) 4818

The 90 kDa molecular chaperones:

biochemistry and possible in vivo functions
P. Csermely
Department of Medwal Chemistry, Semmelweis Umversity, P.O.Box 260,
1-1-1444Budapest 8, Hungary

Molecular chaperones are highly conserved proteins, which help to reach and
maintain the conformational stability of other proteins in the cell. One of the
most abundant chaperone of the eukaryotic cytosol is the 90 kDa heat shock
protein, Hsp90. Hsp90 has been shown to be a rather specific chaperone, aiding
the correct folding of numerous protein kinases and nuclear hormone receptors
involved in various signalling pathways [1]. If Hsp90 function becomes
compromised either by blocking its functions with its specific inhibitor,
geldanamycin, or by massive environmental stress, the affected population
shows a sudden burst of phenotypic changes as a sign of an enhanced adaptation
program. Hsp90 keeps target proteins in a "folding competent state". In contrast
with other chaperones it has two target binding sites: one in its N-terminal, and
another in its C-terminal domain. However, details of its chaperone mechanism
are not clear yet. Ten years ago we have found that Hsp90 has low affinity ATPbinding properties [2]. Later, a direct comparison of the ATP-binding of Hsp90,
and other molecular chaperones, such as Hsp70 having higher affnity
nucleotide binding sites raised some concerns on Hsp90 nucleotide binding.
However, recent studies firmly established Hsp90 as an "active" chaperone,
which function is modulated by ATP both in vitro and in vivo. Hsp90
association with ATP or ADP changes its affinity towards various client
proteins and peptides as well as its complex formation with a number of cochaperone molecules. ATP also affects binding of Hsp90 to filamentous actin in
a modified in vitro motility assay [3]. Other recent findings also indicate a low
affinity, dynamic binding of Hsp90 to various cytoskeletal elements, which
suggests its involvement in the higher organisation of the eukaryotic cytoplasm.
Hsp90 and its co-chaperones, the "foldosome", may participate in a
"microtrabecular lattice"-type meshwork of eukaryotic cells [1].
1. Csermely, P. et al. Pharmacol. Therapeut. 79, (1998) 129
2. Csermely, P. et al. J. Biol. Chem. 266, (1991) 4943
3. Kellermayer M.S.Z. et aL Biochem. Biophys. Res. Commun. 211, (1995)
166

Specificity of interaction between chaperonin containing

TCP-1 (CCT) and its substrates
K. Willison
Institute of Cancer Research,ChesterBeatty Laboratorws,
237 Fulham Road, London SW3 6JB, UK

The chaperonin-containing TCP-I (CCT) assists in the folding of actins

and tubulins in eukaryotic cells. Most chaperonins, such as GroEL in
E.eoli or the TF55/thermosome of Archaebacteria, are composed of 1 or 2
subunit species but CCT is composed of 8 different subunits encoded by
separate genes. CCT purifies as a single hetero-oligomeric complex of
950kDa through multiple chromatographic steps and by antibody affinity
procedures. Consistently, CCT from rabbit reticulocytes, bovine adrenal
medulla and various cell lines, contains 7 polypeptide species in equimolar
amounts (CCT ~, [3, "f, 5, e ~, rl), together with another subunit (CCT0)
which is around half- molar. Testis CCT contains 9-10 polypeptide species
because it has one or two additional testis-specific subunits, depending
upon species, related to CCT~. The Saecharomyces cerevisiae genome
contains 8 CCT genes (CCTI-CCT8), each one being orthologous to the
corresponding gene (Cctet-Cct0) of the mouse. Top views of the torus ring
of CCT from rabbit reticulocyte, bovine testis and human HL60 cells show
8-fold rotational symmetry in the electron microscope. Taken together,
these results are consistent with the idea that each position in the ring is
occupied by a different CCT subunit. We have proposed an unique
arrangement for the 8 CCT subunits within each ring using biochemical
analysis of subunit microcomplexes. Presently we are testing the idea that
the 8 subunits may have different functions in the interaction of CCT with
substrates; specifically, that defined subunits interact with sites on folding
intermediates of 13-actin to facilitate the binding and subsequent release
reactions. We will discuss the use of peptide binding assays to investigate
the nature of the interactions between CCT and [3-actin. Sites on [3-actin
have been identified by this approach, ]3-Actin Sites I-III, and have been
confirmed within the context of the whole actin molecule by site-directed
mutagenesis. Investigations into which particular CCT subunits interact
with these 13-Actin Sites I-III are in progress. Together, these approaches
have allowed us to determine that CCT is a multi-handed complex with
individual subunits making fixed contacts with particular secondary
structural folding intermediates.

s46

Abstracts FEBS'99

Chaperone-assisted protein folding

in the cytosol
F. U. Hartl, S. Teter, W. Houry
M a x - P l a n c k - l n s t l t u t e /or Biochem tstrv,
Ant Klopj~,rspttz 18, D-82152 Martinsrted,

Gepvtany

Although the folded structure of a protein is determined by the

information contained in its amino acid sequence, efficient
realization o f this information in vivo may require assistance
by molecular chaperones and folding catalysts. Folding of
many n e w l y - s y n t h e s i z e d polypeptides in the cytosol depends
on molecular chaperones of the Hsp70 family and on the
cylindrical chaperonins. Little is known about the quantitative
contribution of these chaperone s y s t e m s to overall protein
folding. According to our current model, Hsp70 binds to
nascent polypeptides on ribosomes, preventing misfolding
until all the information required for productive folding is
available. While most proteins appear to fold upon release
from Hsp70 (or other nascent chain-binding chaperones),
certain slow-folding and aggregation-sensitive polypeptides
are subsequently transferred to a chaperonin for folding to the
native state. The chaperonins form large cylindrical complexes
that bind unfolded polypeptides up to ~60 kDa in their central
cavity. In the case o f GroEL, the chaperonin of E. coli, the cofactor GroES caps the opening of the cylinder, resulting in the
displacement o f b o u n d polypeptide into an enclosed folding
cage.
Work is in progress to analyze the substrate flux from Hsp70
to chaperonin in vivo and to identify the authentic substrates
of these chaperone s y s t e m s .

Abstracts FEBS'99

s47

8.3 Protein translocation

Targeting and assembly of membrane proteins
G. yon Heijne
Department of Btochemtstry. Stockholm Universl~L
S-10691 Stockholm. Sweden

In recent years, membrane protein assembly has become an important aspect of

the more general problem of protein targeting and translocation. In addition to
the well-characterized Sec-pathway in E. coli and the SRP/SEC61 pathway in
the ER, the Oxal and TIM 10/12 pathways have been implicated in
mitochondrial inner membrane protein assembly, and it is also possible that the
TAT-pathway in bacteria and chloroplasts may be used by certain membrane
proteins. Finally, some membrane proteins such as the phage M I3 procoat
protein apparently insert "'spontaneously" into the inner membrane. Recent work
on some of these assembly pathways will be reviewed.

Regulation of protein transloeation across the

membrane of the endoplasmic reticulum
B. Dobberstein',K. Schrrder~, M. Pool', B. Mattoglio', E. Hartmannb, T. Rapopor(

ZMBH UniversitgTtHeidelberg. D-69120 Heidelberg.

~Universitat Gottingen, Biochemie II. D-37073 G6ttingen,
' Department of Cell Biology, Harvard Medical School. Boston,
USA

Protein translocation across the membrane of the endoplasmic

reticulum (ER) can occur co- or posttranslationally. Cotranslational
translocation involves targeting of the ribosome/nascent chain
zomplex to the ER membrane and threading of the nascent
polypeptide chain through the pore of the translocon.
targeting is mediated by the signal recognition particle (SRP) and
its receptor, SRP receptor. We will report on the regulation of
targeting by the three GTPase domains contained in SRP and SRP
receptor with particular emphasis on the contribution of the
ribosome.
Franslocation through the membrane can be continuous (secretory
proteins) or discontinuous (membrane proteins). Some proteins can
transiently be retained in the translocon. We will report on the
function of a small translocon associated protein (RAMP4) that
zontacts a nascent type II membrane protein in the translocon and
thereby effects glycosylation efficiency of this protein.

Import of mitochondrial inner membrane proteins

C. Koehlera, D. Leuenbergera, S. Merchantb, N. Ballya,
K. Tokatlidisa and G. Schatza.Biozentrum, University o f Basel
CH-4056 Basel, Switzerland, b UCLA, Los Angeles CA, USA

We have characterized a system in yeast that mediates the

import of multispanning proteins of the mitochondrial inner
membrane from the cytoplasm. This system consists of at least
three hetero-oligomeric protein complexes, two 70 kDa
complexes in the soluble intermembrane space and a 300 kDa
complex associated with the mitoehondrial inner membrane.
One 70 Kda complex consists of Tim9p and Timl0p; the
second 70 kDa complex consists of Tim8p, Tim9p and Tim 13p;
and the 300 kDa complex consists of the integral membrane
proteins Tim22p, Tim54p and of the membrane-associated
proteins Tim9p, Timl0p, Timl2p and Tim21p. Most proteins of
this new import system are essential for viability, and TimSp,
Tim9p, Timl0p and Timl2p are similar in sequence. The 70
kDa complexes have different precursor specificity and pull the
precursor across the outer membrane, whereas the 300 kDa
complex mediates the potential-dependent insertion of the
precursors into the inner membrane. Tim8p is homologous to
two human proteins, DDP1 and DDP2. DDP1, like yeast
Tim8p, is located in the mitochondrial inner membrane space.
Mutation or loss of DDP1 causes a severe X-linked human
disease characterized by deafness, dystonia and blindness. This
disease is the first human disease linked to a defective
mitochondrial protein import system.

Protein transport into chloroplasts:

function, regulation evolution
J. Soil
BotanischesInstitut,Universit~tKiel. D-24098Klel

During evolution, chloroplasts have relinquished the majority &their genes

to the nucleus. The products of the transferred genes are imported into the
organdie with the help of an import machinery that is distributed across the
inner and outer plastid membranes. The protein import systems of
chloroplasts and other organelles operate by a similar set of principles.
Nucleus-encoded proteins destined for the chloroplast are made in the
cytosol with signal sequences that, together with cytosolic factors that
ensure the protein adopts an import-competent confirmation, target the
preprotein to the organellar surface. The protein import machinery of
chloroplasts differs in importam respects from that of other organdies.
Chloroplasts are bound by two 'envelope' membranes, and the outer and
inner envelope each has its own translocation complex, or translocon,
known as Toc and Tic, respectively. Reconstitution experiments of
heterologously expressed Toe and Tic components are used to elucidate
and describe their function in translocation The evolutionary origin of this
machinery is puzzling, because, in the putative predecessors, the
cyanobacteria, the outer two membranes, the plasma membrane, and the
lipopolysaccharide layer lack a functionally similar protein import system.
A 75-kDa protein-conducting channel in the outer envelope of pea
chloroplasts, Toc75, shares -~20 % amino acid identity to a similar sized
protein, designated SynToc75, encoded in the Synechocystis PCC6803
genome. SynToc75 forms a voltage-gated, high conductance channetin
reconstituted liposomes. These findings suggest, that a component of the
chloroplast protein import system, Toc75, was recruited from a preexisting channel-forming protein of the cyanobacterial outer membrane
Furthermore, the presence of a protein in the chloroplastic outer envelope
homologous to a cynobacterial protein provides support for the
prokaryotic nature of this chloroplastic membrane.

s48

Abstracts FEBS'99

Protein export out of the nucleus

C. Dargemont a, B. Ossareh-Nazari ~, M. Rodriguez b, P.
Turpin a, F. Arenzana Seisdedos , R.T. Hay b
aInstitutCurie, UMR144, Paris, France. bSt Andrews University,
Scotland. InstitutPasteur, Paris, France

Bidirectional transport across the nuclear envelop occurs through

nuclear pore complexes. This process requires specific sequences found in
transport substrates. In particular, leucine-rich amino acid sequences
responsible for efficient protein nuclear export (NES) have been identified in
an increasing number of cellular and viral proteins. To characterize the
molecular mechanisms governing NES-dependent nuclear protein export, an
assay that reconstitutes nuclear export in vitro was developed and led to the
identification of the CRM1 protein as the NES receptor. CRM1 ~is a nuclear
protein able to interact with the nucear pore complex and that shares sequence
homologies with the karyopherin 13 family. The CRMI-NES interaction
occurs in a Ran-GTP dependent manner and is inhibited by leptomycin B. N
and C-terrmnal deletion mutants of CRM1 were generated and their ability to
bind NES and Ran-GTP in vitro led to a better characterization of the
functional domains of CRM1.
Protein nuclear export is likely involved in the regulation and
synchronism of both nuclear and cytoplasmic functions. In particular, the
subcellular localization of transcription factors clearly participates to the
control of their activity. In this context, the role of nucleocytoplasmic
transport processes in the regulation of the transcription factor NF-kB was
analyzed. In unsfimulated cells, NF-kB is held in an inactive state in the
cytoplasm by IkB inhibitory proteins which mask its nuclear localization
sequence. However IkBct, when not bound to NF-kB, is transported to the
nucleus through its the ankyrin repeats. In the nuclear compartment, LkBtx not
only abrogates NF-kB/DNA interactions and NFkB-dependent transcription,
but also transports NF-kB back to the cytoplasm. This function of lkBtx is
insured by an NES located in the C-terminal domain of IkBct. In conclusion,
inhibition of NF-kB/DNA binding and the consecutive efficient nuclear export
of this transcription factor by IkBo~ and CRM1 represents an important
mechanism for the control of the expression of NF-kB-dependent genes.
More generally, regulated nuclear export of transcription factors provides an
efficient mechanism to restrict their access to the transcriptional machinery and
turn off a cellular response.

Abstracts FEBS'99

s49

9.1 Molecular mechanisms of secretion

Biogenesis of Neurosecretory Vesicles
W.B. Huttner
Department o f Neurobiology,
University o f Heidelberg, Germany.

Two principal types of neurosecretory vesicles exist, (i) secretory granules

which store neuropeptides, and (ii) synaptic vesicles which contain
neurotransmitters (1).
Secretory granules originate from the trans-Golgi network (TGN). Sorting
of secretory proteins to these vesicles involves their selective aggregation
induced by the lumenal milieu of the TGN and specific structural motifs.
The disulfide-bonded loop of chromogranin B has been identified as one
such motif (2-5). The role of cholesterol in the loop-mediated membrane
binding of the chromogranins and in secretory granule biogenesis will be
discussed (6). Using a cell-free system, the cytoplasmic machinery
mediating secretory granule formation has been studied and found to
involve phosphoinositides (7, 8).
Synaptic-like microvesicles (SLMVs) of neuroendocrine cells originate
from a specialization of the plasma membrane (9). Using a cell-free system,
the cytoplasmic machinery mediating SLMV formation has been studied
and found to include dynamin and the dynamin-interacting protein SH3P4
(10). The role of lipid modification by SH3P4 will be discussed.
(1) Huttner, W.B., et al., Cold Spring Harbor Symposia on Quantitative
Biology, Vol. LX, Cold Spring Harbor Laboratory Press, (1995) 315; (2)
Chanat, E., WeiB, U., Huttner, W.B. and Tooze, S.A., EMBO J. 12, (1993)
2159; (3) KrOmer, A., Glombik, M., Huttner, W,B. and Gerdes, H.-H., J.
Cell Biol. 140, (1998) 1331; (4) Thiele, C. and Huttner, W.B., J. Biol.
Chem. 273, (1998) 122; (5) Glombik, M.M. et al., EMBO L 18, (1999)
1059; (6) Thiele, C. and Huttner, W.B., Seminars Cell Devel. Biol. 9,
(1998) 511; (7) Ohashi, M. et al. Nature 377, (1995) 544; (8) Ttischer, O.
et al. FEBS Lett. 419, (1997) 271; (9) Schmidt, A., Hannah, M.J. and
Huttner, W.B., J. Cell Biol. 137, (1997) 445; (10) Schmidt, A. and Huttner,
W.B., Methods 16, (1998) 160.

Regulation of Exocytosis by the GTPase Rab3

F. Darchen', F. Doussan% A. Clabecq', P.M Lledo%
C. Desnos%B. Poulain'- and J.P. Henry ~
I, CNRS UPR 1929, IBPC, 75005 Paris, France; 2. CNRS UPR 9009
67084 Strasbourg, France; 3, CNRS, IAF, 91198 Gif sur Yvette, France

Rab3 is a monomeric GTP-bindmg protein associated with secretory

vesicles. By using antisense oligonucleotides or recombinant mutated
proteins, Rab3 was found to control regulated exocytosis in neurons and
neuroendocrine cells. Moreover, GTP hydrolysis by Rab3 is rate limiting
in this process. Despite the lack of direct interaction between Rab3 and
the components of the SNARE complex, a GTPase-deficient Rab3 protein
(Q81L) severely delayed the onset of clostridial neurotoxins-induced
inhibition of acetylcholine release in cholinergic neurons of Aplysia.
Since these neurotoxins cannot attack their targets, VAMP and SNAP-25,
once the latter are bound together, this result suggested that GTP-Rab3
stabilized the SNARE complex. The ability of GTP-Rab3 to stabilize the
SNARE complex was confirmed in PC12 cells stably expressing
Rab3aQ81L.
We have also investigated the role of Rab3 in the Caz- dependence of
exocytosis. In Aplysia neurons, intracellular chelation of Ca2+ ions by
EGTA greatly potentiated the inhibitory effect of Rab3Q81L on ACh
release. On the other hand, train- and paired-pulse facilitation, two forms
of short-term plasticity induced by a rise in intraterminal [Ca2+], were
found to increase after injection of Rab3Q81L. In bovine chromaffin cells,
microinjection of antisense oligonucleotides directed to rab3a mRNA
induced a -10-fold decrease of the Ca2+ concentration giving halfmaximal secretory response. These results indicate that Rab3 is implicated
in the Ca ,-+ dependence of exocytosis and could thereby contribute to
short-term plasticity.
GTP hydrolysis by Rab3, which occurs upon stimulation of the secretory
activity, involves Rab3-GAP (GTPase Activating Protein). Rab3-GAP is
mainl~z+.cytosolic, has .a low affinity for Rab3, and is not directly" acUvated"
by Ca ions, suggestmg that the mechanism for GAP activation involves
its translocation from the cytosol to a membrane compartment.
This work was supported by the Association Fran~aise contre les
Myopathies

Molecular aspects of dense-core granule exocytosis:

the role of cysteine string proteins
R.D. Burgoyne and L.H. Chamberlain.
The Physzologtcal Laboratory, University of Liverpool, Lwerpool, UK

The cysteine string proteins (Csps) were demonstrated to be important for

neurotransmitter release from functional studies in Drosophila Csp null mutants
[1]. Following initial suggestions that Csps might act by regulation of Ca2+
channels, they were subsequently found to be restricted in location to synaptic
vesicles in the nerve terminal [2]. We set out to examine the following. Are
Csps expressed on dense-core granules in chromaffin and PC12 ceils? Do Csps
have a direct role in exocytosis? What are the biochemical targets for Csp
action? We have found that Csps are expressed in chromaffin and PC12 cells
where they are present on dense core granules [3]. PC12 cell clones stably
overexpressing Cspl were generated for analysis of Csp function [4]. In these
clones no changes in Ca2. signals following depolarisation were observed
arguing against a major role in regulation of Ca2 channels. In contrast,
exocytosis in permeabilised PC12 cells directly stimulated by Ca2 or GTPTS
was enhanced in the over-expressing clones. These data suggest that Csps have
a direct role in exocytosis. Csps contain a cysteine string domain that is
required for membrane targeting [5] and a J domain. The Csp J domain allows
interaction with and activation of the chaperone protein Hsc70 [6]. Analysis of
the in vitro interactions of Csp and Hsc70 with model unfolded substrates [7]
suggests that Csp itself has a chaperone function. It is possible, therefore, that
Csp acts in exocytosis as a general chaperone required for the maintenance of
correctly folded components of the exocytotic machinery.
[ 1]
Umbach, J.A. et al (1994) Neuron 13,899.
[2]
Mastrogiacomo, A. et al (1994) Science 263,981.
[3]
Chamberlain, L.H., Henry, J. and Burgoyne, R.D. (1996) J. Biol. Chem.
271, 19514.
[4]
Chamberlain, L.H. and Burgoyne, R.D. (1998) Mol. Biol. Cell 9, 2259.
[5]
Chamberlain, L.H. and Burgoyne, R.D. (1998) Biochem.J. 335,205.
[6]
Chamberlain, L.H. and Burgoyne, R.D. (1997) Biochem.J. 322, 853.
[7]
Chamberlain, L.H. and Burgoyne, R.D. (1997) J.Biol. Chem. 272,
31420.

Actin-dependent steps in synaptic

vesicle recycling
Lennart Brodin
The Nobel Institute Jor Neurophysiology, Department of
Neuroscience, Karolinska Instituter. S-171 77 Stockholm, Sweden.

Synaptic neurotransmitter release depends on a rapid recycling of synaptic

vesicles following exocytosis [1]. The actin cytoskeleton has been
implicated in various vesicular trafficking processes [2], but its role in the
nerve terminal is controversial [see e.g. 3, 4]. To examine the role of actin
in synaptic vesicle recycling we used the giant reticulospinal synapse in the
lamprey. Previous studies in this synapse have shown that synaptic vesicle
recycling involves endocytosis via clathrin coated pits in the plasma
membrane area around active zones [5]. Actin-directed compounds were
microinjected into the presynaptic axon, which was then subjected to a
period of action potential stimulation to induce vesicle recycling. The
effects on the synapse was monitored by electron microscopic analysis of
serially sectioned synapses. In control axons, stimulation induced a low
number of clathrin coated pits in the plasma membrane around the active
zones, and actin-like filaments also occurred in this region. Microinjection
of C. Botulinum C2 toxin (which ADP ribosylates G-actin) blocked the
induction of actin filaments and inhibited synaptic vesicle recycling.
Microinjection of GTP'/S caused a massive increase in the number of actin
filaments. The filaments formed a dense matrix around the release sites
which overlapped with clathrin and dynamin-coated endocytic intermediates
that were "alsoinduced by GTPyS. Injection of C. difficile toxin B (which
inactivates rho, rac and cdc42), or of C. b o t u l i n u m C3 toxin (which
inactivates rho) blocked the formation of actin filaments, and caused a
strong inhibition of vesicle recycling. These results suggest that
rho/rac/cdc42 GTPases regulate a perisynaptic actin cytoskeleton which is
essential in synaptic vesicle recycling.
[1] Cremona, O. & De Camilli, P., Curr. Op. Neurobiol. 7 (1997) 323-30.
[2] Geli, M. & Riezman, H., J. Cell Sci. 111 (I998) 1031-37.
[3] Job, C. & Lagnado, L., J. Cell Biol., 143 (1998) 1661-72.
[4] Kuromi, H. & Kidokoro, Y., Neuron. 20 (1998) 917-22.
[5] Shupliakov, O., et al. Science, 276 (1997) 259-63.

s50

Abstracts FEBS'99

Structure and function of SNARE proteins

R. Jahn
Dept. of Neurobiology, Max-Planck-lnstitutefor Biophysical Chemtstry,
D-37077 Gottingeru'Germany

SNARE proteins comprise a new superfamily of small and mostly

membrane-associated proteins that are essential for membrane fusion. The
neuronal variants are among the best characterized family members, they
include the proteins synaptobrevin (VAMP), SNAP-25, and syntaxin 1.
These three proteins form a stable ternary complex that is disassembled by
the ATPase NSF in conjunction with cofactors. Assembly is associated
with major conformational changes and results in an extended four-helix
bundle with all membrane anchor domains extending at one end of the
bundle. Formation of such hetero-oligomeric bundles appears to be the
conserved hallmark of SNARE-proteins, and many family members are
able to interact with each other in a promiscuous fashion. Interference with
assembly by protein fragments or antibodies blocks membrane fusion
supporting the view that it is the assembly reaction that drives membrane
fusion.

Abstracts FEBS'99

s51

9.2 Endocytosis
Endoeytie uptake and intracellular transport of toxins
K. Sandvig ', O. Garred a, T.G. Iversena, P. Nicoziani b, G.
Skretting a, B. van Deursb
aThe Norwegian Radium Hospital, Montebello, Oslo, Norway," hThe
Panum Institute, University of Copenhagen, Copenhagen, Denmark

A number of protein toxins from plants and bacteria consists of two

functionally different moieties, one moiety that binds to cell surface receptors
and another moiety that inhibits protein synthesis enzymatically after entry
into the cytosol. The plant toxin ricin and the bacterial toxin Shiga toxin are
endocytosed and then transported in a retrograde manner to the Golgi and to
the ER before translocation to the cytosol. Protein toxins are useful tools to
study the different pathways involved in their journey to the cytosol. The
plant toxin ricin binds to both glycoproteins and glycolipids with terminal
galactose, and has proven useful to study different forms of endocytosis.
Although molecules involved in clathrin-dependent endocytosis are well
characterized, much less is known about clathrin-independent forms.
Clathrin-independent endocytosis exists on both poles of polarized MDCK
cells, and the one occurring on the apical side is highly regulated. The
requirements for this endocytic process has now been investigated by
studying uptake of ricin from the apical pole after permeabilization of the
membrane at the basolateral pole. In contrast to other previously studied
endocytic uptake mechanism, the clathrin-independent apical endocytosis of
ricin occurs in the presence of GTP),S and seems to be regulated by the small
GTP-binding protein Rho. Transport of ricin from endosomes to the Golgi is
also under regulation, and this transport step can be monitored by measuring
sulfation of a modified ricin molecule. Endosome to Golgi transport of ricin
does not require low pH, but can be increased by compounds that abolish
endosomal acidification. Furthermore, expression of mutant dynamin inhibits
transport of ricin from endosomes to the Golgi. Involvement of clathrin has
now been investigated by using inducible expression of antisense-clathrin
heavy chain. Transport of mannose-6-phosphate receptors to the Golgi
apparatus is known to occur from late endosomes by a rab9~iependent
pathway. We have now investigated ricin transport in Hela ceils where
expression of mutant rub9 can be induced. Mutant rub9 slows down transport
of mannose-6-phosphate receptors i these cells, but has no effect on transport
of ricin to the Golgi apparatus. The data suggest that ricin is transported by a
rab9-independent pathway from endosomes to the Golgi.

Biogenesis, structure and function of membrane

domains in the endocytic pathway of mammalian
cells.
Jean Gruenberg, Feng Gu and Toshihide Kobayashi.
Department of Biochemistry, Sciences II, 30 quai E. Ansermet, 121!
Geneva 4, Switzerland.

Lipids are responsible for the structural integrity of biological membranes,

and confer specific dynamic properties to the bilayer. However, they have often
been considered as passive components of the membrane, beyond their wellknown function as second messengers in signal transduction pathways. One of
the characteristic features of all endosomes along the degradation pathway of
animal cells is that they accumulate internal membranes in their lumen, hence
their multivesicular or multilamellar appearance. We have found that the
biogenesis of multivesicular endosomes occurs on early endosomal membrane in
a process which depends on a putative trans-membrane pH sensor in endosomal
membranes, as well as on the small GTP-binding protein ARF1 and on some,
but not all, components of the COP-I coat. Our studies thus suggest that
membrane invagination within early endosomes depends on ARFI and
endosomal COPs, and that the pH-sensing mechanism signals the onset of the
degradation pathway. Our data also show that internal membranes of late
endosomes contain high amounts of a unique lipid, lyso-bisphosphatidic acid,
and thus form specialized lipid domains within endosomes. Our observations
indicate that these domains are involved in intracellular transport of cholesterol,
as well as protein sorting and membrane trafficking through endosomes. Finally,
our observations also suggest that these specialized endosomal domains are
involved in pathological situations associated with endosomal defects.
References
Clague et al. 1994. J. Biol. Chem. 269, 21-24; Aniento et al. 1993. J. Cell
Biol. 123, 1373-1388; Aniento et al. 1996. J. Cell Biol. 133, 29-41; Gu et al.
1997. J Cell Biol. 139, 1183-1195; Kobayashi et al. 1998. Nature 392, 193197.

Inhibition of clathrin-coated pit assembly

by an Epsl5 mutant
A Benmerah*, M. Bayrou*, H. Boleti*, N
Bensussan, A Dautry-Varsat*.

Cerf-

* Umtb de Biologie des Interactions Cellulalres. URA-CNRS 1960

lnstltut Pasteur. Paris. France. CJF 1NSEtL~/ 97-10. Paris. France

A recently identified component of clathrin-coated pits, Epsl5, is

constitutively associated with the AP-2 complex. Epsl5 is required for
receptor-mediated endocytosis but its precise function remains unknown
Interestingly, Eps 15 contains three EH (Eps 15-Homology) domains also
found in proteins required for the internalization step of endocytosis in
yeast. Results presented here show that EH domains are required for the
plasma membrane targeting of Eps 15. Furthermore, when cells expressed
an Epsl5 mutant lacking EH domains, the plasma membrane punctate
distribution of both AP-2 and clathrin was lost, implying the absence of
coated pits This was further confirmed by the fact that dynamin, a
GTPase found in coated pits, was homogeneously redistributed on the
plasma membrane and that endocytosis of transferrin, a specific marker
of clathrin-dependent endocytosis, was strongly inhibited. Altogether,
these results strongly suggest a role for Eps 15 in coated pit assembly and
more precisely a role for Epsl 5 in the docking of AP-2 onto the plasma
membrane This hypothesis is supported by the fact that a GFP fusion
protein encoding the ear domain of a-adaptin, the AP-2 binding site for
Epsl5, was efficiently targeted to plasma membrane coated pits Epsl5
dominant negative mutants of endocytosis were further used to study the
pathway of entry of intracellular bacteria, Chlamydia, in their host cells.

Regulation of endocytosis
B. van Deursa, K. Rodal b, F. Vilhardt", M. Stahlhut", G.
Skretting b, A. Llorente b, O. Garredb and K. Sandvig b
aThe Panum Institute, Universtty of Copenhagen, Denmark and bThe
Norwegian Radium Hospital, Montebello, Oslo, Norwc~v

Our recent studies show that cholesterol is required for the invagination of
clathrin-coated pits. Thus. when cholesterol was extracted by methyl-[3cyclodextrin, internalization of transferrin, but not of ricin, was strongly
reduced. EM analysis revealed that invaginated caveolae disappeared in the
cholesterol-depleted cells. By contrast clathrin-coated pits at the cell surface
increased in number and concentrated transferrin receptors to the same extent
as control cells, but they were remarkably flattened. Invagination of coated
pits and transferrin uptake could be recovered in serum-free medium without
cyclodextrin, the recovery was inhibited by the cholesterol synthesis-inhibitor
lovastatin, but did occur when a water-soluble form of cholesterol was present
in addition to lovastatin. Interestingly, the flattened coated pits in cholesteroldepleted cells were not distributed at random but appeared in microdomains
of 2-4, suggesting that preexisting coated pits act as nucleation sites for the
binding of clathrin and formation of new coated pits.
Endocytosis in various cell types studied so far seems to depend on Nethylmaleimide (NEM)-sensitive proteins and is inhibited by NEM. However,
in MDCK l cells, NEM induces macropinocytosis at the apical surface. Thus
the uptake of ricin and HRP was strongly increased in NEM-treated cells and
EM studies with Ruthenium Red. a "dye" binding to plasma membrane
polysaccharides and thus allowing an unequivocal distinction between
surface-associated and truly internalized structures revealed that the effect of
NEM on the apical plasma membrane domain was a marked ruffling followed
by closure of the vacuoles formed by the ruffles. The ruffling activity
apparently involves protein kinase C and phospholipase D while
phosphoinositide 3-kinase seems to be involved in the final closure step.

s52

Abstracts FEBS'99

10.2 Functions of glycosylation

Higher plants developed structurally different
motifs to recognize foreign glycans
W.J. Peumans and E.J.M. Van Damme
Laboratoryfor Phytopathologyand Plant Protectton, KULeuven,
W De Croylaan 42, B-3001 Heverlee, Belgium

Many plant species accumulate carbohydrate-binding proteins

commonly referred to as lectins or agglutinins. Until recently, plant
lectins have been considered a heterogeneous and complex group of
proteins differing from each other with respect to their molecular
structure, sugar-specificity and biological activities. However,
biochemical analysis and molecular cloning of a few hundred lectins
enabled to subdivide the majority of all currently known plant tectins
into seven families of structurally and evolutionary related proteins.
Within each of these families the overall structure of the lectin domains
as well as the 3-D structure of the sugar-binding motif are apparently
strictly conserved. Since according to the available data there is no
similarity between the 3-D structure of the different lectin families
plants must have developed at least seven structurally different
carbohydrate-binding motifs. Though not completely understood yet,
the specificity of most plant lectins seems to be directed against foreign
glycans. The binding sites of most plant lectins are most
complementary, indeed, to N- and O-linked glycans from higher and
lower animals, which is in good agreement with the presumed defensive
role of plant lectins against phytophagous invertebrates and herbivorous
animals. A closer examination allows several important conclusions
with regard to structure/specificity relationships within the whole group
of plant lectins. First, some lectin families exhibit a highly conserved
specificity whereas others cover a broad range of specificities. Second,
some sugars are recognized by two or more structurally different
carbohydrate-binding motifs. Third, plants are apparently strongly
'interested'
in mannose- and galactose/N-acetylgalactosaminecontaining glycans.

Molecular analysis of animal lectins that mediate cellular

recognition events
Kurt Drickamer
Department of BiochemJstry. Umversityof Oxford. Oxford 03(I 3QU UK

Many of the biological roles of complex carbohydrates are mediated

through recognition by specific receptors known as lectins [1]. Calciumdependent (C-type) animal lectins target biological functions such as
endocytosis and cell adhesion based on the locations and structures of
their saccharide ligands [2]. The selectin cell adhesion molecules and the
hepatic asialoglycoprotein receptor are examples of C-type lectins that
recognize endogenous oligosaccharides. Other C-type lectins, such as
serum mannose-binding protein and the macrophage mannose receptor,
mediate an innate immune response to pathogens by distinguishing the
surface sugars of microorganisms from those of the host.
Analysis of rat semm mannose-binding protein reveals that multiple
carbohydrate-recognition domains (CRDs) in the protein oligomer are
arranged in order to maximize their ability to interact with surface
oligosaccharides of potential pathogens while avoiding interference from
endogenous mammalian oligosaccharides.
Studies using chimeras
between serum and liver forms of the protein indicate that collagenous
domains attached to the CRDs are essential for oligomer formation and
complement fixation. The basis immunodeficiency caused by mutations
in this region has been investigated.
The structural basis for binding of sialyl-Lewis x and related
oligosaccharides to the CRDs of the selectin cell adhesion molecules has
also been studied by creating mutants of marmose-binding protein that
mimic the selectins. The primary recognition site is the fucose moiety,
with supplementary contacts via the galactose and electrostatic
interactions with the charged portion of the ligand. In contrast, binding to
sulphatide appears to be largely an electrostatic interaction.
1. Drickamer, K and Taylor, ME (1998) Trends Biochem Sci. 23, 321
2. Weis, WI, Taylor, ME and Drickamer, K (1998) ImmunoL Rev 163, 19

Regulation of selectin ligand glycosylation

D. Vestwebera, M.-C. Huang a, T. Marquardt b, K. Liihna
alnstitute o f Cell Biology, ZMBE, and bChildren's Hospital,
University of Miinster, Germany
P-selectin glycoprotein ligand-I (PSGL-1) and E-selectin ligand-1 (ESL-I) are
the two major selectin ligands on mouse neutrophils. Transfection experiments
demonstrate that each ligand requires oO,3-fucosylation for selectin-binding.
We have used mice deficient for Fuc-TIV and/or Fuc-TVI1 (Obtained from Dr.
John Low, Ann Arbor, USA) to examine how these enzymes generate selectinbinding glycoforms of PSGL-1 and ESL-I in mouse neutrophils. Selectinbinding was analysed by affinity isolation experiments using recombinant,
antibody-like forms of the respective endothelial selectins. We observe
essentially normal binding of E- or P-selectin to PSGL-I expressed by Fuc-TIV
deficient neutrophils, but f'md that PSGL-1 expressed by Fuc-TVII deficient
neutrophils is not bound by E- or P-selectin. By contrast, E-selectin binds
with normal efficiency to ESL-I on Fuc-TVII deficient neutrophils, but exhibits
an 80% reduction in its ability to bind ESL-1 isolated from Fuc-TIV deficient
neutrophils. Our data imply that in mouse neutrophils and their precursors,
Fuc-TVII exclusively directs expression of PSGL-1 glycoforms bound with
high affinity by P-selectin. By contrast, Fuc-TIV preferentially directs
expression of ESL-1 glycoforms that exhibit high affinity for E-selectin.
Selective modification of PSGL-1 and ESL-1 by the two fucosyltransferases
could be mimicked in stably transfected CHO-Pro "5cells, but not in CHO-dhfrcells.
Leukocyte adhesion deficiency type II (LAD II) is an inherited disorder of
unknown nature that affects fucosylation of glycoconjugates. Children with
this disorder are severly handicapped and suffer from immunodeficiency due
to the lack of selectin ligands. We and others have found that the fucosylation
defect in LAD II fibroblasts can be corrected by addition of L-fucose to the
culture medium. This prompted us to initiate dietary fucose therapy on a
patient with LAD II. Oral administration of fucose to the patient restored the
ability of his neutrophils to bind to P-selectin. Binding was Ca2+-dependent
and was completely abolished by antibody blockade of PSGL-1. Recurrent
episodes of infection and fever, continuously observed before therapy, did not
occur anymore since the onset of therapy.

Bioactive glycans in inflammation

Risto Renkonen
Haartman Institute, University o f Helsinki, Finland

L-selectin-dependent lymphocyte extravasation has been shown to be

an initial event of acute heart allograft rejection in rats. Upon screening
600 endomyocardial biopsies (EMBs), taken at different time points
after heart transplantation in man, we identified 91 samples having
histological signs of acute rejection. Rejection and non-rejection EMBs
were analysed for the presence of properly glycosylated, i.e., sulfated
sialyl Lewis x (sLex) decorated L-selectin ligands. Two anti sLex (2F3
and HECA-452) and one anti 6-sulfation (MECA-79) monoclonal
antibodies were used. Non-rejecting heart endothelium did not express,
or expressed only weakly, sulfated or sLex decorations of L-selectin
ligands. On the contrary, these epitopes were readily detectable on
endothelium of capillaries and venules at the onset and during acute
rejection episodes. The endothelial expression of L-selectin ligands
decreased to background levels as the rejection resolved. Our data
demonstrate a complete correlation between the level of expression of
the sulfated sLex decorated ligands on the one hand and the histological
severity of acute heart allograft rejection on the other hand. Our ex vivo
experiments show that some multivatent fucosylated glycans possess
organ-spesific antiiflammatory potency. These data suggest that
functionally active endothelial L-selectin ligands are instrumental in
lymphocyte extravasation at the onset and during acute rejection
episodes.

Abstracts FEBS'99

Sialic acid binding iectins (siglecs) in the immune and

haemopoietic systems
P. R. Crocker
Departmentof Biochemistry,Umversityof Dundee,Dundee, Scotland, UK

Siglecs are a distinct subset of sialic acid-binding membrane proteins

belonging to the immunoglobulin superfamily. Well-characterised members
include sialoadhesin expressed on macrophages, CD22 on B cells, MAG on
myelin forming cells and CD33 on myeloid cells of the haemopoietic system.
Each of these proteins consists of an extracellular region made up of an Nterminal V-set Ig domain and varying numbers of C2-set domains ranging
from sixteen in sialoadhesin to one in CD33. The sialic acid binding region
of the V-set domain complexed with 3'sialyllactose has been characterised by
X-ray crystallography [1]. The expression patterns and binding properties of
siglecs point to diverse biological functions involving cell-cell interactions
and intracellular signalling.
The ability of a given sigiec to mediate cell-cell interactions may be
compromised by cis-interactions of the lectin binding site with neighbouring
sialic acids in the glycocalyx. Sialoadhesin, with seventeen Ig domains, can
be seen by electron microscopy to position the sialic acid binding site up to
30 nm from the plasma membrane and this seems to be important in its
ability to mediate cell-cell adhesion. In the case of CD22, which specifically
binds t2,6-1inked sialic acids, the high levels of this linkage on CD22positive resting B cells seems to prevent the majority of them from using
CD22 as an adhesion molecule. However, a minor subset of B cells appears
to be competent for CD22-dependent binding in trans, and recent evidence
supports the idea that this B cell subset is involved in CD22-dependent
homing to the bone marrow, where high levels of CD22 ligands are
expressed by sinusoidal endothelial cells.
To date, the identification and characterisation of siglecs has come
about through a combination of diverse experimental approaches~ The human
genome project provides DNA sequence for identification of potential new
siglecs. Progress in this area will be discussed, in particular the recent
characterisation of siglec-5 [2] expressed on mature myeloid cells.
[1] May, A. P. et al Mol Cell 1, (1998) 719
[2] Comish, A. L., et al. Blood 92, (1998) 2123.

s53

s54

Abstracts FEBS'99

10.2 Structure and biosynthesis of glyco-conjugates

Mapping the Binding Specificity of Glycopeptides
by NMR

The role of glycans in glyeoprotein folding

A. Helenius ~, E. S. Trombetta b, K. Cannon b, C. Ritter" and
M. Molinari"

Bemd Meyer, institute for Organic Chemistry,

University of Hamburg, 20146 Hamburg, Germany

Institute of Biochemistry, ETHZ, Zurich, Switzerland

~Dept. of Cell BioL, YaleSchoolof Medicine, New Haven, CT, USA

Addition of N-linked glycans allows nascent and newly synthesized

glycopolypeptide chains to interact with two endoplasmic reticulum
chaperones; calnexin (CNX) and calreticulin (CRT) and a thiol redox
enzyme, ERp57. Together with glucosidases I and II, UDP-Glc:glycoprotein
glucosyltransferase, a newly discovered soluble UDP-phosphatase and
ERp57, these promote proper folding and disulfide oxidation, prevent
premature oligomerization, affect degradation and support quality control of
glycoproteins. We have analyzed the folding of influenza hemaggtutinin HA,
VSV G protein, and the Semliki forest virus E 1 and p62 in some detail. Using
a thermoreversible folding mutant of VSV G protein (ts045), it was possible
to obtain in vivo evidence for conformation induced reglucosylation and
calnexin binding. The results are all consistent with a 'lectin-only' model for
CNX/CRT function in which the key interaction between substrate
glycoprotein and chaperone is through N-linked glycans. To investigate how
the system distinguishes between folded and unfolded glycoproteins, we
analyzed the substrate specificity of UDP-Glc:glycoprotein
glucosyltransferase, the enzyme serving as the folding sensor in the
CNX/CRT cycle. Various conforrnational forms of bovine pancreatic RNase
B were tested as substrates for the isolated enzyme in vitro. We found that it
used the partially folded RNase B*S protein but not the native or minimally
perturbed forms such as RNase B*S. The enzyme was only marginally active
on fully denatured, reduced and alkylated Rnase B. Moreover, using dimers
of folded and misfolded RNase B, we could show that when the enzyme
recognizes a folding defect it only reglucosylates local glycans.

The glycosylation of peptides can induce severe changes in the 3D

structure of the peptide backbone. We could show in several cases
that carbohydrates induces linkage specific conformations in the
peptide.
When peptides or glycopeptides are interacting with receptor
proteins, like antibodies, binding epitopes of a glycopeptide cannot
easily be mapped. Using an NMR based technique that was recently
developed by us, we can assign the binding epitope directly from the
spectra of a mixture between ligand and protein. The new procedure
can also be used to identify bioactive compounds from mixtures
w~thout the need to separate the individual components. Using the
epitope mapping on mucin peptides and glycopeptides that bind to a
monoclonal antibody SM3, we could show and differentiate the individual bJnding contributions of the carbohydrate portion and the
peptide portion. Applying the same technique to the interaction of
the CD4 protein with peptides and glycopeptides of the HIV indicates
that binding epitopes can easily be assigned by NMR spectroscopy.
The method is very sensitive in that only about 2 nmol of protein are
required.

MUCIN BIOSYNTHESIS
H. Clausen, E.P. Bennett, H. Hassan, T. Schwlentek
Faculty of Health Sctences, School of Denttstrp. Umversityof
Copenhagen, Notre Alle 20. DK-2200, Copenhagen N. Denmark

Mucin-type O-linked protein glycosylation is initiated by the transfer of Nacetylgalactosamine (GalNAc) to serine and threonine amino acid residues
catalysed by UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase
activities (GalNAc-transferases). Eight members of a large mammalian
family of homologous GalNAc-transferases with different peptide acceptor
substrate specificites and expression patterns have been identified and
characterized. The data indicate that the repertoire of GalNAc-transferases
in cells is the main factor determining the attachment sites of O-glycans,
and suggest that O-glycosylation of proteins is a highly regulated process
with cell type specificity. The repertoire of GalNAc-transferases vary with
cell differentiation and malignant transformation, and it is suggested that
changes in GalNAc-transferase expression relate directly to changes in
mucin glycosylation in cancer. The cell membrane mucin, MUC1, is a
prime target for immunotherapy of breast, prostate, and ovarian cancers,
and in vitro methods for chemoenzymatic production of glycopeptide
vaccine candidates have been developed. The processing of O-glycans also
involve large glycosyltransferase gene families, and expression of different
members of these allow for differential regulation of elongation/branching
status of O-glycans. Progress in the characterization of the processing step
of O-glycans will also be presented.
Supported by funds from the 4'h EU Framework.

Abstracts FEBS'99

s55

11.1 Lipids in menbrane function

The physiopathologic significance of the
transmembrane redistribution of aminophospholipids
Jean-Marie Freyssinet
lnstitut d'H~matologie et d'lmmunologie - Facult# de M~decine,
Universit~ Louis Pasteur, 67085 Strasbourg, France

The plasma membrane of non-stimulated mammalian cells presents an

asymmetric distribution of its constitutive phospholipids. Aminophospholipids, anionic phosphatidylserine (PS) and neutral phosphatidylethanolemine
(PE), are mainly concentrated in the inner leaflet. The mechanism governing
this organization involves a flippase, a Mg2"-ATPase termed aminophospholipid translocase with respect to its well-characterized function but for
which the corresponding gene remains to be unambiguously identified. When
stimulated by a variety of effectors leading to an elevation of cytosolic Ca 2,
the membrane phospholipid asymmetry is frequently lost in which case PE and
more particularly PS occur in the exoplasmic leaflet and are also accessible at
the outer surface of shed membrane microparticles. The mechanisms
responsible for PS/PE externalization remain also to be firmly characterized. A
non-specific scramblase has been proposed to fulfil this function, but the rate
of phosphatidylcholine scrambling is significantly higher than that of PS when
reconstituted into liposomes. Furthermore, the human scmmblase gene is
strongly homologous to a mouse leukemogenesis-associated gene, which
makes it difficult to pinpoint the exact role of this candidate. Members of the
ATP-binding cassette (ABC) family of transporters have been shown to act as
phospholipid translocases in mammalian cells or cell lines and in yeast, but the
action of some of them may be restricted to labelled phospholipid analogues.
Once accessible, PS and probably PE become multifunctional effectors or
determinants. The exposure of PS and the shedding of membrane
microparticles are hallmarks of apoptotic cells. PS acquires a procoagulant
potential due to its ability to promote the assembly of the characteristic enzyme
complexes of the blood coagulation cascade, and constitutes a recognition
signal for phagocytosis. Activated cells are thought to be rapidly engulfed,
which may not be the case for derived fragments. The persistence of exposed
PS which can elicit various responses with procoagulant, proinflammatory or
autoimmune consequences is certainly not neutral. Furthermore, shed
microparticles carry antigens specific of the cells they stem from and are a
source of aminophospholipid substrates for secretory phospholipase A2. Such
membraneous structures can be considered true carrier of biologic information
able to mediate transcellular active or passive exchanges depending on their
composition, the latter depending on the nature of the stimulus at their origin.
Scott syndrome, an inherited bleeding disorder characterized by deficiencies of
PS exposure and membrane vesiculatlon, represents an invaluable knockout
model in humans for the assessment of mechanisms controlling the transmembrane redistribution of PS and PE, with a possible pharmacologic impact.

Biosynthesis of phosphatidic acid in yeast

G. Daum and K. Athenstaedt
bt~titut fi~r Biochemie mid Lebensmittelchemie. Techm~che Unt~ermtat.
Petersgasse 12/2, A-8010 Gta2. Austria

Phosphatidic acid (PA) is a key intermediate in the formation of

phospholipids and triacylglycerols. In yeast as in mammahan cells two
different pathways for PA biosynthesis are known: (i) Glycerol-3-phosphate
(G-3-P) is acylated in two steps to PA with the intermediate formation of
lysophosphatidic acid (LPA). (ii) Dihydroxyacetone phosphate (DHAP) is
acylated to I-acyI-DHAP, which is reduced in an NADPH-dependent
reaction to yield LPA, that is further converted to PA. In the yeast
S a c c h a r o m y c e s cerevisiae highest specific activity of G-3-P and D~HAP
acylation can be detected in lipid particles, a compamnent with a
hydrophobic core mainly formed from triacylglycerols and steryl esters
surrounded by a phospholipid monolayer membrane with only a few
proteins embedded. The second site of acylation of both precursors are the
microsomes. Mitochondria harbor only DHAP acyltransferase but lack
G-3-P acyltransferase activity [1, 2].
Using yeast mutant strains we demonstrated that in Iip~d pamcles
one enzyme is capable of catalyzing the first step of acylation of both
precursors G-3-P and DHAP. The same enzyme is also present in the
microsomal fractmn but microsomes contain additional G-3-P/DHAP
acyltransferase activity. Thus, at least three different enzymes seem to be
involved in the first step of G-3-P and/or DHAP acylation. NADPHdependent I-acyI-DHAP reductase (ADR) is located in lipid particles and
microsomes but not in mltochondria. Similar to acyltransferases. ADR
activity is present in both the lipid particle and microsomal fraction. Recent
studies with a yeast mutant demonstrated that the latter compartment
contains at least two ADR isoenzymes. Redundancy of enzymes revolved in
PA synthesis in the yeast appears to guarantee efficient formation of this
important lipid intermediate.
[1] Athenstaedt, K. and Daum, G., J. Bacteriol. 179 / 1997) 761 l
[2] Athenstaedt, K. et al., J. Bacteriol. (1999) in press
Supported by project 11491 of the FWF to G.D..

Rapid transbilayer movement of lipids in yeast cells

A. Herrmanna, U. Marx a, T. Polakowski b C. Lang b
"Humboldt-Universitdt Berlin, Inst. f Biologic, D-10115 Berlin, Germany
bTechnische Universttdt Berlin, Inst. f Biotech., D-13355 Berlin,
Germany

Protein-mediated phospholipid transbilayer movement has been shown to occur

in the plasma membrane of a variety of eukaryotic cells. In wild-type yeast cells
the involvement of the Drs2 protein which belongs to the subfamily of highly
related S. cerevisiae type IV ATPases in the redistribution of
phosphatidylserine from the exoplasmic to the cytoplasmic leaflet of the plasma
membrane is controversially [1,2]. To differentiate between the various routes
of analogue internalization, we studied the transbilayer movement and
distribution of phospholipids in the plasma membrane of the endocytosisdeficient S. cerevisiae end4 mutant. Evidence is presented that the fluorescent
phospholipid analogues of pbosphatidylcholine (C6-NBD-PC) and of
phosphatidylserine (C6-NBD-PS) became rapidly internalized. Both analogues
redistributed to the cytoplasmic leaflet with a half-time of <15 min at 0C. The
plateau of internalized analogues was about 70%. Probably, transbilayer
movement is protein-mediated since the flip-flop of both analogues was very
slow in Iiposomes composed of plasma membrane lipids. Rapid analogue
internalization was not abolished upon depletion of intracellular ATP by about
90%. Only for C6-NBD-PC a moderate decrease of the plateau of internalized
analogues of about 20% was observed while that of C6-NBD-PS was not
affected. The Drs2 protein plays only a minor role, if at all, for the rapid
transbilayer movement of analogues in S. cerevisiae e n d 4 cells. In S. cerevisiae
end4 Adrs2 cells harboring both an end4 allele and a d r s 2 null allele about 60%
and 50% of C6-NBD-PC and C6-NBD-PS, respectively, became internalized
within 15 min. The preferential orientation of C6-NBD-PS to the cytoplasmic
leaflet is in qualitative agreement with the sequestering of endogenous
phosphatidylserine to the cytoplasmic leaflet as assessed by binding of annexin
V. Virtual no binding of annexin V to spheroplasts of the parent wild-type
strain or mutant strains was observed. Likewise, no difference in the exposure
of endogenous aminophospholipids to the exoplasmic leaflet between these
strains was found by labelling with TNBS. Thus, lipid asymmetry, at least of
aminophospholipids, was preserved in S. cerevisiae e n d 4 cells independent of
the presence of the Drs2 protein.
[1] Tang et ah, Science 272, (1996) 1495.
[2] Siegmund et al., J. Biol. Chem., 273, (1998) 34399.

Membrane fusion activity of alphaviruses:

Specific roles for sphingolipids and cholesterol
J. Wilschut a, J.M. Smita, J. Corver a, A. Ortiz a, R. Bittman b

aLab of Physiol Chem, Univ of Groningen, Groningen, Pays-Bas

bDept of Biochem, Queens College, CUNY, Flushing, NY, USA
Alphaviruses, such as Semiiki Forest virus (SFV) and Sindbis virus (SIN), are
enveloped positive-strand RNA viruses. SFV and SIN infect their host cells via
a membrane fusion reaction, through which the viral genome gains access to the
cell cytosol. It is well-established that the cell entry mechanism of SFV involves
uptake of virus particles by receptor-mediated endocytosis and subsequent
fusion of the viral envelope with the membrane of the endosomal cell
compartment, induced by the acidic pH within the lumen of the endosome. For
SIN, there is controversy about the route of cell entry, several published
observations suggesting that this virus may fuse directly with the cellular plasma
membrane. In a model system, both SFV and SIN fuse efficiently with
liposomes, fusion occurring on the time scale of seconds. The kinetics of these
fusion processes can be followed conveniently on the basis of dilution of
fluorescent pyrene-phospholipids from biosynthetically labeled virious into the
target liposomes. For both SFV and SIN, fusion is strictly dependent on a
mildly acidic pH (- pH 5.0), strongly suggesting that not only SFV but also SIN
enters cells through receptor-mediated endocytosis and fusion from within
acidic endosomes. The virus-liposome fusion process is nonleaky, and
presumably involves a distinct hemifusion intermediate, since it is inhibited by
lysophosphosphatidylcholine and stimulated by free fatty acids. As the
liposomes do not contain a protein or carbohydrate receptor, virus-receptor
interaction is not a mechanistic requirement for the expression of membrane
fusion activity by SFV and SIN. On the other hand, fusion does require the
presence of cholesterol and sphingolipids in the liposomes. Cholesterol is
involved in low-pH-dependent binding of the viruses to the target membrane, as
evidenced by direct binding measurements. However, cholesterol-mediated
binding does not suffice for completion of the fusion event. Fusion appears to
be specifically catalyzed by sphingolipids. At low pH, the E2/E1 heterodimeric
envelope glycoproteins of SFV and SIN dissociate with formation of a trypsinresistant trimeric form of El, the fusion protein of these viruses. The trypsinresistant trimeric phenotype of E1 arises immediately afler binding of the virus
to the liposomes. We propose that the El ttimer formed in the presence of
sphingolipids represents the fusion-active conformation of the viral spike.

s56

Abstracts FEBS'99

11.2 Lipids in signal transduction

Signalling through phospholipase D
S. Cockcrofi
Department of Phystolo~, University College London,
Rokefeller Building, 1 University St.. London WCIE 6JJ, UK

Phospholipase D is an enzyme that hydrolyses phosphatidylcholine to

phosphatidic acid. The basal activity of phospholipase D in
mammalian cells is very low, yet the enzyme can be activated in most
cell types, rapidly and transiently, by a wide variety of stimuli
including hormones, neurotransmitters, growth factors and cytokines.
Two mammalian PLDs have been cloned, PLDI and PLD2 and both
proteins can be regulated by ARF. ARF proteins are GTPases which
bear sequence homology to both heterotrimeric GTP binding proteins
and also to the monomeric GTP binding proteins of the Ras family.
The human family of ARF comprises of five isoforms which can be
grouped into three classes based on sequence homology, ARFsl and 3,
ARFs 4 and 5 and ARF6. ARF proteins have been found to have a
number of disparate functions including the regulation of constitutive
membrane traffic in both the endocytic and exocytic pathway,
assembly of coat proteins, maintenance of organelle integrity,
regulated exocytosis and delivery of paxillin to focal adhesions. All
ARF proteins are also capable of stimulating the activity of PLD1.
Structural analysis of ARF1 defines the PLD1 effector region to the
a2-helix, part of the 13-strand and N-terminal helix and its ensuing
loop. ARF mutants with increased or decreased ability to activate PLD
have been utilised to examine which of the many functions of ARF are
mediated by phospholipase D activation.

Structural aspects of phospholipase C signaling

M. Katan
CRC Centre for Cell and Molecular Biology. Chester Beatt)
Laboratories. Fulham Road, London SW3 6JB. UK

The intracellular signaling second messengers diacylglyceml and lnositol

1,4,5
trlsphospbate
(1P3)
are
generated
by
action
of
phosphatidylinositol-specific phosholipase C (P1-PLC) on membrane
phosphoinositldes. Determination of the crystal structure of one of the PIPLC isozymes, PLC-~SI, together wzth structure-function analysis provided
new insights into catalytic and signaling roles of PI-PLCs.
Based on multidomain structure of PLCrl (incorporating catalytic domain
and modular PH-, EF- and C2 domains) the domain organization and
evolutionary hnks with other eukaryotic PLC& [3 and y isozymes as well as
bacterial PI-PLCs have been determined. The studies also revealed structural
basis of substrate recognition and mechanism of catalysis common to all
eukaryotm PI-PLCs. Since the interaction of PI-PLCs with the membrane is
not only required for access to its substrate but also for interactions with
regulatory molecules, a number of experimental approaches were used to
study these interactions. In case of PLCrl they involve the PH-domain, C2domain and the hydrophobic ridge within the catalytic domain. The PH
domain, through binding to PIP2 in the membrane, is the mare deterrrunant
of membrane attachment that can be regulated by cellular concentrations of
PIP2 and IP3. The hydrophobic ridge is likely to penetrate membrane and
provides negative rather than positive contribution to high rates of
hydrolysas, The C2 domain, which could bind multiple calcium ions, is not
likely to function as calcium-dependent phospholipid binding domain. Some
insights into molecules that could act on membrane bound PLCSI to increase
its activity have also been obtained and allowed considerations of structural
basis for this "inactive" to "active" transition. This transition is likely 1o
involve changes in relative orientation of the active s~te towards the
membrane and/or more extenswe membrane interactions.

Phospholipase A2 in signal transduction

in astrocytoma cells
M. Sanchez Crespo, M. Hernandez, Y. Bayon, M.L. Nieto
IBGM, CSIC, Facultad de Medicina, 47005 Valladolid, Spain

Astrocytoma cells contain significant amounts of cytosolic phospholipase

Az (cPLAz) functionally linked to G-protein coupled receptors for
thrombin, lysophosphatidic acid and acetylcholine (muscarinic M3), as well
as receptors for ligands of other families such as secretory phospholipase
A2 (sPLA2) and TNF-a. All of these agonists activated cPLA2 as judged
from the appearance of both fre arachidonate (AA) in the cell culture
supernatant and the phosphorylated slow-migrating form of cPLAz in
western blots; however, the agonists differed regarding their ability to
activate MAP kinases. Thrombin and sPLAz activated WI2-MAP/ERK, eJun N-terminal kinase (JNK), and p38 MAP kinase, whereas TNF-a did
not activate p42-ERK kinase. Studies directed to assess the temporal
pattern of activation of the MAP kinases relative to cPLAz and
experiments with pharmacological inhibitors showed the involvement of
JNK and p38 in response to both thrombin and TNF-a, and the
requirement for p42-MAP/ERK in response to sPLA2. Analysis of the AA
metabolites present in the cell medium showed that most label appeared as
free AA, whereas a 25% of the label from cells incubated with TNF-a for
periods as long as 24 hours eluted as cyclooxygenase products in RPHPLC. Unlike thrombin, TNF-ct was found to produce a rapid and
persistent activation of the transcription factor nuclear factor rd3 (NF-r,.B),
and a time-dependent induction of cyclooxygenase-2 (COX-2). In
summary, stimulation of G-protein coupled receptors in 1321N1
astrocytoma cells produces a transient activation of AA release without
further oxidative metabolism to eicosanoids. In contrast, stimulation of the
TNF receptor family induces a transcriptional activation of COX-2, most
likely related to the activation of NF-r,B, which accounts for the
conversion of AA to eicosanoids.

Lysosomal ceramide is not involved in

stress-induced apoptosis
T. Levade ~, B. Srgui*, C. Bezombesb, E. Uro*, J. Medin ~, N.
Andrieu-Abadie', M. Chatelut , R. Salvayre*, J.P. Jaffrrzou b
INSERM U 466, CHU Rangueil, and bINSERME 9910, Institut C,
Rrgaud, Toulouse(France);Universityof Illinois, Chicago, IL, USA
A major lipid signalling pathway in mammalian cells implicates the
generation of ceramide from the ubiquitous sphingolipid sphingomyetin
(SM) [1,2]. Hydrolysis of SM by a sphingomyelinase present in acidic
compartments has been reported to mediate, via the production of ceramide,
the apoptotic cell death triggered by stress inducing agents [2]. We have
shown that natural ceramide is unable to escape lysosomes [3]. In the
present study, we investigated whether the cerarnide formed within or
accumulated in lysosomes can indeed trigger apoptosis. We show that
stimulation of SV40-transformed fibroblasts by TNFet or an anti-CD40
resulted in apoptosis equally well in cells derived from control individuals
and from a patient affected with Farber disease (a genetic defect of acid
ceramidase activity leading to lysosomal accumulation of ceramide).
Furthermore, induction of apoptosis using anti-CD95, TNF0q
daunorubicin, and ionizing radiation was similar in control and Farber
disease lymphoid cells. In all cell types, apoptosis was preceded by a
comparable increase of intracellular ceramide levels. Retroviral-mediated
gene transfer of acid ceramidase in Farber fibroblasts, which led to complete
metabolic correction of the ceramide catabolic defect, did not affect the cell
response to TNF~ and anti-CD40. We also demonstrate that neither TNF~
nor anti-CD95 induced the degradation to ceramide of a natural SM that had
been first introduced selectively into acidic compartments. In addition, while
short-chain ceramides induced apoptosis, loading cells with natural ceramide
through receptor-mediated endocytosis did not result in cell death.
Altogether, these data strongly suggest that lysosomal ceramide is not
involved in the signaling pathway of stress-induced apoptosis.
Refs: 1. Hannun, Y.A. (1996) Science 274, 1855-1859
2. Testi, R. (1996) TIBS 21,468-471
3. Chatelut et al. (1998) FEBS Lett. 426, 102-106

Abstracts FEBS'99

NUCLEAR PHOSPHOLIPASE C 0PLC) SIGNALLING.

Lucio Cocco, R. Stewart GUmour*, Sue (7oo Rhee and Francesco A.
Manzoli. Instituteof Anatomy,Universityof Bologna,Italy;The Universityof
Auckland, School of Medicine, N.Z.*; Laboratoryof Cell Signaling,NHLBI,
NIH, Bethesda, MD.

We have demonstrated that in Swiss 33"3 fibroblasts the mitogenic response to

IGF-I involves the participation of the whole phospboinositide (PI) cycle in the
nucleus. A key step in this signalling pathway is the activation of a nuclear
specific phospholipase C (PLC) isoform ([~D. The activity of this isoform, whose
nuclear localisation is dependent on a cluster of basic residues in the COOHterminal domain, increases 2-3 fold within minutes of stimulation by IGF-I
leading to a massive production of diacylglycerol and to the subsequent
translocation of PKCcc within the nucleus. A wider implication for the nuclear PI
cycle comes from studies on the differentiation of Friend erythroleukemia cells.
When they are induced to erythroid differentiation in the presence of both
dimethylsulfoxide (DMSO) and the anti-turnout drug tiazofurin the nuclear PLC
13~ is down-regulated. These observations hinting at a key role of PLC 13j both
during erythroid differentiation programme and IGF-I induced mitogenesis have
been strengthened by experiments in which PLC 131 has been overexpressed or
completely down-regulated by expressing antisanse mRNA for PLC 131. In Swiss
3T3 cells stably transformed with an antisense PLC 131 construct, the abolition of
the PLC 131 gene product, as determined by Western Blots, induces a loss of the
mitogenic responsiveness to IGF-I showing a direct relationship between nuclear
PLC 131 evoked signals and IGF-I induced cell growth. This is reinforced by the
evidence that in cells overexpressing nuclear PLC [It the mitogenic response to
IGF-I is dramatically increased respect to wild-type cells. The overexpression of
nuclear PLC 13z in Friend cells impairs the erythroid differentiation induced by
DMSO. The down-stream step of the above described nuclear PLC signalling
seems to be constituted by transcription factors such as AP-I in Swiss 3T3 cells
stimulated with IGF-I and NF-E2 in Friend erythroleukaemia cells. The
activation of the two transcription factors appears to be dependent on the
expression and activity of PLC 131. The issue of the regulation of the nuclear
PLC 131 has been investigated by means of mutants in the region of the COOHterminal domain containing the consensus sequence for MAPkinase. Indeed
Swiss 3T3 cells transfected with these mutants lack both the activation of nuclear
PLC 131 and mitogenicity upon IGF-I stimulation. All in all these data assign a
key role to the regulation of nuclear PLC 13~ activity and expression and
indicate that modulation of nuclear PLC 13t has an important function in
switching cell programming from a proliferative to a differentiative state.

s57

s58

Abstracts FEBS'99

12.1 Mitochondrial biogenesis function and pathologies

Tight control of gene expression and respiration in mammal
mitochondria
G. Attardi, Y. Bai, G. Villani, M. Greco, P. Fernandez-Silva,
C. Ausenda, J. A. Enriquez, M.-X. Guan, A. Chornyn
Divtston of Biology, Cahforma Institute of Technology, Pasadena. CA 911~
USA

The increasing evidence that mutations of mitochondrial DNA (mtD~

are frequently associated with diseases and accumulate with aging has raised
issue of what are the genetic and functional thresholds operating in hun
mitochondria. The development of novel approaches in this laboratory
opened the way to investigating this important question. Thus, the analysk,
human cell lines containing different proportions of the wild-type and mul
versions of a given mitochondrial tRNA gene or different amounts o f a partict
wild-type tRNA has indicated that, in cultured human cells, there is only a two
three-fold excess of at least some of the tRNAs over the levels required to sup[
a normal rate of mitochondrial protein synthesis. Furthermore, the constructior
mouse cybrid cell lines carrying in a constant nuclear background a vary
number of copies of wild-type genes for the ND5 subunit of the respirat
NADH dehydrogenase has allowed the determination of the thresholds operat
at various levels of expression of this gene. Thus, it has been shown that, in w
type cells, the copy numbers of ND5 gene and of ND5 mRNA are only ab
twice the minimal level needed to support a normal rate ofND5 subunit synthe
Furthermore, this synthesis is rate-limiting for the assembly of the functio
NADH dehydrogenase required to support the normal rate of respiration a
consequently, rate-limiting for respiration. Similar conclusions have been reacl
in experiments in which wild-type cells were nearly completely depleted
mtDNA by three-day exposure to a low concentration of ethidium bromide,
analyzed during the subsequent re-establishment of the normal complemenl
mtDNA.
In the area of control of respiration, the use of a novel methodology us
intact cells has revealed that, in a variety of human cell types, the cytochrorr
oxidase (COX) capacity is in low excess (16-40%) relative to that requirec
support the endogenous respiration rate. In other experiments, the COX capa~
has been shown to be nearly limiting (7-22% excess) for state 3 (ADP-controll
respiration.
The evidence obtained in the experiments described above has clef
indicated that the control of gene expression and respiration in mammal
mitochondria is much tighter than is generally assumed.
PROTEIN TRANSLOCATION
INTO MITOCHONDRIA
W. Neupert, lnstitur fitr Physiologische Chemic,
Universitiit Miinchen, Goethestr. 33, D-80336 Miinchen,

The biogenesis of mitochondria involves the translocation of several hundreds of

different proteins from the cytoplasm into the mitochondria. These proteins are
encoded by nuclear DNA, synthesized on cytosolic ribosomes and then transported
to the various rmtochondrial subcompartments, the outer membrane, the intermembrane space (IMS), the inner membrane and the matrix space. Translocation
machineries which facilitate this process are located in both the outer and the inner
membranes, the TOM and TIM complexes, respecttvely. Further assisting components are present in the IMS and the matrix.
Recognition of preproteins, their partial transfer across the outer membrane and
the insertion of resident outer membrane proteins are mediated by the TOM complex. The TOM complex from N. crassa was purified to homogeneity. Upon reconstitution into lipid vesicles it proved to constitute an active translocase for
various preproteins forming cation selective, high conductance channels. Isolated
TOM complex particles are about 140A in diameter and appear to contain two
pores per particle.
Further translocatlon across the outer membrane and movement into and
through the inner membrane is mediated by one, of at least two, import machineries, the TIM complexes, and requires a membrane potential. The Tim17-Tim23
machinery facilitates translocation of presequence-targeted preproteins. Tim44,
mt-Hsp70 and Mgelp, hydrophilic components attached to the complex at the matrix side of the inner membrane, drive import into the matrix in an ATP-dependent
manner. The Tim22-Tim54 complex represents a second independent translocation
machinery which mediates the insertion of members of the mitochondrial carrier
family into the inner membrane. Three homologous proteins of the intermembrane
space, Tim9, Tim l 0 and Tim 12 interact directly with the incoming carrier proteins
at the TOM complex. They appear to chaperone their movement from the outer
membrane to the inner membrane. These small, divalent metal binding proteins
form complexes which together with Tim22-Tim54 facilitate the insertion of the
earner proteins directly into the inner membrane.
The Oxal complex mediates the insertion of a subset of proteins, some nuclearencoded, others mitochondrially encoded, into the inner membrane. These proteins
reach their correct orientation via a membrane potential-dependent export step
from the matrix. The Oxal translocase in the inner membrane mediates this export
step in a proton motive force requiring reaction.

Mitoehondrial assembly in yeast

Les A. Grivell
Sectionfor MolecularBlolog3,. Institute of Molecular Cell Biology,
Um~rstiy of Amsterdam, Kruislaan 318, 1098SMAmsterdam, The
Netherlands

The yeast Saecharomyces cerevisiae is likely to be the first organism for which
a complete inventory of mitocbondrial proteins and their functions can be
drawn up. A survey of the 340 or so proteins currently known to be localised in
yeast mitochondria reveals the considerable investment required to maintain
the organelle's own genetic system, which itself contributes seven key
components of the electron transport chain. Translation and respiratory
complex assembly are particularly expensive processes, together requiring
around 150 of the proteins so far known. Recent developments in both areas
will be reviewed and approaches to the identification of novel mitochondrial
proteins discussed.

Mitochondriai disoders: in and out of the magic circle

M. Zeviani
Divisionedi BiochimJcae GenetJca, lstituto NazionaleNeurologico"CarloBesta"
wa Celona, 11 Mdano, Italy

In contrast with the remarkable wealth of information concerning mtDNA

mutations, our knowledge on nuclear genes responsible for OXPHOS
disorders is scanty. A defect in communication between the nuclear and
mitochondrial genomes has been hypothesized in autosomal dominant or
recessive disorders associated with multiple mtDNA deletions. Three loci for
an autosomal dominant form of multiple deletions, have been identified on
chr. 10q, 3p, and 4q, while mutations in thymidine phosphorylase, located
on chromosome 22q13.32-qter, are responsible for an autosomal recessive
form. The most common disorder due to nuclear gene mutations is an
infantile encephalopathy (Leigh' syndrome, LS), characterized by a severe
involvement of the brainstem and cerebellum. A generalized defect of
cytochrome c oxidase, COX, is common in LS. No mutation in any protein
subunits of COX has ever been identified in LS ~cx~ patients. Using
complementation assays based on the fusion of LS ~ex) cell lines with
several rodent/human rho hybrids we demonstrated that the COXphenotype was rescued by the presence of a normal human chromosome 9.
Sequence analysis of SURF-l, a candidategene on chr. 9, revealed
mutations in numerous DNA samples from LS ~cx) patients, indicating that
SURF-1 is a LS ~ex'~gene. A mitochondrial etiology related to the energy
pathway has also been discovered in a number of nettrodegenerative
diseases. One example is Friedreich's ataxia, whose responsible protein,
called frataxin, is involved in the intramitochondrial handling of iron. Iron is
an important component of the prosthetic groups of cytochromes, and of the
sulfur-iron clusters of respiratory complexes. Another example is an
autosomal recessive form of hereditary spastic paraplegia (HSP) which has
been linked to loss-of-function mutations in a gene encoding a novel protein,
named Paraplegin. Paraplegin is highly homologous to the yeast
mitochondrial ATPases, AFG3, RCA1, and YME1, which have both
proteolytic and chaperon-like activities at the inner mitochondrial membrane.
These examples indicate that genes controlling the formation, activity, and
turnover of the respiratory chain complexes can be responsible for
OXPHOS disorders in humans.

Abstracts FEBS'99

s59

Mitochondria in Apoptosis
Guido Kroemer
CNRS, ERSI984, 19 rue Guy M6quet, F-94801 Villcjmf, France
The hypothesis that mitochondria control cell death has passed through
successive phases of neglect, disdain, suspicion, and (partial) acceptance.
Although the details are still controversial, it is aggreed on that, in response to
most pro-apoptotic signal transduction pathways or lethal damage pathways,
mitochondrial membrane permeability is compromised, leading to the disruption
of essential mitochondrial functions and/or the seIective release of soluble
mitochondrial intermembrane (not matrix) ~roteins (SIMPs). Several among
these SIMPs have potential apoptogenic properties: apoptosis inducing factor
(AIF), because it can translocate to the nucleus where it causes chromatin
condensation and large scale (50 kBp) DNA fragmentation; pro-caspases 2,3,
and 9 because they participate in the caspase activation cascade; and cytochrome
c because it interacts with Apaf-I to activate caspase-9. If these proteins, in
particular caspases, become activated, they give rise to typical apoptotic cell
death. In contrast, when caspases are inhibited (or when their activation is
prevented due to the depletion of the Apaf-1 co-factor ATP), cells die from a
bioenergetic catastrophe without acquiring the apoptotic morphology.
What determines cell death thus is not always the action of SIMPs. Rather, cell
death is determined by the underlying cause of SIMP release: mitochondrial
membrane permeabilization. Of note, it appears that both anti-apoptotic Bcl-2like proteins and pro-apoptotic Bax-like proteins act on mitochondria to inhibit or
favor membrane permeabilization, respectively. The apoptogenic activity of Bax
involves its translocation from the cytosol to the outer membrane, followed by a
conformational change (perhaps induced by interaction with proteins from the
pro-apoptotic "BH3-only" branch of the Bcl-2 family). It has been suggested that
Bax would be itself sufficient for membrane permeabilization and that
oligomerization of this pore-forming protein would allow to build up channels
sufficiently large to release SIMPs. However, the Bax-mediated release of
SIMPs is inhibited by cyclosporin A, a ligand of mitochondrial cyclophilin D,
and by bongkrekic acid, a ligand of the adenine nucleotide translocator (ANT),
indicating that Bax-mediated outer membrane permeabilization requires, at least
at some point, interaction with sessile mitochondrial proteins from the inner
membrane. Moreover, these findings suggest the involvement of the permeability
transition pore complex (PTPC), in the regulation of membrane permeability.
This scenario has important implications for the understanding of pathologyrelated dysregulations in apoptosis, as well as for the design of therapeutic
strategies aimed at correcting such imbalances in cell death regulation.

s60

Abstracts FEBS'99

12.2 Proton pumps, redox-coupled systems and ATPases

Cytochrome bct Complexes: Possible Mechanisms of
Proton pumping in light of the X-ray structures.
Edward Berry
Lawrence Berkeley National Laboratory. Berkeley, CA USA

Catalytic mechanism of bovine heart cytochrome c oxidase

S. Yoshikawa a, K. Shinzawa-Itoh a, T. Tsukihara b

The cytochrome bct complex comprises the middle segment of the

mitochondrial respiratory chain, oxidizing ubiquinol and reducing
cytochrome c. In the process 2 H+/e- translocated across the mitochondrial
membrane, contributing to the electrochemical potential gradient of protons
which is a source of energy for transport and ATP synthesis.
Homologous membrane protein complexes in bacteria perform the
same function for these cells, and the related b6f complex of cyanobacteria
and chloroplasts oxidizes plastoquinol and reduces plastocyanin or
cytochrome c6 in the photosynthetic electron transport chain.
Proton translocation is achieved in the bct complexes by asymmetric
arrangement of the active sites for H+-producing and H*-consuming redox
reactions, as proposed in Peter Mitchell's chemiosmotic hypothesis. More
specifically it probably occurs by a modified version of his "protonmotive Q
cycle mechanism" which accounts for the observed stoichiometry and a
number of other peculiar phenomena. Thus on one level we understand the
mechanism of the enzyme without a need for structural information.
There are now five structures for vertebrate mitochondrial bct
complexes in the protein database, and a fungal structure is expected soon.
The b6f complex is yielding, albeit more slowly, to structural investigations
by x-ray and electron crystallography. What do these structures add to our
understanding of the function of the enzyme? What do they add to our
knowledge of membrane protein architecture?
Location of two binding sites for quinone and quinone-analog
inhibitors, in protonic equilibrium with opposite faces of the membrane and
within electron transfer distance of cytochrome b heroes, supports the
modified Q cycle. Details of proton transfer from these sites to the
respective aqueous phases are being worked out. The unexpected mobile
role of the Rieske protein extrinsic domain suggests an evolutionary past in
which this domain was a soluble periplasmic protein. Structural details
allow a more informed discussion of the mechanism for enforcing bifurcated
electron transport at the Qo site, which is essential to achieve the observed
stoichiometry.

Cytochrome c oxidase is the terminal oxidase of cell respiration which reduces

02 to H20 coupled with proton pumping. The most important information
for elucidation of the catalytic mechanism of this enzyme is redox coupled
conformational changes at high resolution. After solving X-ray structure of
bovine heart eytochrome c oxidase in the fully oxidized state at 2.8 /~
resolution[l], we have been trying to improve resolution of the X-ray crystal
structures of the enzyme in various oxidation and ligand binding states.
Recently, we have obtained the structures of the fully oxidized, fully reduced,
azide-bound and CO-bound forms at 2.3 A, 2.35 A, 2.9 A and 2.8 A
resolutions, respectively. In the fully oxidized enzyme, a bridging peroxide
was found between the two metals, Fea3 and CUB in the 02 reduction site.
One of the three histidine imidazoles which coordinate to CUB was covalently
linked to a tyrosine near the 02 reduction site to fix the tyrosine-OH close
enough to form a hydrogen bond with 02 bound at Fea3. The tyrosine was
linked to a hydrogen bond network leading to the molecular surface on the
matrix side. These findings suggest that 02 reduction is triggered by the
hydrogen bond formation between the tyrosine and 02 bound at Fea3 to give a
hydroperoxide intermediate, not g-peroxo intermediate as has been suggested

Cytochrome c oxidase: Proton pumping by electrostatic

repulsion
H. Michel
Max-Planc/,*hlstttutftir Btophystk, Hetnrich-Hoffinann-Str 7. 60528
Frankfilrt, Germany

Triggered by the results of recent X-ray crystallographic analyses, published

data concerning the coupling of individual electron transfer steps to proton
pumping have been reanalysed. The widely accepted view that two protons
are pumped during each of the last two electron transfer steps [1 ], which are
the P---~F and F--+O transitions, is challenged. The input parameters used in
ref. 1 to show that the P-->F and F--+O transitions are coupled to proton
pumping exclusively, are erroneous. Based on the available structural,
spectroscopic, and mutagenesis data a detailed mechanistic model, carefully
considering electrostatic interactions has been presented [2]. In this model
each of the four reductions of heine a during the catalytic cycle is coupled to
the uptake from the inner side of one proton via the D-pathway. These
protons, but never more than two, are temporarily stored in the regions of the
heme a and a3propionates, and are driven to the outside by electrostatic
repulsion from protons entering the active site during turnover. The first
proton is pumped by the uptake of one proton via the K-pathway during
reduction of the binuclear site. Atomic structures are assigned to each
intermediate. The classical P(eroxy)-state is assumed to be already an
oxoferryl state in agreement with recent results using resonance Raman
spectroscopy [3]. Alternative proton pumping cycles based on the same
principles are discussed.
[1] M. Wikstrrm, Nature, 388, (1989), 776.
[2] H.Michel, Proc. Natl. Acad. Sci. U.S.A., 95, (1998), 12819.
[3] D.A. Proshlyakov et al., Biochemistry, 35, (1996), 8580.

aDepartment of Life Science, HiraejiInstitute of Technology, Kamigohri

Akoh Hyogo 678-1297. Japan, blnstitutefor Protein Research, Osaka
Untversity, YamadaOkaSuita 565-0871. Japan

[2].
A redox coupled conformational change in Asp51 of subunit I and a hydrogen
bond network connecting Asp51 with the matrix surface only in the fully
oxidized state indicated a redox coupled switching in the accessibility of the
carboxyl group of Asp51 to the bulk water phase on one side to the other
accompanied by a possible pK shift in the carboxyl group. The hydrogenbond network system including Asp51 did not include the 02 reduction site
suggesting an indirect coupling between proton pumping and 02 reduction in
the enzyme.
[1]T. Tsukihara et al. Science, 269 (1995) 1069.
[2]W. S. Caughey et al. in The Enzymes (Boyer P. D., ed) Academic Press,
New York vol. 13 (1976) 299.
ATP synthase's motor function
Peter Dimroth
Mikrobiologisches Instimt, EidgenOssische Technische
Hochschule, ETH-Zentrum, CH-8092 Ziirich, Switzerland
The ATP synthase is constructed of an extrinsic domain, Ft, which harbours
the catalytic sites for ATP production and a membrane-integral domain, Fo,
which transforms the free energy of an electrochemical gradient of protons
or Na+ ions into a counterrotation of the rotor versus the stator. The motor
module consists of a single a subunit, being part of the stator and a ring of
12c subunits, being part of the rotor. When ATP is synthesised, the coupling
ions move through the stator channel onto a binding site on the rotor from
where they are released to the opposite side of the membrane after the rotor
has turned. We propose a functional model for the generation of rotational
torque. 1) An empty, negatively charged rotor site is electrostatically
attracted by the positive stator charge. 2) The electrical potential facilitates
the thermal escape of the rotor site into the direction of the stator channel. 3)
The rotor site is occupied with the coupling ion from the stator channel. 4)
This allows further rotation of this site through an area of low dielectric. 5)
By this movement, the coupling ion gets access to the opposite side of the
membrane and dissociates.
The model is consistent with recent results on structure and function of the
Na+-translocating ATP synthase of Propionigenium rnodestum.

Abstracts FEBS'99

s61

13.1 Neurotransmitter receptors

Structure and function of glycine receptors
H. Betz, B. Laube, N. Griffon, J. Kuhse, V. Schmieden
Max-Planck-lnstitut for Himforschung, Abt. Neurochemie,
Deutschordenstr. 46, 60528 Frankfurt, Germany

The inhibitory glycine receptor (GIyR) is a pentameric

chloride channel protein, which exists in several
developmentally and regionally regulated isoforms in the
CNS. These result from the differential expression of four
genes encoding different variants (al-ct4) of the ligandbinding subunit of the GlyR. The stoichiometric assembly of
these 0~subunits with the structural 13 subunit is governed by
"assembly cassettes" within the extracellular domains of
these proteins and creates chloride channels of distinct
conductance properties. GIyR gating is potentiated by Zn 2+,
a metal ion coreleased with different neurotransmitters. Sitedirected mutagenesis has unraveled major determinants of
agonist binding and Zn2+ potentiation.
Different motor disorders in man and mouse result from
mutations in GIyR subunit genes. The closely related spastic
and spasmodic phenotypes in mouse cause myoclonus and
hyperexcitability after the second week posmatally and are
due to insertional and point mutations in the [~ and ctl
subunits, respectively. Human hereditary startle disease or
hyperekplexia has been associated with point mutations
affecting the agonist affinity of the GlyR. In mouse, some of
these phenotypes have been recently rescued or mimicked by
transgenic approaches. In addition, deletion of the GIyR
anchoring protein gephyrin has been found to cause a similar
phenotype.

Molecular insights into GABAj~ receptor physiology

B. Bettler,, Nervous ~,stem Research, Novartis Pharma AG.
K-125.6.08, CH-4002 Basel, Switzerland

Receptors for extracellular nucleotides

R.A. North a, F.A. Rassendrcn b, A. Surprenant a, C. Virginio b,
A. MacKenzie a
alnstitute of Molecular Physiology, University of Sheffield, UI~ hIGH,
UPR 2142 CNRS, 34396 Montpellier, France, CGlaxo Wellcome
Research and Development, 37135 Verona, Italy.

P2X receptors are ligand-gated ion channels activated by the binding of

extracelhilar adenosine 5'-triphosphate (ATP). Seven subunits are known,
and channels can form by homo- or hetero-polymerization. Most of the
protein lies on the extmcellular aspect of the plasma membrane; the second
of the two transmembrane domains contributes residues to the ioinic pore.
When recording from P2X2 receptors expressed in human embryonic kidney
ceils or from sensory neurons in rat nodose ganglion, the ionic pore opened
by brief applications (< 1 s) of ATP is cation-selective, and significantly
permeable to calcium; it has only a low permeability to larger organic
cations such as N-methyl-D-glucamine (PNMnG/PN~ 0.05). However, with
prolonged ATP application (10 - 60 s) the channel becomes readily
permeable to large cations (PNM~/PN~ 0.5), and fluorescence microscopy
shows entry of the even larger divalent cationic marker YO-PRO-1. This
pore dilatation was observed also for the P2X4 receptor, and it was much
enhanced in P2X., receptors carrying point mutations in the second
transmembrane segment (N333A). Progressive dilatation of the ionconducting pathway during prolonged activation reveals a hitherto
unexpected mechanism by which ionntropic receptors may alter neuronal
function.

Dopamine receptors signaling in cell proliferation and

"behavior
E. Borrelli, Y. Bozzi, H. Courvoisier,V. Heidt, C. Iaccarino, C.
Mathis, R. Picetti, T. A. Samad, A. Usiello, D. Vallone
IGBMC, INSERM/CNRS/ULP, BP 163, 67404 lllkirch Cedex, France

GABAB receptor~ were first recognized 18 years ago and it is 25 years since
the GABAB receptor agonist baclo|en was introduced into the treamlent of
spasticity. However the lack of a molecular understanding of the GABA,
receptor system made it impossible to fully exploit its therapeutic potential.
It was not until 1997 that the development of the high-affinity antagonist
[1251]CGP64213 allowed the isolation of GABAaRIa (BRla) using an
expression cloning approach (Kaupmann et al., 1997). Subsequently the
GABAsRIb cDNA was isolated using homology screening. GABA~RIa
and GABABRIb derive from the same gene by N-terminal alternative
splicing. Database searches with the GABAaRI sequence information led to
the discovery of GABABR2 (Kaupmann et al., 1998a). The distribution of
GABABRI and GABABR2 transcripts in the brain, as studied by in situ
hybridization, is largely overlapping and qualitatively parallel those of
GABAB agonist and antagonist binding sites, suggesting that GABAaR I and
GABABR2 constitute the majority of native GABAB binding sites. Although
the cloned GABAB receptors showed many of the expected properties in
terms of structure and pharmacology, they only reluctantly reproduced the
signalling properties of native receptors in heterologous cells (Kaupmann et
al., 1998b). The strong overlap of the in sito hybridization patterns indicated
that GABABR1 and GABABR2 are co-expressed in many neuronal
populations and that a co-expression was possibly needed for robust
functional activity. Indeed while neither GABABRla/-b nor GABABR2
alone efficiently activated Kir3 channels their co-expression in HEK293
cells and Xenopus oocytes yielded robust GABA evoked currents.
lmmunoelectron microscopy using specific antibodies provided further
evidence for an assembly of heteromeric GABAB receptors in vivo by
showing an extensive co-localization of GABABRI and GABABR2 proteins
at Purkinje cell dendritic spines. This represents the first demonstration of
hetero-assembly among G protein coupled receptors.
Kaupmann et al., Nature 386, 1997, 239.
Kaupmann et al., Nature 396, 1998a, 683.
Kaupmann et al., Natl Acad Sci USA 95, 1998b, 14991.

Dopamine D2 receptors are abundantly expressed in the brain and in the

pituitary gland. The physiological function of these receptors in vivo has been
addressed using genetically engineered mice in which the expression of these
receptors has been altered (D2R-/- mice). The analysis of the phenotype of
D2R -/- mice has allowed us to show that these receptors have key roles in
the regulation of the locomotor behavior as well as in the coordination of
movements. Furthermore, at the pituitary level we have shown that D2
receptors regulate lactotroph's proliferation rate and in their absence female
mice develop pituitary tumors of lactotroph origin [1]. Always at the pituitary
level dopamine also controls the identity of melanotroph, which in the
absence of D2Rs start to process proopiomelanocortin not only into ct-MSH
and 13-endorphin but also into ACTH [2]. Importantly, we have previously
shown that D2R-/- mice although present an unaltered locomotor response to
morphine they do not show morphine-induced place-preference [3]. This
indicates the loss of the rewarding properties of morphine in the absence of
D2Rs. In contrast, cocaine administration gives different results. Indeed,
D2R -/- mice show a lack of cocaine-induced locomotion. This indicates
different neurobiological mechanisms for the activity of these drugs and that
D2R is clearly involved in both responses. The analysis at the molecular level
of the brain of D2R -/- mice upon cocaine and morphine treatments will be
discussed as a tool to decipher the neurochemical pathways involving D2
receptors in response to drugs of abuse.
[1] Saiardi A.et al. Neuron 19, 1997:114.
[2] Saiardi A. and Borrelli E. Mol. Endo. 12, 1998:1132
[3] Maldonado R.et al Nature 388, 1997: 586.

s62

Abstracts FEBS'99

13.2 Receptors in the immune system

A. Lanzavecchia, G. Iezzi and A. Viola

FUNCTION OF ADAPTOR PROTEINS IN SIGNAL TRANSDUCTION FROM THE B CELL ANTIGEN RECEPTOR
J. Wienands,B. Wollscheidt,V. Rolli, W. Schamel, Y.-W. Su, H. Jumaa,

Basel Institute for Immunology, Basel, Switzerland

P Nielsen M. Reth Dep. of Molecular Immunology,BiologyIII, Univ.

Freiburgand MPI for Immunobiology,Freiburg,Germany

The dynamics of T C R triggering and T cell activation

In the course of the T-APC interaction, few peptide-MHC complexes trigger

and downregulate in a serial fashion many TCRs. This process of serial
triggering ensures the sustained signaling which is required for T cell
activation.
Monovalent ligation OF TCR is sufficient for triggeringand the potency of
an agonist, i.e. its capacity to trigger many TCR with time, depends from an
optimal kinetic window that is rather fast and which can be adjusted by
CD4/CD8 coreceptors. Ligation of the costimulatory receptor CD28 on T
cells, while not affecting the process of TCR serial triggering and
downregulation,

amplifies the

signalling process

by

recruiting raft

microdomains that contain LcK and LAT to the site of TCR engagements.
This mechanism is particularly important for naive T cells that express low
levels of rafts, LAT and Lck at the plasma membrane. The duration of
antigenic stimulation determines the fate of antigen-stimulated T cells, i.e.
their clonal expansion, the development of memory and effectors
phenotypes

and ultimately their death. Prolonged TCR and cytokine

signalling offers a window of opportunity for epigenetic changes in cytokine

genes and is essential to determine in a stochastic fashion the cytokine
production phenotype.

Regulation o f l y m p h o c y t e activation by I T I M bearing receptors and their activating isoforms

E. Vivier
Centre d'Immanologie de Marseille-Luminy and
Institut Universitalre de France
A family of cell surface receptors involved in the control of cell activation
has recently emerged. These receptors are characterized by the presence of
1 to 4 intracytoplasmic Immunoreceptor Tyrosine-based Activation Motifs
(ITIMs) based on the consensus (I/V/L/S)xYxx(L/V). ITIM-bearing
receptors belong to one of two receptor families: the immunoglobulin- and
the C2 lectin proteins. ITIM-bearing receptors are expressed on
hematopoieitc cells as well as non-hematopoietic cells. ITIM-bearing
receptors require co-aggregation with protein tyrosine kinase-dependent
receptors in order to mediate their inhibitory function. Upon engagement,
the Tyrosine residue present in 1TIM is phosphorylated and allows the
association with SH2-containing intracytoplasmic phosphatases.
Phosphatases recrmted in vivo by /TIM-bearing receptors belong to 2
categories: the protein tyrosine phosphatases, SHP-1/SHP-2 and the
polyphosphate inositol pbospbatase, SHIP. Whereas FcTRIIB is the only
SHIP-associating ITIM-bearing receptors, all other 1TIM-bearing
molecules bind to SHP-1 and/or SHP-2. We focused our attention on
Killer-cell Ig-like Receptors (KIRs), which serve as NK and T lymphocyte
receptors for MHC Class Ia molecules, on the CD94-NKG2A/B
heterodimers, which serve as NK and T lymphocyte receptors for MHC
Class Ib molecules (e.g. HLA-E), as well as on PIR-B, which is
expressed on mouse B and myeloid cells, and showed that these receptors
are ITIM-bearing inhibitory receptors. Isoforms of ITIM-bearing receptors
have been identified, and are characterized by their lack of intracytoplasmic
ITIM, as well as their activating properties. We have identified
KARAP/DAP-12, a 12 kDa transmembrane polypeptide bearing an ITAM
(Immunoreceptor Tyrosine-based Activation Motif) which associates with
KARs (Killer-cell Activating Receptors), the activating isoforms of KIRs.
Genetic dissection of IT/M-beating receptors and KARAP will contribute
to the precize understanding of this novel regulatory pathway in vivo.

The B cell antigen receptor (BCR) complex consists of the membranebound immunoglobulin molecule (mIg) and the Ig-ct/Ig-I~heterodimer
involved in ligand-binding and signal transduction, respectively. We
analysed the composition of affinity purified BCR by blue native gel
electrophoresis. Only one band reacting with anti-lg and anti-Ig-ct
antibodies was detected, showing that the BCR is forming a complex of
defined size the stoichiometry of which we are currently analysing.
One of the most rapidly phosphorylated proteins, detected after ligation
of the BCR or exposure of B cells to pervanadate, is the B cell specific
adaptor protein SLP-65/BLNK, showing similarity to the T cell adaptor
protein SLP-76. Both proteins are forming complexes with other
adaptor proteins like Grb2 and Vav and are essential for lymphocyte
activation. The phosphorylated SLP-65 protein is recruiting the protein
tyrosine kinase BTK to the complex and this may result in increasing
PLC-y phosphorylation and activity. Employing the hormone regulated
Cre recombinase MerCreMer we developed a method for inducible
gene expression in mamalian cells. By inducibely expressing the BCR
we demonstrate that the phosphorylation of SLP-65 and Syk is
dependent on BCR expression. These data suggest that SLP-65 and Syk
is reorganised by the BCR into an active signalling complex. The
implication of these data for the signalling function of the pre-BCR and
BCR will be discussed.

Abstracts FEBS'99

s63

13.3 Phosporylation/dephosphorylation in regulatory processes

The cAMP dependent protein kinase. Structural
approach and role in Dictyostelium discoideum
development
M. V~ron
Unit~ de R~gulatlon enzymatique des Activit~s ceUulaires,
Institut Pa~eur, Paris, France

cAMP dependent protein kinase (PKA) is present in all eukaryotic

organisms. The first protein kinase to be discovered, it was also the first for
which the 3D-structure was resolved by X-ray crystallography. Although all
PKAs show high sequence homology, differences between PKAs from higher
and lower organisms have allowed us to propose a functional role for some
protein domains not directly involved in substrate binding and/or catalysis. The
relevance of these observations for other important kinases including MAP
kinase and Src whose crystal structures have recently been determined will be
discussed.
PKA plays a pivotal role in the developmental process of the cellular slime
mold Dictyostelium discoideum. Mutants lacking R subunit or with
overexpressed C subunit prematurely develop into spores, while mutants
lacking the C subtmit can grow but not develop. The role of PKA in
Dictyostelium development will be discussed in view of its connection with a
two-component system regulating phosphodiesterase in the spore pathway.

Role of the PDKI/PKB/GSK3 cascade in insulin signaltransduction

P Cohen

MRC Protein Phosphorylation Unit, Umversi(v of Dundee,

MSI/WTB Complex, Dow ,Street, Dundee DDI 5EH. Seotland

A protein kinase cascade has been dissected that plays a key role
in mediating the insulin-induced stimulation of glycogen synthesis.
In this cascade, insulin stimulates the activation of protein kinase
(PKB) which then inactivates glycogen symhase kinase-3 (GSK3),
leading to the dephosphorylation and activation of glycogen synthase
and hence its the stimulation of glycogen synthesis. The insulin-induced
activation of PKB requires its phosphorylation at two residues,
Thr308 and Ser473, and is prevented by inhibitors of
phosphatidylinositide (Ptdlns) 3-kinase. A protein kinase that
phosphorylates Thr308 has been identified and termed
3-phosphoinositide-dependent protein kinase-1 (PDK1) because it
only phosphorylates PKB in the presence of Ptdlns(3,4,5)P3
(the product of the Ptdlns 3-kinase reaction) or Ptdlns(3,4)P2.
In this talk, the likely identity of the Ser473 kinase will be revealed and
the mounting evidence that suggests that a wider role for the
PDK1/PKB/GSK3 cascadein insulin signal transduction will be reviewed.
Reference.
[1 ] Cohen, P. (1999). Phil.Trans.Roy.Soc.Lond.B 354, 485-495

Nuclear targeting of Ser/Thr protein phosphatase 1

M. Bollen, M. Beullens, A. van Eynde, V. Vulsteke, I.
Jagiello, Q. Jin, H. Ceulemans, W. Stalmans
Afdeling Biochemie, Facultett Geneeskunde, Katholieke Universiteit
Leuven, B-3000 Leuven, Belgium

Ser/Thr protein phosphatases of type 1 (PP1) consist of a catalytic

subunit and one or two non-catalytic subunits that control the activity and
substrate specificity of the holo-enzyme and bring the phosphatase in close
proximity of its physiological substrates. Close to 20 mammalian regulatory
proteins of PP 1 are currently known.
NIPPI is an inhibitory subunit (351 residues) of a major nuclear species
of protein phosphatase 1 (PP1NNwP0. We have identified two closely
spaced phosphatase-binding sites in the central domain (residues 191-210)
of NIPP1. One site represents an inhibitory stretch of basic residues. The
second, non-inhibitory site (RVTF) conforms to a 'RVXF'-consensus
sequence that also mediates the binding of other regulatory subunits to PP1.
The two serines flanking either side of the RVXF-motif are established
phosphorylation sites for protein kinases A and CK2, respectively.
Phosphorylation of these two serines abolished the binding of the RVXFmotif to the catalytic subunit and decreased the inhibitory potency of
NIPP1191-210
NIPP1 also targets PP1 to RNA. The RNA-binding is mediated by a
novel RNA-binding motif in the carboxyterminal 22 residues of NIPPI. In
the absence of RNA, this RNA-binding motif inhibits PP1 and may thus
prevent the dephosphorylation of proteins by PPIN~wPI when the
holoenzvme, is not bound to RNA. The synthetic RNA-binding motif and
NIPP1224"351 also displayed a single-strand endoribonuclease activity with a
specificity similar to that of the bacterial RNase E. However, full-length
NIPP1 and NIPP1143-351 were not able to cleave RNA, indicating that the
endoribonuclease activity of N1PP 1 is restrained by its central domain.
Four different transcripts can be generated from the human NIPPI gene
(PPPIR8). One transcript (NIPPIa) encodes full-length NIPP1. The other
transcripts are expected to encode NIPP1143-351 (NIPPII3 and NIPP15) or
NIPP1224-351 (NIPP1y/Ardl).

Regulation of cellular functions by NO/cGMP

F Hofrnarm, M. Sausbier, A. Pfeifer, M. Biel
Institut ~ r Pharmakologie und Toxikologie, TU Miinchen,
Biedersteiner
Str 29, 1)-80802 M~inchen, Germany

Cyclic GMP has been identified as the physiological mediator of a variety of

substances including nitric oxide (NO), peptide hormones, toxins, and
organic nitrates. Despite the recent progress in understanding the diverse
biological functions of NO and cGMP, it has been difficult to identify the
cGMP-effectors that mediate the NO/cGMP signal in a given tissue. We
studied the physiological function of cyclic nucleotide gated cation channels
(CNG) 1 and cGMP-depandent protein kinase I (cGKI)2 and II (cGKII),3
which are major receptor proteins for cGMP in vertebrates by introducing a
null mutation into the murine CNG3, cGKI and cGKII gene.
CNG3 is essential for day light vision but non-essential for reproduction
whereas cGKI regulates smooth muscle contractility. Inactivation of cGKI in
mice abolishes NO/cGMP-dependent relaxation of smooth muscle resulting
in severe vascular and intestinal dysfunctions without affecting the cAMP
signalling cascade. Inactivation of cGKI attenuates cGMP-dependent inhibition of platelet activation and leads to microthrombosis without affecting
cAMP-induced prevention of platelet aggregation. Thus, cAMP and cGMP
signal via independent pathways with cGKI being the specific mediator of
the NO/cGMP effects in murine smooth muscle.
1. Biel, M. et al. (1994) "Another member of the cyclic nucleotide-gated
channel family expressed in testis, kidney and heart" Proc. Natl. Acad.
Sci. USA 91, 3505-3509
2. Pfeifer, A. et al. (1998) ,,Defective smooth muscle regulation in cGMP
kinase I-deficient mice" E M B O J. 17, 3045-3051
3. Pfeifer, A. et al. (1996) ,,Intestinal secretory defects and dwarfism in mice
lacking the cyclic GMP-dependent protein kinase II gene" Science 274,
2082-2086

s64

Abstracts FEBS'99

Signaling angiogenesis via p42/p44 MAP kinase cascade

Jacques Pouyss~gur, Edume Berra, Emmanuel GothiG Julie
Milanini, Gilles Pages, Darren Richard and Francesc Vinals.
Centre de Biochimie-CNRS UMR 6543, Parc Valrose 06108
Nice, France - pouysseg@unice.fr
Vascular Endothelial Growth Factor (VEGF), a potent agonist
secreted by virtually all cells, controls migration and division of
vascular endothelial cells. Disruption of one VEGF allele in mice has
revealed a dramatic lethal effect in early ernbryogenesis, suggesting
a key role in vasculogenesis. We first analysed the regulation of
VEGF mRNA in normal and transformed CCL39 fibroblasts and
second, we dissected the VEGF promoter to identify the signaling
pathway(s) controlling the expression of this gene in response to
growth factors, oncogenes and hypoxic stress. We demonstrated
that the p42/p44 MAP kinase signaling cascade controls VEGF
expression at least at two levels. In normoxic conditions, MAPKs
activate the VEGF promoter at the proximal (-88/-66) region where
Spl/AP2 factors bind. Activation of p42/p44 MAPKs is sufficient to
turn on VEGF rnRNA. At low 02 tension, Hypoxia Inducible Factor1~ (HIF-lc0, a limiting factor rapidly stabilised, phosphorylated and
translocated to the nucleus, plays a key role in the expression of
several genes including VEGF. We demonstrated
that
p42/p44MAPKs stoichiometrically phosphorylate HIF-lcc in vivo and
that HIF-l-dependent VEGF gene transcription is strongly enhanced
by the exclusive activation of p42/p44MAPKs. Finally we
demonstrated that the regulation of p42/p44MAPK activity is critical
for controlling proliferation and growth arrest of vascular
endothelial cells at confluency. These results point to at least three
major targets, Spl and HIF-I(x for the VEGF promoter and DNA
synthesis for endothelial cells, whereby p42/p44 MAP kinases exert a
determinant action on angiogenesis.

Abstracts FEBS'99

s65

13.4 Growth factor receptors

Generation and Control of Phosphotyrosine-mediated
Signals

Vascular Endothelial Growth Factors and Receptors involved

in Angiogenesis and Lymphangiogenesis

A. Ullrich
Max-Planck-lnstaut fur Biochemie,, 82152 Martinsried, Germany

Karl Alitalo and collaborators Molecular/Cancer Bmlogy Laboratory,

Haartman Institute and Department of Biomedicine, University of Helslnki,
POB 21. 00014 Helsmki, FINLAND

P h o s p h o t y r o s i n e - m e d i a t e d signals play a central role in control and

regulation o f f u n d a m e n t a l biological processes such as cell g r o w t h ,
differentiation, movement, communication, survival and maintenance of
metabolic homeostasis. Recent findings shed new light on the process o f
receptor tyrosine kinase (RTK) activation and signal definition. In extension
to the established m e c h a n i s m s o f ligand induced homodimeric receptor
complex formation, new data highlight heterodimeric receptor aggregation as
a powerful means o f signal diversification and expansion. Promiscuous
receptor interactions involve different ligand binding kinetics and generate
divergent receptor phosphorylation sites that could allow enhanced or
modified signal generation. Besides activation by a specific ligand, a newly
defined R T K function involves signal integration o f a variety of stimuli,
including calcium-dependent responses in neuronal cells, activation of Gprotein-coupled receptors or cellular stress such as UV irradiation. On the
basis of existing evidence for such crossactivation pathways, RTKs must be
considered as representing critical foci and switch points for multiple
environmental and internal stimuli. A novel mechanism o f phosphotyrosine
signal generation involves, in contrast to previously established models,
constitutively active rather than inducible tyrosine kinases that are in a
d y n a m i c e q u i l i b r i u m w i t h c o n s t i t u t i v e l y active p h o s p h o t y r o s i n e
phosphatases (PTP). Receptor tyrosine kinase activation i.e. release o f
activity occurs then by inactivation of tyrosine phosphatases through
oxidation or physical disruption o f RTK-PTP interactions. Example and
consequences of this new model will be discussed.

Angiogcnesis, the formation of new blood vessels from preexisting ones, and the
permeabday of blood vessels are regulated by vascular endothehal growth factor (VEGF) via
its two known receptors Fhl (VEGFR-I) and KDR/FIk-1 (VEGFR-2). The Fit4 receptor
tyrosme kmase ts related to the VEGF receptors, hut does not bind VEGF and its expression
becomes restricted mainly to lymphatic endothelia during development. Heterozygous Fit4
knock-out locus containing a LacZ cassette in place of the Flta first exon gives staining
lymphatic endothelium and homozygous knock-outs die due to lailare of proper vascular
formation. We have purified the Flt4 ligand, VEGF-C, and cloned its cDNA from human
prostatic carcinoma. Whde VEGF-C Ls homologous w~th other members of the
VEGF/platelet derived growth factor family, it is made as a precursor protein having an
extended N-terminus and a C-terminal half containing extra cysteine-rlch motifs characteristic
of a protein component of silk. VEGF-C is proteolytically processed, and brads and acUvates
Fh4, which we rename as VEGFR-3. Transgemc mice expressing VEGF-C under a basal
keratin promoter developed a hyperplastic lymphatic vessel network m the skin. VEGF-C is
thus a novel regulator of endotheha. However, proteolytically processed VEGF-C was also
capable of stimulating VEGFR-2 and was weakly anglogemc. VEGF-C also reduced vascular
permeability, but its point mutant, which actwated only VEGFR-3 did not. VEGF-D is
closely related to VEGF-C, proteolytlcally processed and binds to the same receptors. Thus,
VEGF-C appears to be an angmgenic and lymphangiogenic growth factor. Another related
novel growth factor, VEGF-B was also cloned and found to be co-expressedwith VEGF and
VEGF-C m heart, muscles and less m other tissues. VEGF-B bound VEGFR-1, formed ceil
surface-assocmted, disulfide-hnked homodimers and heterodimers with VEGF when
coexpressed. Our results suggest that VEGF-B has a role in endothelial cell growth and
angmgenesis, particularly in skeletal muscle and heart.

Tie, one of the receptor tyroslne kmases we have cloned, is expressed m mouse
hematopoleuc stem cell fracaons and m all studied fetal endothelial cells. Both Tie and its
hgand are also highly expressed m several human megakaryoblast~cleukemia cell lines. In
transgemc mice the Tie gene promoter directs endothehal specific expression of heterologous
genes. Mouse embryos homozygous for a disrupted Tie allele developed severe edema, their
microvasculature was ruptured and they died between days 13 5 and 14.5 of gestation Thus
Tie is required during embryonic development for the sprouting of new vessels, particularly in
the regions undergomg anglogenic growth of capillaries, but it is not essential for
vasculogenesis. Our results thus demonstrate an increased complexity of signaling for
endothelial cell proliferation, migration, differentiation and surwval. Knowledge of these
signals is essential for the control of anglogenesis in a variety of diseases including cancer.
For a review, see: Korpelainen, E. and Ahtalo, K.: Signaling anglogenesls and
lymphanglogenesis. Current Opinion in Cell Biology 10: 159-164, 1998

Control of Invasive Growth by PRGF Receptors

Paolo M. Comoglio
Institute for Cancer Research, Umversity of Tormo Medwal School,
Strada Prownciale 142. 10060 Candlolo (Tormo), Italy

Hepatocyte growth factor (HGF), also known as "scatter factor" is a

mesenchimal cytokine which triggers in epithelial cells a unique
biological program leading to "invasive-celt-growth". This phenotype
results from the integration o f apparently independent biological
responses to HGF, among which are cell proliferation, survival, motility,
invasion o f surrounding extracellular matrices and induction o f cell
polarity. In physiological conditions, the coordinated accomplishment of
the underlayed genetic programs leads to the formation of tubular
structures by epithelial organs (the so-called branched morphogenesis)
[I]. The HGF receptor is encoded by the oncogene MET, the prototype
of a family of tyrosine kinase receptors which includes the genes RON
and SEA The three receptors feature unique signal transduction
properties as their cytoplasmic tails contain a two-tyrosine multi
functional docking site that binds and activates multiple SH2-containing
intracellular signal transducers [2]. During development the coordinated
control of "invasive growth" by the HGF/Met pair is essential, as knockout experiments involving either the ligand or the receptor result in
embryonic lethality due to defects in liver and placenta. Deregulated
activation of the "invasive-growth" phenotype by the M E T oncogene
confers to cancer cells invasive and metastatic properties [3]. M E T is
found overexpressed and amplified in a number o f human metastatic
tumors. A direct genetic connection between activation of M E T and
human cancer has been recently established by the finding of germline
and somatic mutations in the receptor catalytic domain of patients
affected by papillary renal carcinomas. The recent progresses which
have been made in dissecting the signaling mechanisms through which
the MetA-IGF receptor controls "invasive-growth" will be summarized.
In addition, the molecular mechanisms responsible for constitutive
activation of the oncogene will be discussed.
[1] Boccaccio C. et al., Nature, 391, (1998), 285
[2] Ponzetto C. et al., Cell, 77, (1994), 261
[3] Bardelli A. et al., Proc.Natl.Acad.Sci.USA, 95, (1998), 14397

Combinatorial Signal Transduetion by Oncogenie Receptor,,

Yosef Yarden
The Weizmann Institute o f Science, Rehovot 76100, Israel

An epidermal growth factor (EGF) motif is shared by multiple growth factors,

whose signals are transmitted into cells by a group o f transmembrane tyrosine
kinases o f the ErbB/HER family. In mammals the ErbB module includes four
receptors, E r b B - l / E G F - r e c e p t o r , that binds at least 6 ligands, and two
receptors for neuregulins (NRGs), ErbB-3 and ErbB-4. Importantly, no
k n o w n ligand binds with high affinity to ErbB-2, but this protein is the most
oncogenic member and its overexpression in h u m a n cancer correlates with
poor prognosis.
A combination o f in vitro experiments and gene targeting in mice
implies that ErbB proteins and their ligands are repeatedly used in midgestation as a versatile signaling module controlling several cell lineages.
This versatility stems from alternative utilization o f components o f the
signaling module, and it allows an enormously large potential for signal
diversification. Perhaps the major generator o f diversity is the ability o f each
ligand to induce rapid dimerization o f specific ErbB proteins. Apparently, all
ligands are bivalent: their high affinity site selects the primary receptor,
whereas the broad specificity o f the low affinity site displays preference for
ErbB-2.
Each o f the ten possible homo- or heterodimeric ErbB complexes is
e n d o w e d with specific signaling properties. The most potent receptor
complex is a heterodimer between the kinase-defective ErbB-3 and the ligandless oncoprotein ErbB-2. In addition, heterodimeric receptor complexes are,
in general, more potent than their homodimeric counterparts. We attributed
the mechanism underlying these differences to the sorting process o f ligandactivated receptors. Because sorting of endocytosed receptors to degradation
in the lysosome terminates signaling, receptors that constitutively recycle back
to the cell surface (e.g., ErbB-3) are more potent. We identified the sorting
protein as c-Cbl, a proto-oncogenic phosphotyrosine-binding protein whose
RING finger allows it to tag incoming receptors with ubiquitin. This ligation
targets receptors to a combined lysosomal/proteasomal degradation step. The
relevance o f Cbl-mediated sorting to oncogenesis by autocrine loops, as well
as to i m m u n o t h e r a p y o f h u m a n c a n c e r by m o n o c l o n a l antibodies to
HER2/ErbB-2 will be discussed.

s66

Abstracts FEBS'99

13.5 The multifaceted function of G-proteins

Effectors and Regulators of Small G-Proteins
P. N. Lowe
Biologtcal Chemistry Umt, Glaxo WellcomeMedicines Research
Centre, Gunnels WoodRoad, Stevenage, SG1 2NY,, England

The Ras Superfamily of small G proteins includes the Ras, Rho and Rab
families. They act as molecular switches controlling key cellular
pathways, which can be altered in disease states. Thus, they have
potential as targets for chemotherapeutic agents. In their active GTP
botmd form, they bind to protein effectors. Often one small G protein
can bind multiple unrelated effectors and hence control several
pathways. They are down-regulated by an intrinsic GTPase, stimulated
by GTPase-activating proteins (GAPs) and upregulated by exchange
factors (GEFs). Inhibitors of small G protein action could be designed to
have one of several mechanisms, e.g. interference with post-translational
modification, with the protein:protein interaction with effectors or with
the GTPase cycle. The latter two mechanisms have the potential for a
high level of target selectivity. Approaches being taken to aid their
evaluation will be described. We have developed biophysical techniques
using fluorescence, isothermal titration calorimetry and scintillation
proximity to quantify and characterize protein:protein interactions such
as Ras with Raf or RasGAPs; Rho/Cdc42/Rac with RhoGAP, PAK,
ACK and WASP. Analysis ofmutagenesis and structural studies
suggests that inhibitors could achieve high selectivity for modulating a
specific effector-driven pathway. Studies on the structure and
mechanism of GAPs, by ourselves and others, show that Ras and Rho
GAPs increase the intrinsic GTPase by contributing a critical arginine
residue to the active site. We have investigated the role of this arginine
in stabilizing the putative transition state. These data suggest that
modulation of GTPase activity by small molecules could be an attractive
target.

Ras, Ral, R a p l , what is the connection

Johannes L. Bos
Lc~boratoo'for Ph~stologlcal Chemt.~tryand Centrefor Biomedical Genetics.
Utrecht Universio. Untversifettsweg 100, 3584CG Utrecht. The Netherlands.

Ras is the paradigm of a family of 13 small GTPases that share similarities in

the effector domain, suggesting a relatlonshlp in function. For Ras, this
function has been studied extensively, revealing a role for Ras m many
biological processes. Ral is another member of the Ras family, of which the
function is still largely elusive. Recently, it was found that Ral-specific guanine
nucleotide exchange factors (RalGEFs) are direct effectors of Ras. Indeed, Ral
mediates signals from Ras towards the induction of gene expression and cell
cycle progression. For full oncogenic transformation the RalGEF pathway cooperates with other Ras effector proteins, i.e. Rafl and phosphatidylinositol-3
kmase (PI-3K) [1]. We recently identified an interesting connection between
the Ral and the PI-3K pathway in the transcription factor AFX. This factor is
directly phosphorylated by PKB, a downstream target of PI-3K and by a
putative kinase induced by the Ras-RalGEF-Ral pathway [2].
Another member of the family. Rapl (or K-revl) was identified as a revertant
of Ras-induced cell transformation. This resulted in the hypothesis that Rapl
may be an antagonist of Ras signalling, but more recent evidence indicates that
Rapl is involved in a pathway distinct from Ras. Rapl is very rapidly
activated by a large variety of growth factors, suggesting some common
function in signal transduction. Several Rapl-specific GEFs have been
identified, one of which, Epac, was found to be directly regulated by cAMP,
providing a novel cAMP-target protein [3].
[1] R.M.F. Wolthuis and J.L. Bos. Ras caught m another affair: the exchange factors
for Ral. Curr.Opin.Dev.Bioh 9( 1999)112-117
[2] G.J.P.L. Kops. N.D. de Rulter, A.M.M de Vries-Smits, D.R. Powell, J.L. Bos and
B.M.T. Burgering. Direct control of the Forkhead transcription factor AFX by protein
Kinase B. Nature in press
[3] J. de Rooij, F J.T. Zwartkruis, M.H G. Verheijen, R.H. Cool, S.M.B. Nijman, A.
Wutinghofer and J.L. Bos. Epac is a Rap I guanme-nucleotide-exchange factor directly
activated by cAMP. Nature 396(1998)474-477.

Mechanism
proteins?

of ARFI activation:

a model

for small

P. Chardin, B. Antonny, S. Beraud-Dufour, J. Cherfils*, E. Macia,

S. Robineau, S. Paris and M. Chabre.
1PMC, CNRS, 660 route des Lucioles, 06560 Valbonne, France.
*LEBS, CNRS, 91198 Gif-sur-Yvette, France.
ARFs are small GTP-binding proteins involved in vesicular traffic. ARFI in its
active GTP-bound form recruits protein complexes (the <<coats>~) that act as
scaffolds to drive the budding of small vesicles from donor membranes. Although
ARF1 is myristoylated at the N-ter, it appears mostly soluble in the GDP-bonnd
<<resting,>state. As for most other G proteins, GDP-> GTP exchange induces major
conformational changes in 2 regions, named Switchl and 2, allowing the specific
interaction of the GTP-bonnd form with effector proteins. In ARFI, it also kicks a
N-terminal amphiphilic helix out of the hydrophobic pocket where it is bound in
the GDP form, promoting a GTP-dependent interaction of this helix with
membranes. Thus, GDP-> GTP exchange on ARFI coordinates its membrane
association with the recruitment of the <<coat>,, suggesting that ARFI-GTP
contributes to pull the membrane into the matrix of ,~coat,, at the same time that it
helps to polymerize it, to bud the vesicle. Spontaneous GDP release from ARFI is
extremely slow. Activation of ARF1 in-vivo is catalyzed by guanine nucleotide
exchange factors (GEFs). One of them: ARNO, has a N-ter coiled-coil for homodimerization, a central "Sec7" domain responsible for the exchange activity and a
C-ter PH domain that allows the recruitment of ARNO on membranes containing
PIP3 and/or PIP2. The structure of the "Sec7" domain consists of 10 helices, with a
striking hydrophobic groove, the binding site for ARFI, as shown by site-directed
mntagenesis. In Sec7 domains, a glutamic acid plays an essential role in the
mechanism of exchange probably by destabilizing Magnesium and the [3phosphate, thus favoring the dissociation of the nucleotide. GEFs for Ras or Rho
family proteins have different structures and based on structural and mutagenesis
data, do not seem to have a conserved glutamic acid that could play a similar role.
Although the Sec7 domain can promote GDP dissociation from a truncated form
of ARFI ([AI7]ARFI) in solution, ARNO promotes an efficient nucleotide
exchange on full-length myrARFl only in the presence of lipid vesicles. An
interaction of the myristoylated N-ter amphiphilic helix of ARF1 with lipids is
required to allow exchange. The membrane surface also helps to concentrate
ARF1 and its GEF at the same place and probably to position them in an optimal
orientation, but has minor effects on the catalytic activity of the Sec7 domain ~perse>~. Membrane recruitment has also been found to play an important role for Ras
and Rho exchange factors.
Finally, mono-unsaturated
DiAcylGlycerols (secondary
products
of
PhosphoLipase D activity) stimulate the activity of an ArfGAP that promotes GTP
hydrolysis on ARFI, suggesting that the interaction of this ArfGAP with
membranes is also essential to control the return of ARFI to its GDP-bound
,<resting>~state.
Structural and Mechanistic Studies on Ran Regulation
of Nuclear Transport
I. Vetter, R. Hillig, L. Renault, J. Kuhlmann. J. Becker,
C. Villa, A. Wittinghofer
Max-Planck-lnstitut, 44139 Dortmund, Germany

The GTP-binding protein Ran is the major regulator of nucleo-cytoplasmic

transport. A number of regulatory and effector proteins for Ran have been
identified in higher organisms and it is currently the focus of intensive
research to understand the requirements for the import and export of proteins,
R N A s and protein-RNA complexes in and out of the nucleus. The guaninenucleotide exchange factor (GEF) for Ran, RCC1, is exclusively localized in
the nucleus, while the GTPase-Activating protein RanGAP is only found in the
cytoplasm. This separation of activities creates a gradient of RanGTP across
the nuclear pore which might be the major driving force for the system. A
number of importins and exportins have been described, which are involved in
binding the import or export target proteins, respectively. RanBPI has a
specialized function: its interaction with RanGTP helps dissociate the
import/export effector complexes and mediates downregulation by RanGAP.
The three-dimensional structures of Ran. RanGAP and RCCI have been
solved. These structures will be described, together with the mechanism of
GEF catalyzed nucleotide exchange of Ran determined from kinetic
investigations and of the mechanism of GAP function. The interaction of Ran
with RanBP1 will also be described both from biochemical studies using
various spectroscopic techniques and from structural studies derived from the
X-ray structure of the complex of Ran*GppNHp with RanBDI from RanBP2.
This structure shows the conformational change of Ran from the GDP-bound
to the triphosphate-bound conformation and the involvement of the C-terminal
DEDDDL motif of Ran in nucleo-cytoplasmic transport.

Abstracts FEBS'99

Regulation of adenylyl cyclase by G proteins

S.R. Sprang, J.J.G.Tesmer, R.K.Sunahara, A.G. Gilman

European Molecular Biology Laboratory,

Meyerhofstrasse 1, D-69117 Heidelberg, Germany.
All known mammalian isoforms of adenylyl cyclase (AC) are stimulated by
the alpha subunit of the heterotrimeric G protein Gs, and (with the exception
of type IX) by the diterpine forskolin. We have determined, by x-ray
crystallography, the three-dimensional stmcture of the complex between
GTP-gamma-S-bound Gs-alpha, and the catalytic domains of type V (Nterminal, or C1 domain) and type II (C-terminal or C2 domain) AC. In
addition, we have analysed the stmctures this complex bound to a variety of
competitive and non-competitive nucleotide inhibitors of AC together with
magnesium, manganese and zinc cations. The switch II helix of Gs-alpha
forms the core of the interface between the G protein and the C2 domain of
AC. The binding sites for forskolin and that for ATP are located at pseudodyad-related sites within the shallow groove between the C1 and C2 domains.
The C2 domain presents residues that specifically recognize the adenine ring
of the substrate, whereas the C1 domain contains the two aspartate residues
that ligate the two magnesium ions required for catalysis. The structure of the
active site, conservation of catalytic aspartate residues, and metal specificity of
the magnesium binding site suggest that AC and the Poll family of DNA
polymerases share common ancestory. From a comparison of the structures
of G-protein-bound catalytic domains of AC and the inactive C2 domain
homodimer, we infer that Gs-alpha and forskolin stimulate AC in a
synergistic fashion by inducing a small domain rotation that aligns the
residues of the C1 and C2 domains and thereby produce a productive catalytic
site. In addition to this hypothetical structural change, we have observed that
the elements of the C1 and C2 domains collapse upon the substrate when it
binds to the catalytic site. We propose that these two conformational changes
provide vehicles for positive and negative regulation by G-protein alpha
subunits and other modulators.

s67

s68

Abstracts FEBS'99

14.1 Ionic channels

TREK-I is a polymodal K + channel sensor
mechanical, chemical and thermal stimuli.
E. Honor6

of

lnstitut de Pharmacologie MolJculaire et Cellulatre, CNRS

UPR 4ll, Sophia Antipolis, 06569 Valbonne, France.

TREK-1 is a member o f the novel structural class o f K + channels

with four transmembrane segments (TMS) and two pore domains (P) in
tandem. TREK-1 shows a strong expression in various tissues including
heart and brain. It is opened by membrane stretch, as well as membrane
crenators including arachidonic acid (AA). Moreover, TREK-1 is opened
by clinical doses o f inhalational anaesthetics. Intracellular acidosis mimics
the activation induced by A A and volatile anaesthetics. Lowering
intracellular pH shifts the pressure-activation relationship towards
positive values and leads to channel opening at atmospheric pressure. The
pHi-sensitive region in the carboxy-terminus o f TREK-1 is the same that
is critically involved in mechano-gating as well as A A activation. TREK-1
is also opened by heat which synergizes with pressure activation. A
convergence occurs between mechanical, chemical and thermal stimulations
and the carboxy terminus o f TREK- 1 is critical for polymodal activation.

Diseases due to chloride and potassium channel mutations

Thomas J. Jentsch
Zentrumfiir Molekulare NeurobiologieHamburg(ZMNH), Universttiit
Hamburg, Martinistr. 85. D-20246 Hamburg

Ion channels play central roles in the regulation of electrical excitability, in

transepithelial transport, and in the ionic homeostasis of intra- and
extracellular compartments. Correspondingly, there is increasing evidence
that mutations in ion channel genes underlie several inherited human diseases.
The CLC gene family of chloride channels has at least nine members in
humans, and three of its genes underlie human disease. We could show that
mutations in the skeletal muscle chloride channel C1C-1 cause animal and
human myotonia, a disease charaterized by muscle membrane
hyperexcitability and defective relaxation. Mutations in the renal channel
C1C-Kb leads to Bartter's syndrome, a disease characterized by massive salt
loss, implicating CIC-Kb in renal salt reabsorption. Mutations in the renal
chloride channel CIC-5 cause Dent's disease, a disorder characterized by
proteinuria and hypercalciuria, which in turn leads to kidney stones. We could
show that CIC-5 localizes to the endocytotic pathway in the proximal tubule
where it probably provides an electic shunt for the pumping of the H +ATPase. This underscores the importance of intracellular chloride channels
for vesicle trafficking and sorting.
Recently we have also investigated the role of potassium channels in
human disease. While KCNQ1 mutations cause the long QT syndrome,
mutations in either K C N Q 2 or K C N Q 3 cause Benign Familial Neonatal
Convulsions (BFNC), a neonatal form of epilepsy. Both proteins combine to
form a common heteromeric channel. The loss of channel function in BFNC
patients is only slight as the corresponding mutations lack a dominant
negative effect. By contrast, mutations we found in KCNQ4, a novel member
of this gene family, cause dominant deafness (DFNA2) via a dominant
negative effect. K C N Q 4 is expressed in sensory outer hair cells of the
cochlea, where it may be important in signal transduction.

subunits of voltage-gated K + channels

O. PongsI , T. Leicher1, R. B~hdeg1, R. Waldsch(ltzI , J. Wolfart1, J. Stormz,
P. Giese3, D. Isbrandt1
1 Instltutfdr NeuraleSignalverarbeptung,ZMNH,Martlnistrasse52, 20246Hamburg,
Germany
2 Bygmngfor prekliniskmedJsin,Instituteof Physiology.PB 1103Blindem.0317Oslo
Norway
3 UmversltyCollegeLondon.Departmentof Anatomy& DevelopmentalBiology,
GowerStreetLondon,WC1E6BT. UnitedKingdom
Voltage-gated potassium (Kv) channels of the Shaker related family are
hetarooligomedc complexes being assembled from membrane-spanning
Kwt and auxiliary Kvl?>subunits. We have cloned the human genes for six
Kvot subunits and for three Kvl~ subunits. Three of the Kvct subunits
(KCNA1, 6, 5) are clustered at chromosome 12p13. Mutations in the
KCNA1 open reading frame may be correlated with one form of ataxia,
mutations in the KCNA5 gone with an acquired LQT syndrome. Kvl~
subunits may increase the surface expression of Shaker related Kv
channels. In addition, Kv,l and Kvl~3 subunits may confer rapid
inactivation on certain Kv channels. Thus, the activity of certain rapidly
inactivating A-type Kv channels depends on the presence of Kvi~l/Kve,3
subunits. We show by analyzing Kvl?,l k.o. mutants that Kvl?,l-dependent
A-type Kv channels contribute to action potential repoladzation and
frequency-dependent action potential broadening. Loss of Kvl?,l function
may lead to a decreased slow afterhyperpelarisation (sAHP) amplitude,
probably because of an altered Ca2+-homeostasis. The observed changes
in learning and memory abilities of the Kvl~l k.o. mica may be correlated
with the observed alterations in CaZ*-homeostasis and sAHP-amplitude.

Structure, function and molecular pharmacology

of voltage-gated sodium channels
W.A. Catterall
Dept of Pharmacology, Universttyof Washington, Seattle WA 98195, USA

The voltage-gated sodium channels are composed of a large tx subunit of

260 kDa in association with a 131 subunit of 36 kDa and, in neurons, a disulfidelinked 132subunit of 33 kDa. The o~subunit is the pore-forming subunit. ]31 and
~2 are single membrane-spanning glycoproteins containing immunoglobulin-like
folds in their extracellular domains. They interact with the ct subunit through their
extracellular domains and modulate channel expression and gating. The
immunoglobulin-like folds have the structures of cell adhesion molecules and
interact with extracellular proteins like tenascin which may be involved in
localization or immobilization of sodium channels.
The t~ subunits of sodium channels are organized in four homologous
domains (I through IV) which each contain six transmembrane alpha helices (S 1
through $6) and additional membrane-associated sequences. The $4 segments
contain positively charged residues which serve as voltage sensors for channel
activation and move outward under the influence of the electric field to initiate
activation. The $5 and $6 segments and the short membrane-associated segments
between them (SS1/SS2) form the pore.
The fast inactivation of the open sodium channel is mediated by closure of
a hinged-lid-like inactivation gate formed by the short intracellular loop between
domains III and IV of the a subunit. The hydrophobic motif IFM within this loop
serves as the inactivation particle. This motif moves from a cytoplasmic location
into the channel structure during inactivation and becomes inaccessible to
chemical modification. The three-dimensional structure of the core of the
inactivation gate, including the IFM motif, has been determined by NMR
spectroscopy and forms the basis for a mechanistic interpretation of site-directed
mutagenesis studies of the inactivation process. The inactivation gate folds into a
receptor region formed by the IIIS4-$5 loop, the IVS4-$5 loop, and the
iotracellular end of the IVS6 segment.
Local anesthetics and related drugs block the pore of sodium channels by
binding to a receptor site formed by amino acid residues in transmembrane
segment $6 in domain IV. Site-directed mutations of critical amino acids in this
segment (F1764A and YI771A) greatly reduce the affinity for local anesthetic
block and specifically disrupt high affinity binding to the inactivated state. Many
different structural classes of sodium channel blocking drugs interact with this site
in the pore. In contrast, peptide scorpion toxins which alter gating of sodium
channels bind to the extraeellular ends of $4 segments and trap them in either an
activated or non-activated state, o~-scorpion toxins trap the IVS4 segment in its
inward position and slow or prevent inactivation; ~-scorpion toxins trap the IIS4
segment in its outward position and greatly enhance activation. Voltage sensortrapping may be a general mechanism of action of peptide toxins which affect ion
channel gating.

Abstracts FEBS'99

s69

Epithelial sodium channel (ENaC):

structure-function relationships
B. C. Rossier
Institut de pharmacologie et de toxicologic
Rue du Bugnon 27, CH-IO05 Lausanne, Switzerland

Amiloride-sensitive electrogenic sodium transport, mediated by ENaC,

is the rate-limiting step for sodium reabsorpuon by epithelial cells that
line the distal renal tubule, the distal colon, the ducts o f salivary and
sweat glands (control o f sodium balance, blood volume anl:lblood
pressure) and lung epithelium (control o f lung fluid clearance)1. ENaC
belongs to the recently identified ENaC/degenerin channel sul2erfamily.
E N a C is a heterotetrameric protein composed o f t ~ o e, one ~ a n d one
subunits surrounding the channel pore (a[30~y) z. Each subunit has
two t r a n s m e m b r a n e domains (M1 and M2) with short amino- and
carboxy-termini in the c y t o p l a s m and a large extracellular loop with a
p r e - M 2 hairpin loop. Functional domains have been identified in: i)
the aminotern~nus that is involved directly or indirectly in channel
gating kinetics ~ ii) the large extracellular loop with two cysteine-rich
domains (CRD-1 and CRD-2), implicated iq the efficient transport o f
assembled channel to the plasma m e m b r a n e '~ iii) the pre-M2 and M 2
(amiloride binding and ion permeation) ~
iv) the carboxyterminus,
involved in retrieval o f the channel from the plasma membrane and its
degradation . 1 will describe in vitro and in vivo experiments that define
the physiological relevance o f functional domains i), ii) and iv) in the
c o n t r o l o f blood pressure and lung fluid clearance
1.
Oart.~. H. & Palmer. 1, Epithelial sodtum channels: function. ~tructure, and
regulation. Physlol Rev 77. 359-396 (1997)
2.
Firso~. D.. Gautschi. I. Merdlat. AM.. Rosster. B.C & Schdd. L The
heterotetrameric architecture of the epithelial sodium channel (ENaC). E.IIBO J
17. 344-352 (1998).
3.
Grunder. S. et ell A mutation causing pseudoh)poaldosteronlsm t)pe I
identifies a con~er;ed gl~clne that is mvol'~ed m the gating of the epltheha[
sodium channel E31130 J 16. 899-907 (1997).
4.
Flrso~. D.. Robert-N]coud. M., Gruender. S. Schlld, L. & Rossier. B.C.
Mutational Anal)sis of C)steine*rich Domain of the Epithelium Sodium
Channel (ENaC). J BtoL Chem 274. (m press) (1999).
5.
SchAd. L.. Scneeber~er, E.. Gautschi. I. & Flrsov. D Identification ot" Amino
Acid Residues in the or. ~. and y Subunits of the Epithelia[ Sodium Channel
/ENaC) Involved m Amdoride Block and Ion Permeation.J. Gen P/o's~ol 109.
t-12 (1997).
6.
Staub. O et al WW domains of Nedd4 bind to "theproline-nch PY motif in tile
epLthelial sodium channel deleted in Liddle's s~ndrome E.IIBO J I$. 23712380 (1996)

s70

Abstracts FEBS'99

14.2 ABC transporters

Membrane topology and functional domains of the
human multidrug resistance-associated protein, MRP1
B. Sarkadi a, E. Bakos a,b, G. Szak~lcsa, A. Vgradib,

YEAST ABC PROTEINS: A TALE OF SEX, STRESS

and DRUGS. K. Kuchler.Deparunentof MolecularGenetics, Universityof
Vienna, ViennaBio Center,Dr. Bohr-Gasse9/2,A.1030Vienna.Austria.
Phone"431-4277-61807; FAX:431-4277-9618; e-mail:kaku@moLunivieac.at

aNatl. Inst. Haematol. Immunol., Membr. Res. Group and

blnst. Enzymology, Hung. Acad. Sci., Budapest, Hungary

Multidrug resistance is a major problem in cancer chemotherapy.

The human multidrug resistance protein (MRP1) causes such a
resistance by an ATP-dependent extrusion of hydrophobic drugs and
various glutathione conjugates from the tumor cells. Structurally
MRP1 shows many similarities to P-glycoprotein (MDR1), but in
addition to an MDRl-like core domain, MRP1 contains an N-terminal
membrane-bound (TMDo) and a cytoplasmic linker (Lo) region. In
order to understand the role of these regions we have studied the
functional properties of various truncated MRP1 constructs. We have
also produced chimera proteins by extending the human MDR1 with
the N-terminal regions of MRP1. The engineered proteins were
expressed in a baculovirus-insect (Sf9) cell system and the functional
properties of the protein variants were examined in isolated membrane
vesicles. According to these studies, full-length MRP1 showed a
specific, vanadate-sensitive, ATP-dependent transport of leukotriene
C4 (LTC4) and N-ethylmaleimide glutathione (NEM-GS). In the same
membrane preparations, by using labelled 8-azido-ATP, we could
follow an LTC4-stimulated, vanadate-dependent nucleotide occlusion
by MRP1. We found that neither the MRP1 core, nor the MRP1MDR1 chimeras showed such an MRPl-specific transport or
nucleotide occlusion activity, although in the chimeric proteins the
function of MDR1 was preserved. The co-expression of the MRP1
core with its N-terminal region, including L0, fully restored function.
Surprisingly, a truncated MRP1 mutant, lacking the entire TMD 0
region, still showed full transport and nucleotide occlusion activities.
These experiments indicate a functional re-association of separately
expressed domains of MRP1, and suggest that the interaction of the
MRP1 core and L 0 regions are required for the specific transport
function of this protein. MDR1, MRP1, and the newly discovered
MDR and MRP homologues seem to have a basic role in the
protection of our cells and body against xenobiotics, as well as in
hindering the effective treatment of malignant tumors by
chemotherapeutic drugs. Thus establishing the structure-function
relationships in these proteins may significantly help our
understanding of these basic cellular transport functions.

Regulation of ATP-sensitive K-channel activity by

the regulatory ABC transporter subunit, the
sulphonylurea receptor.
Stephen J. Tucker
University Laboratory of Physiology, Oxford University, Parks Road,
Oxford. OX1 3PT, United Kingdom

ATP-sensitive potassium (KATP) channels are found in a wide variety of

tissues including pancreatic beta-cells, cardiac, skeletal and smooth muscles and
in some neurons. They are involved in coupling the metabolic state of the cell to
its electrical activity. In the pancreatic beta-cell this links the plasma glucose
concentration to insulin secretion. The beta-cell KATP channel is a tightly
associated octameric complex of four Kir6.2 potassium channel subunits and
four sulphonylurea receptor (SUR1) ABC-transporter subunits. Kir6.2 subunits
form the channel pore, while SURI acts as a regulatory subunit, endowing the
KATP channel with sensitivity to the stimulatory effects of MgADP and Kchannel openers, and to the inhibitory actions of sulphonylureas. Recent
advances will be discussed which have shed light on the mechanisms by which
ATP interacts with the Kir6.2 subunit of the channel to inhibit the channel and
also the mechanism by which sulphonylureas interact with SURI to induce
closure of the KATP channel pore.

The completion of the Saccharomyces cerevisiae genome sequencing revealed

the existence of 30 distinct ATP-binding cassette (ABC) genes in this
unicellular eukaryote. According to phylogenetic tree analysis, these proteins
can be classified into six subfamilies.Several membersof the PDR5 subfamily
are implicated in the development of pleiotropic drug resistance (PDR) to
cytotoxic drugs. For instance, overexpressionof the Pdr5 efflux pump leads to
a pronounced PDR phenotype, mediating resistance to dozens of rather
hydrophobic compounds. In addition, recent results show that Pdrl2, a close
homologue of Pdr5, confers hyperresistance to water-soluble carboxylate
anions such as weak organic acids but not to hydrophobic drugs. Thus, yeast
ABC pumps are structural and functional homologues of P-glycoproteins and
MRPs implicated in multidrugresistance development m cancer cells.
Our main goals are to understand the molecular switches initiating PDR
development, as well as to unravel the mechanism(s) of ABC transporter
function. These goals are pursued by studying the biogenesis, regulation of
expression, intracellular trafficking and turnover of ABC proteins such as Pdr5,
Snq2, Pdrl2, Pdrl0, Pdrl5. We were also interested to dissect the molecular
basis for the broad drug substrate specificity and inhibitor susceptibility of the
Pdr5 antifungal azole pumps, as well as its homologues from pathogenic fungi.
For instance, we have demonstrated that certain point mutations in PDR5 not
only can modulate its azole transport specificity, but simultaneously abolish or
even enhance Pdr5 susceptibility to inhibition by PDR reversal agents.
Although the physiological functions of many yeast ABC proteins are still
an enigma, our recent data show that certain PDR genes represent targets of
cellular stress response pathways, since their expression is tightly regulated by
a number of adverse conditions. In fact, biochemical and genetic experiments
demonstrated a remarkable complexity concerning the regulation of ABC
genes within the PDR network. For example, expression of the SNQ2 and
PDR15 genes is individually regulated by the stress regulators Yapl and
Msn2/Msn4, respectively, while both genes are regulated at the same time by
Pdrl/Pdr3, the key factors controlling PDR development. In my talk, I will
provide an update on yeast ABC proteins. I shall discuss their purported and
known cellular functions, including their role in cellular detoxification and
PDR, their function as stress response genes and the molecular cross-talk
between stress response and PDR development.

Abstracts FEBS'99

s71

15.1 Growth regulators and signalling in plants

Signalling leading to plant morphogenesis in the
Rhizobium-legume interaction
T. Bisseling, A. Emons, H. Franssen, D. Gadella, R. Geurts
Dept of Plant Sciences, Wageningen University. The Netherlands

The interaction of bacteria and plants leads to a dedifferentiation of root

cortical cells. These cells become reprogrammed and develop into a new organ,
the root nodule. This organ hosts the bacteria in an intracellular manner and there
the bacteria are able to reduce atmospheric nitrogen. The first host cells that
respond to Nod factors, lipochitooligosaccharide signal molecules secreted by
the rhizobia, are the root hairs and the root pericycle cells. In root hairs Nod
factors cause a rapid change in the actin skeleton that leads to morphological
changes of the hairs that can facilitate the bacterial infection of the plant. The state
of the art of our knowledge on Nod factor perception and signal transduction
leading to the changes in the configuration of the actin skeleton will be discussed
The Nod factor induced response in the root pericycle leads to the activation of a
plant gene, ENOD40, that encodes a peptide and that might also be active at the
RNA level. The potential role of ENOD40 in facilitating cell divisions in the
cortex by which new primordia axe formed from which nodules will develop,
will be discussed.

Hormonal control of cell division cycle in plant

D. Dudits, T. Pastemak, T. M6szhros, F. Ayaydin,
Zs. Kelemen, P. Miskolczi, GV. Horv,lth, K. Kelemen,
Z. Magyar, A. Feh6r

GAI: gibberellin response regulator

N.Harberd, L. Hynes,K. King,J. Peng,D. Richards
MolecularGeneticsDepartment,JohnInnesCentre,Colne5
Lane, Norwich,NR4 7UJ, UK

Recent decades have seen large increases in world wheat grain yields. This
so-called 'Green Revolution' is in part due to the widespread adoption of
varieties containing the semi-dwarfing Rht mutant alleles, and of associated
changes in agricultural practise. These yield increases have enabled wheat
productivity to keep pace with the demands of the rising world population.
Like Rht mutant alleles in wheat, the gai mutant allele of Arabidopsis
confers a semi-dominant dwarf phenotype, and a dramatic reduction in
responsiveness to the plant growth hormone gibberellin (GA). The
Arabidopsis GA1 and RGA genes have been cloned. These genes encode
closely related proteins (GAI and RGA) that are members of a class of
putative transcriptional regulators defined by the Arabidopsis SCR product.
Recently, we have found that the DNA sequences o f Rht (and D8, the maize
orthologue) are closely related to those of GA1/RGA. Analysis of mutant D8
and Rht alleles suggests that the wild-type proteins (GAI, RGA, D8, Rht)
are bi-partite in structure, with an N-terminal GA-specificity domain, and
a C-terminal putative transcriptional regulator domain. These proteins
appear to act as repressors of stem elongation, whose activity is opposed by
GA. The mutant proteins are altered in ways that make growth repression
relatively resistant to the effects of GA. The possible biotechnological
significance of these findings will be discussed.

Abscisic Acid Signalling In Arabidopsis

J. Giraudat, N. Beaudoin, F. Gosti, J. Leung, S. Merlot,
F. Parcy, C. Serizet
lnstitut des Sciences Vdg~tales, C N.R.S , 91190 Gif-sur- Yvette, France

Institute ofPlant Biology. BRC, HAS, 6726 Szeged, Hungary

Plant growth regulators (auxins, citokinines, abscisic acid) can directly

influence the cell division in different plant organs and in cultured cells. The
discovery of set of cell cycle control proteins (cyclins, cyclin-dependent
kinase (CDK) Rb, inhibitors) and the corresponding genes provides
molecular tools for analysis of the link between the hormonal signal
transduction and cell cycle control mechanisms. Treatment of alfalfa cells or
leaf discs with 2,4-D activates CDK genes and histone H1 phosphorylation
activity in p 13~ct -bound protein fraction. In alfalfa at least three CDK-related
kinase complexes are involved in G2/M transition. The cdc2MsMB; D and F
kinases are differentially activated by 2,4-D. The cdc2MsF kinase is active in
mitotic cells, localised in preprophase band, nuclear ring in early prophase,
mitotic spindle and phragmoplasts. The complex formation between this
mitotic kinase and the D-type cyclin (cye Ms4) could be demonstrated in
yeast two-hybrid system, GST pull-down assay after in vitro
transcription/translation or by immunodetection of kinase partner in the in
vitro or in vivo complexes. Kinetin treatment of alfalfa cells in combination
with auxin increased the kinase activity. Abscisic acid reduced the histone HI
phosphorylation activity. Experiments are in progress to determine the
potential role of alfalfa retinoblastoma and CDK inhibitors in mediation of
hormonal effects on cell division in cultured cells.
[1] D. Dudits et al. In: Molecular and Cell Biology of the Plant Cell Cycle,
eds. J.C. Ormrod and D. Francis, Kluwer Academic Publishers, (1993)
pp. 111
[2] D. Dudits et al. In: Plant Cell Division, eds. D. Francis, D. Dudits and D.
Inz6, Portland Press, London (1998) pp. 21

The plant hormone abscisic acid (ABA) is a key regulator of seed

maturation and germination, and mediates adaptive responses to
environmental stress. Although the nature of the ABA receptor(s) remains
unknown, substantial progress has been made recently in the
characterisation of more downstream elements of the ABA signalling
pathways. In particular, genetic screens based on the inhibition of seed
germination by applied ABA have led to the isolation of several
Arabidopsis mutants with altered ABA sensitivity. The abil-1 and abi2-1
mutations markedly reduce ABA responsiveness in both seeds and
vegetative tissues. We showed that the ABI1 and A B I 2 genes encode
homologous proteins that belong to the 2C class of protein
serine/threonine phosphatases (PP2C). The a b i l - I and abi2-1 mutations
are dominant. Hence it remained unclear whether or not ABI1 and ABI2
contribute to ABA signalling, and in case they do regulate ABA
responsiveness whether they are positive or negative regulators of ABA
action. We isolated seven loss-of-function alleles of the ABI1 gene as
intragenic revertants of the a b i l - I mutant. All these suppressor mutations
translate into missense mutations within the PP2C domain of ABII, and
the seven revertant alleles encode ABI 1 proteins that lacked any detectable
phosphatase activity in vitro. In contrast to the ABA-resistant a b i l - 1
mutant, all the intragenic revertants were more sensitive to ABA than the
wild type. The revertant alleles were recessive to the wild-type ABI1 allele
in enhancing ABA sensitivity, indicating that this phenotype actually
results from a loss-of-function in ABI 1. These results thus provide genetic
evidence that the wild-type ABII phosphatase is a negative regulator of
ABA responses. To identify novel loci controlling ABA sensitivity, we
performed genetic screens for phenotypic suppressors and enhancers of
the abil-1 mutation. In both screens we recovered alleles of mutants that
were previously known to affect ethylene signalling. This prompted us to
investigate the cross-talks between the signalling pathways for these two
plant hormones. Our current results will be presented.

s72

Abstracts FEBS'99

Genetic manipulation of jasmonic acid biosynthesis

Jose J. Sfinehez-Serrano
Centro Nacwnal de Biotecnologia CSIC, Madrid, Spare

The plant hormone jasmonic acid (JA) plays regulatory roles on plant
development and responses to environmental stresses, such as mechanical
wounding or pathogen attack. Upon wounding, increased de n o v o synthesis
results in higher endogenous JA levels, which trigger the activation of
wound-inducible genes. Jasmonates are synthesized through the octadecanoid
pathway from linolenic acid, the most prevalent fatty acid in plant membrane
lipids. Free linolenic acid is substrate of a lipoxygenase (LOX), which
converts it with positional specificity to either 9- or 13-hydroperoxy linolenic
acid. Allene oxide synthase (AOS) uses the latter hydroperoxide as a substrate
yielding an unstable allene epoxide which after cyclation gives rise to 12-oxophytodienoic acid. Alternatively, hydroperoxide lyase (HPL) action on 13hydroperoxy linolenic acid diverts the pathway to products which are not
precursors for JA. Conversion of linolenic acid to 12-oxo phytodienoic acid
is likely to occur in chloroplasts. Further processing to JA involves a
cytoplasmic reductase and ~-oxidation, likely to occur in peroxisomes.
To further understand the role of JA in plant development and in the response
to wounding, we have isolated and characterised potato cDNAs encoding
some of the enzymes potentially involved in the biosynthesis of this hormone,
namely a plastidial 0)3 fatty acid desaturase, two distinct 13-LOX, HPL and
an AOS homologue. Their patterns of expression along plant development
and upon wounding and JA treatments have been investigated. The majority
of genes coding for JA-biosynthetic enzymes are transcriptionally activated by
wounding and some of them are also activated by JA, allowing a positive
feed-back regulation of the biosynthetic pathway. Transgenic potato plants
have been obtained in which the expression of these genes is greatly reduced
due to co-suppression or antisense-mediated depletion of their mRNAs. The
effects of reducing the levels of these enzymes have been analysed in the
transgenic lines in terms of plant development and tuberisation, JA content,
and responses to wounding and pest and pathogen attack.

Abstracts FEBS'99

s73

15.2 Plant organ development

The Control of Proximal-Distal Pattern Formation
during Early Ovule Development in Arabidposis
K. Schneitz, S, Balasubramanian. U. Schiefthaler, D.Chevalier,
A. Redweik, P. Sieber
Institute of Plant Biology, University Of ZOrich,
Zoliikerstrasse 107, CH-8008 ZOrich, Switzerland

The ovule represents the major female reproductive organ in higher plants.
Furthermore, it has recently been established as an excellent model system to study
organogenesis at the genetic and molecular level. V~ and others have isolated alarge
number of mutants with a defect in ovule development and the ongoing genetic and
molecular characterization of these mutants provides further insight into the
mechanisms underlying several aspects of ovule development such as primordium
outgrowth, specification of identify, pattern formation and cellular differentiation
[tl.
Early ovule development is in part characterized by the progressive establishment
of three proximal-distal (P-D) pattern elements during the outgrowth of the ovule
primordium [2}. The distal element gives rise to the nucellus which forms the
megaspore and eventually the embryo sac containing the egg cell. The central
element develops into the chalaza with the inner end outer integuments, the
progenitor tissues of the seed coat, and the proximal element gives rise to the
funiculus or stalk with the vascular strand. What genes control the formation of this
pattern and how do they accomplish it ? We have identified and cloned a novel gene,
(N,ZZ) ,which controls the formation of the distal or nucellar pattern element.
Plants lacking NZZ function fail to form a nucellus and variably affect the
development of the chalaza, the NZZtoous encodes a 314 aa basic protein with no
homology to entries in the databases. Mutations in the hemeobox gene BELL (BELl)
lead to the development of outgrowths of previously unknown identity in place of the
outer integument. We provide evidence that these outgrowths are of nucellar
identity indicating that the central element is assigned a new distal identity in bell
mutants. In the case of a simultaneous absence of NZZ and BELl function in the
central element this domain experiences a shift to a more proximal identity and
gives rise to a funicutus-like structure. Our genetic and molecular analysis of nzz
and bell mutants indicates that N;EZ and BELl control P-D patterning in the distal
two-thirds o1 the ovule primordium through mutual antagonistic interactions.
[1] Schneitz et aL, Trends Plant Scl. 3, (1998) 468.
[2] Schneitz et al., Development, 125, (1998) 2555.

s74

Abstracts FEBS'99

15.3 Plant defense mechanisms against biotic and abiotic stresses

Plant disease resistance genes:
structure, function and evolution
J.D.G. Jones, C. Thomas, K. Hammond-Kosack, 3. Parker,
E. van der Biezen, W. Durrant, T. Romeis, P. Piedras
The Samsbury Laboratory, John lnnes Centre, Norwich NR4 7UH, UK
Various mechanisms are used by plants to resist pathogens. One involves
recognition of pathogen-specified molecules by the plant. Disease resistance
(R) genes confer recognition of pathogen races carrying the corresponding
"avirulence" (Avr) genes. This observation has prompted research into the
basis of R gene -dependent recognition, and the subsequent signalling and
defence responses.
Four classes of "gene-for-gene" R genes have been isolated. All except
the protein kinase-encoding Pro gene, encode leneine rich repeats (LRRs), a
protein motif associated with protein/protein interactions. The largest class
appears to be cytoplasmic, with LRRs at the C terminus and a consensus
nucleotide binding site CNBS)for interaction with ATP or GTP towards the N
terminus NBS/LRR genes confer resistance to viruses, bacteria, fungi and
nematodes. This class probably recognizesAvr gene-dependent molecules that
enter the plant cell. Two other classes ofR gene carry extracytoplasmic LRRs
and probably recognize A vr gene specified molecules delivered from outside the
plant cell. Xa21 encodes eLRRs and a cytoplasmic protein kinase domain,
whereas Cf-2. Cf-4. Cf-5 and Cf-9 carry membrane anchored eLRRs. Many R
genes have been isolated, attention has shifted to what and how their products
recognize, and how signals are transduced to initiate defence mechanisms.
We study tomato Cf-2, Cf-4, Cf-5 and Cf-9 genes conferring resistance
to specific races of Cladosporium fulvum. Cf-4 and Cf-9 genes also confer
responsiveness to the Cfulvum Avr4 and Avr9 peptides respectively. We also
study the Arabidopsis RPP1 and RPP5 genes for Peronospora resistance.
Sequence analysis at the Cf-9, Cf-4 and orthologous Ctt3 loci has
permitted comparison of 11 Cf-9. Most variation is seen at the amino acids
that flank the conserved leucines in the LXXLXLXX beta strand/beta turn
region. These amino acids are believed to be solvent exposed and may confer
specificity. DNA sequence show that sequence novelty has been generated by
sequence exchange between Hcr9s, either by crossovers or gene conversion,
and my mutation and divergent selection (Parniske et al, Cell 91: 821-832).
Recent data on the role and mechanism of resistance gene product
function will be reported.
Recognition and signal transduction in pathogen defense
D Scheel, B Blume, T Kroj, T Nurnberger, M Tschope,
H Zinecker, and U zur Nieden
Instttute of Plant B~oehemtstrv, IIemberg 3, D-06120 Halle, Germany
Plants recognize potentially pathogenic organisms through receptors that
specifically bind signal molecules, so-called elicitors, derived from pathogen
or plant surface structures and thereby initiate signaling cascades activating a
multicomponent defense response Cultured parsley cells and a glycoprotein
elicitor from hyphal cell walls of the plant pathogenic oomycete,
Phytophthora sojae, are the experimental system used to investigate the
molecular mechanisms underlying recognition and signaling events The
oligopeptide fragment, Pep-13, of this glycoprotein stimulates a defense
response in cultured parsley cells that is identical with the reaction to the
glycoprotein and very similar to the response of intact plants to infection
with the pathogen Pep-13 binds specifically to a 100 kDa plasma membrane
receptor and thereby initiates a signal transduction cascade Early elements
of the plant response are ion fluxes across the plasma membrane, transient
increases in cytosolic calcium levels, activation of mitogen-activated type
protein kinases and the production of reactive oxygen species, the oxidative
burst, followed by defense gene activation and phytoalexin accumulation.
Data will be presented that demonstrate a causal relationship between early
and late elicitor reactions and establish a network of signaling events

Perception of elicitors of Cladosporiumfuivumby tomato

and other solanaceous plants
P.J.G.M. De Wit, M.H.A.J. Joosten, R. Laugh.
Wageningen Agricultural University, Department of Phytopathology,
Binnenhaven 9, 6709 PD Wagenmgen, NL
Many Cladosporiumfulvum (CJ) resistance genes and matching fungal
avirulence (Avr)genes have been cloned in recent years. We have isolated Avr
genes that encode elicitors of the hypersensitive response (FIR) in tomato
plants carrying the complementary Cfgenes. The Avr9 gene encodes a small
peptide (AVR9) which induces HR in tomato plants carrying resistance gene
Cf-9. AVR9 is a cystine-knotted peptide which contains three disulfide
bridges and three antiparalle115 sheets. An alanine scan proved that all
cysteine bridges and Phe21 are required for FIR activity [1]. A high affinity,
membrane-located binding site for AVR9 was found which, however, does
not seem to be the Cf9 protein itself. Most probably AVR9 binding and HR
induction requires a complex of proteins of which the Cf9 protein is only one.
AVR4 is also a small secreted cysteine-rich peptide with 4 disulfide bridges,
which are all required for HR induction on tomato plants carrying resistance
gene Cf-4 [2]. Both AVR9 and AVR4 do not seem to be virulence factors.
However, other extracelhilar proteins isolated from C. fulvum, such as ECP2,
are virulence factors. By a targeted search, tomato plants were found that
respond with FIR to ECP2 and presumably carry a resistance gene that we
have designated Cf-ECP2 [3 ]. Several other ECPs were found which induce
a HR in some genotypes of tomato and even in some genotypes of Nicotiana
species. This indicates that solanaceous plants have a very versatile
surveillance system to recognize non-self proteins.
[1] Mahe, E. et al., J. Pept. Res. 52 (1998) 482.
[2] Joosten, M.H.A.J. et al., Plant Cell 9 (1997) 367.
[3] Laug6, R. et al., Prec. Natl. Acad. Sci. USA 95 (1998) 9014.

How do plants survive dehydration?

D. Bartels
BotanischesInstitut,Universit~itBonn
Kirschallee l, D-53115 Bonn,Germany

The
resurrection
plant
Craterostigma
plantagineum
(Faro.
Scrophulariaceae) is being used as an experimental model system to
understand pathways leading to desiccation tolerance. Analysis of gene
expression in this plant has shown that desiccation tolerance involves many
different pathways. A major pathway during dehydration is the synthesis of
LEA proteins. Many genes encoding LEA proteins have been isolated.
There is plenty of evidence to suggest that the synthesis LEA proteins is a
major factor of desiccation tolerance. In order to address the function of
LEA proteins transgenic plants were created which overexpress LEA
proteins and results will be discussed.
The synthesis of LEA proteins is regulated on the transcriptional level and a
major focus is the isolation of transcription factors which regulate several
down-stream genes. Two putative transcription factors have been isolated:
one (HDZIP-I) is structurally closely related to the family of the
homeodomain leucine zipper gene family and the other one (HSF-I) to the
family of the heat shock transcription factor. Their molecular analysis will
be presented.
Besides the plant hormone abscisic acid lipid based signalling molecule
are suggested to rapidly to convert metabolism in the unstressed planl
towards a metabolism which is directed towards the synthesis of desiccation
protective substances such as LEA proteins. The different signal molecules
will be discussed.

Abstracts FEBS'99

s75

Genetical and molecular analyses of cold acclimation in

Arabidopsis thaliana
J Salinas
Departamento de Alejora Gen~ttcay Btotecnologia, I VL-~, ( "arretera de
la Coruha, Km 7, 28040 Aladrtd, Spare

Plants from temperate regions increase their freezing tolerance in

response to low-nonfreezing temperatures This process, known as cold
acclimation, involves a number of biochemical and physiological
changes including increased levels of soluble sugars, proteins, amino
acids and organic acids, as well as the modification of membrane lipid
composition. Most of these changes seem to be regulated through
changes in gene expression. We have used both molecular and genetical
approaches to investigate the signal transduction pathways that control
gene expression during cold acclimation Arabidopsts thaliana, a plant
offering all the possibilities for genetic and molecular studies, and
showing cold acclimation response, has been our model system. By
means of the molecular approach we isolated a number of genes (RCls,
CAB, CBFs) whose expression is induced during cold acclimation. The
analysis of their promoter regions in transgenic plants has revealed the
existence of at least three signal transduction pathways that mediate low
temperature reponses Two of them correspond to those already proposed
that are dependent on ABA and CBF1, the transcription factor which
binds to the low temperature response element CCGAC The third one is
a new pathway which is independent of both ABA and CBF1. On the
other hand, we have obtained mutants affected in the regulation of R C I
gene expression Since the expression of RC1 genes is regulated by low
temperatures and also by other related treatments as ABA, dehydration or
high salt, the analysis of these mutants not only should allow a better
understanding of the signalling pathways that activate gene expression
under low temperatures, but also to study how these pathways cross-talk
and interact with others to coordinate gene expression under different
environmental stresses

s76

Abstracts FEBS'99

16.1 Enzymatic mechanisms and catalysis

Serine oligopeptidases: structure and catalytic mechanism
L. Polg~ ~, Z. Szeltner ~, V F'til6pb
qnstitute of Enzymology,HungarianAcademyof Sciences, I 113
Budapest, Hungary,bDepartmentof Biological Sciences,Universityof
Warwick, CoventryCV4 7AL, U.K.

There is a group of serine peptidases,

unrelated to the trypsin and subtilisin
families, that cannot hydrolyze
peptides
containing about
30
residues or more. One such enzyme
is prolyl oligopeptidase involved in
the maturation and degradation of
peptide hormones and neuropeptides.
We have recently solved its 1.4 A
resolution crystal structure, which
contains a peptidase domain with an
cdi3-hydrolase fold, and its catalytic
triad (Ser554, His680, Asp641) is
covered by the central tunnel of an
unusual [3-propeller. This domain
makes the enzyme an oligopeptidase
by excluding
large structured
peptides from the active site.
The kinetic and structural results demonstrate important dissimilarities
between prolyl oligopeptidase and the enzymes of the pancreatic and subtilisin
classes. Thus. the pH-dependence of the specificity rate constant is more
complicated, due to an arra3 of charged residues near the catalytic site. The
rate limiting step of the catalysis is a conlbrmational change rather than a
chemical step. One component of the oxyanion binding site is the OH group of
a tyrosine residue (Tyr473), which is absent from the active sitc of other
classes of serine peptidases. Kinetic investigations with Tyr473Phc variant
have shown that the oxyanion binding site utilizes some of the binding energ)
of the substrate tbr stabilizing the transition state. The mechanisnl of
stabilization is substrate- and pH-dependent.

Catalytic and Kinetic Mechanism of an Enantioselective

Bacterial Epoxide Hydrolase
Rick Rink, Jeffrey Lutje Spelberg, and Dick B. Janssen
Biochemical Laboratory, Groningen Biomolecular Sciences and
Bzotechnology Institute, University of Groningen,
9747 AG Groningen, The Netherlands

Epoxide hydrolases catalyze the hydrolytic cleavage of epoxides to diol

and are promising biocatalysts for the kinetic resolution of racemic mixtures c
epoxides, yielding enantiopure epoxides or diols which can be used for ff
synthesis of a variety of fine chemicals. The epoxide hydrolase of the epichk
rohydrin-utilizing bacterium Agrobacterium radiobacter AD1 converts a variety
substrates with moderate enantioselectivity, including styrene epoxide an
derivatives thereof [ 1,2]. Based on sequence similarities, site-directed mutagenes
experiments, and X-ray crystallographic studies, it was concluded that the enzyrr
posesses an ct/[3-hydrolase fold structure with a catalytic triad composed of
['~-'I~0--~Ic-H H~O

Y-J"

"

~R.S)~po~de

OHOH

~ _ . (-h~,?,~-~ . r - ~ - ! - ~
.;g;,~..Y--J" "
U-J" "
(S)~pox,aa

(R)~

nucleophilic aspartate, a histidine, and a 'charge relay' aspartate.

Based on the X-ray structure, we hypothesized that ring opening is probab]
facilitated by protonation of the epoxide oxygen by 2 Tyr residues located in the c~
domain of the enzyme. Using site-directed mutagenesis, these tyrosines weJ
separately or both replaced by phenylalanines. The single Tyr mutants were sti
active, but had increased K~ values for styrene oxide, whereas the double mut~
was inactive. Stopped flow fluorescence experiments with (R)-styrene oxk
revealed that the rate of formation of covalent intermediate was slowed down in tt
Tyr mutants. More important, the mutants showed an improved enantioselectivi~
for three different styrene oxide derivatives, which was due to a decrease in kcat fi
the (S)-enantiomer but not for the (R)-enantiomer, leading to higher E values.
The results provide further insight in the catalytic mechanism of cpoxk
hydrolases and indicate that reducing the rate of protonation by site-directe
mutagenesis may improve their enantioseleetivity.
[1] Rink et al. (1998) Biochemistry 37:181191-18127.
[2] Lutje Spelberg et al. (1998) Tetr. Asymm. 9:459-466.

Stereospecificity and catalytic efficiency of or-proton

exchange reactions catalysed by B6-enzymes
J.P.G. Malthouse
University College Dublin, Dublin, Ireland.

In this talk I shall discuss how pyridoxal phosphate-dependent

enzymes discriminate between exchanging the pro-2R and pro-2S ct-protons
of glycine and the a-protons of L and D-amino acids. Pyridoxal phosphatedependent enzymes catalyse a wide range of reactions which can involve
cleavage of the a-carbon bond of amino acids. It has been argued that for
optimal catalytic efficiency the a-carbon bond to be cleaved should be
orthugonal to the plane of the imine-cofactor x-electron system. The reaction
catalysed will depend on whether the amino acid R-group, ct-carboxylate or
a-proton is orthogonal to the plane of the imine-cofactor ~-electron system.
Therefore the stereospecificity of the exchange of the a-protons of L and D
amino acids or of the pro-2R or pro-2S protons of glycine will depend on
how the earboxylate and R-groups are bound relative to the plane of the
imine-eofaetor ~-electron system. For the exchange of ct-protons of amino
acids we would expect the degree of stereospecificity to depend on the
efficiency of binding of the amino acid R-group and ct-carboxylate group.
The catalytic efficiency and stereospecificity of the exchange of the ctprotons of a range of amino acids have been studied with several catalysts
including tryptophan synthase [1], serine hydroxymethyltransferase [2[,
aspartate aminotransferase and a catalytic antibody [3]. In this talk I will
describe how such studies have shown how the different structural features of
pyridoxal phosphate-dependent enzymes and their substrates contribute to the
efficiency and stereospecificity of catalysis and a-proton exchange.

References
[1] Milne & Malthouse, Biochem. J., 314, (1996) 787.
[2] Fitzpatrick & Malthouse, Eur. J. Biochem., 252, (1998), 113.
[3] Mahon et al., Febs Lett., 427, (1998), 74.

The interaction of ester substrates with the active site of

carbonic anhydrase.
S. Lindskog, B. Elleby and B. Sjrblom
Department of Biochemtstry, Ume&University. Umed, Sweden

While the physiological reaction catalyzed by the zinc-containing enzyme

carbonic anhydrase is the reversible hydration of carbon dioxide, the enzyme
can also act on other carbonyl systems, such as esters and aldehydes.
However, the molecular details of ester hydrolysis are not well understood,
and the precise location of the ester substrate in the active site is not known.
Some clues have come from studies of site-specific mutants of human
isozyme II. The 4-nitrophenyl acetate hydrolase activities of more than 100
mutants at 26, or more, sequence positions have been measured. In some
cases, enhanced esterase activities have been observed. Enhancement factors
exceeding 2 have been found for certain mutants at positions 7, 65 and 200
located in the active site cavity.
To obtain further indications concerning the location of ester substrates in
the active site, we have investigated effects of selected mutations on the
substrate specificity. Rates of hydrolysis of 4-, 3-. and 2-nitrophenyl acetate
and 4-nitrophenyl propionate catalyzed by wild-type and mutant forms of
human carbonic anhydrase II were measured. The results show that the
mutations TyrV-+Phe and Ala65---~Leu lead to activity enhancements with all
the investigated substrates, but there is no significant effect on the specificity.
In contrast, some mutations at sequence position 200 have large specificity
effects. For example, while the mutation Thr200--*Gly results in a 3-fold
increase of the rate of hydrolysis of 4-nitrophenyl acetate, the activity is
enhanced 10 times with 3-nitrophenyl acetate and 380 times with 2nitrophenyl acetate. These results are interpreted in terms of the removal in
the mutant o f a steric interference between the 2-NO2 group, in particular,
and the side chain of Thr200. Mutants involving residues lining a
hydrophobic pocket near the catalytically essential zinc ion have also been
investigated. The most pronounced specificity effect was found for the
Val143--~Gly mutant. This mutation leads to a 6-fold decrease of the rate of
hydrolysis of 4-nitrophenyl acetate but a 20-fold increase of the activity with
the propionyl ester as substrate. These results suggest that the side chain of
Val143 interferes sterically with the aeyl moiety of 4-nitrophenyl propionate.
Based on these results, we have constructed a hypothetical model of the
location of these ester substrates in the enzymic active site.

Abstracts FEBS'99

Mutational analysis of specificity, mechanism and stability

in amylolytic enzymes from two different structural families
B Svensson'.KS Bak-JenserLJ Sauer.H M~i. MT Jens~. TE Gortschalk.KW Rodenburg.
T Chnstensenb. nw SIgu~kJold.N Payre=.H Drlguez.S Cottaz.N Aghajart~,R Haser
~Depf of Chem , Carlsberg Laboratory, Copenhagen, eDept, of,Btochem., August Krogh lnst .
Unsv. of Copenhagen. ~Cermav. CNRS,Grenoble, [BCP, CNRS, Lyon

Amylolytlc enzymes catalyze cleavage of glueosidic bonds in starch and related

sugars Around 200 pnnmry structures, 20 enzyme speeifictties, and several crystal
structures are known. These multidomam enzymes occur in five protem families, of
which the glycoside hydrolase family 13 of (l~/ct)8-barrel proteins is largest. Based
on experimental and modelled three-dimensional structures of enzyme-substrate
analogue complexes and sequence alignments, residues and regions were proposed
as targets for mutational analysts of structure/function relationships and protein
engineering to modulate the specificity of barley a-amylase, a member of family 13
The binding cleft contains 10 contiguous subsltes aocornodating substrate glucosyl
res,dues By mutagenesis at the extreme ends, i e. in subsites -6 and +4, or in subsite
+1, adjacent to the catalytic site, higher than wild-type activity towards insoluble
starch, and altered action pattern on oligosaccharide substrates were achieved. This
may be utilized in generation of larger oligosaccharide products from high molecular
weight substrates Germinated barley contains two a-amylase isozymes of 80%
sequence ~dent~ty and distinct stability and substrate specificity properties. Only
tsnzyme 2 recognizes the endogenous barley =-amylase/subtilisin inhibitor (BASI)
of the [3-trefoil fold protein famdy. Guided by sequence differences of lsozyme 1
and 2, salt-bridge mutants were prepared in isozyme 1 having altered stability and
the sensitiv,ty to BASI was introduced or removed by site-directed mutagenesis in
different tsozyme hybrids. The biochemical properties of the various mutants and
the parent tsozymes are discussed on the basis of the crystal structures [1,2].
Asperg~llus n i g e r glucoamylase of family 15 contains a catalytic (a/a)6barrel domain connected via a v e ~ long highly O-glycosylatcd peptJde linker to a
starch granule binding domain Engineering by homologue hnker-replacement
affected the conformational stabdity of the catalytic and the starch binding domains,
even though the domains as such contained no mutatmns The simultaneous binding
of double headed synthetic oligosaccharide inhibitors targeted at the catalytic site
and at a binding site on the surface of the starch binding domain, respectively,
suggested that the linker confers flexible domain cooperation [3]. This demonstrates
that mobility in solution and interaction between domains are important for the
natural function of the multidornain enzyme A simdar arduteeture ~s found in
certain cellulolyhc, chitruolytic, and xylanolytic enzymes. The present data illustrate
conceptual tools useful in analysis of the mutual impact of domains on stabilit_~ and
enzymatic function of polysaocharide degrading enzymes
[1] Kadziola et al., J. Mol. Biol., 278, (1998) 205. [2] Vall6e et al., Structure. 6,
(1998) 649. [3] Sigurskjold et a l , Biochemistry, 37, (1998) 10446.

s77

s78

Abstracts FEBS'99

16.2 Metal-assisted catalysis

Structure and function of methyl-coenzyme M reductase
for methanogenic archaea
R.K. Thauer

Methane activation by methane monooxygenase from

Methylococcus capsulatus

Howard Dalton and Claire Lesieur

Max-Planck-lnstltut fur terrestrische Mlkrobiologte, Karl-von-Frisch-Str ,

D-35043 Marburg, Germany

Methyl-coenzyme M reductase catalyzes the final reaction step in the

formation of methane by methanogenic archaea [1]. The 300,000 Da yellow
protein is composed of three different subunits in an ct2~272 arrangement and
contains tightly but not covalently bound two tool of the nickel porphinoid
coenzyme F430. The enzyme is active only in the Ni l+ reduction state. The
structure of the oxygen sensitive enzyme has recently been resolved to 1.45 A
resolution for the enzyme from Methanobacterium thermoautotrophicum [2].
The electron density map suggested five modified amino acids in subunit ct at
or very near the active site region: 1-N-methylhistidine=257, 5-(S)methylarginine '~271, 2-methylglutamine ~4, S-methylcysteine a452
and
thioglycine a445. We have now confirmed these unusual modifications by
chemical analysis, and present evidence that the methyl group of the four
methylated amino acids is biosynthetically derived from the methyl group of
methionine, most likely via S-adenosylmethionine [3].
[I ] Thauer, R.K. Microbiology 144, 2377-2406 (1998).
[2] Ermler, U., Grabarse, W., Shima, S., Goubeaud, M. & Thauer, R.K. Science
278, 1457-1462 (1997).
[3] Selmer, T., Kahnt, J., Goubeaud, M., Shima, S., Grabarse, W., Errnler, U. &
Yhauer, R.K. (unpublished results)

Heme-thiolate proteins catalysis:

how to selectively generate NO?
D. Mansuy
UMR 8601 CNRS, Univer$it~ Paris V,
4.5 rue des Saims-P~res,73270 Paris Cedex 06, France

Heme-thiolate proteins such as cytochromes P450 and NO-synthases

(NOS) are all characterized by the presence of iron-protoporphyrin IX bound
to the protein by an iron-cysteinate bond. Ubiquitous NO-synthases axe
responsible for the biosynthesis of NO from selective oxygenation of Larginine. When compared to cytochrome P450-dependent monooxygenases,
NOS involve a supplementary prosthetic group, tetrahydrobiopterin (BH4),
to be catalytically active. The role of BH4 in NOS has been studied by
comparing the oxidations of various N-hydroxy-guanidines, including N~hydroxyarginine, by recombinant NOS, with or without BH 4, and by
cytochromes P450.
Both BH4-free NOS and microsomal cytochromes P450 catalyze the
oxidative cleavage of a wide variety of compounds containing a C=N(OH)
bond by 0 2 and NADPH, with formation of the corresponding ureas and
cyanamides and nitrogen oxides [ 1].
The two classes of enzymes exhibit highly similar characteristics : (i) a
very broad substrate selectivity, (ii) a relatively low catalytic efficiency
(kcat/Km between 0.01 and 0.1 min-l.p2d-1), and (iii) a strong inhibition by
superoxide dismutase.
By contrast, BH4-containing NOS only catalyze the oxidation of a
limited number of compounds involving a C=N(OH) bond with formation of
NO. For instance, NC-hydroxy-arginine and -homo-arginine act as such
substrates whereas NC0-hydroxy-nor-argininedoes not [2]. Formation of NO
from those substrates is fast (kcat_-50-600 rain-l), and is not inhibited by
supcroxide dismutase.
The presence of BH 4 in NOS appears to be crucial for favoring the
selective oxygenation of few substrates by NOS Fe(II)-O2 over their non
selective, Of-dependent oxidation via the oxidase function of NOS.
[I]
[2]

A. Jousserandot et al., Biochemistry, 37, (1998), 17179.

C. Moali et al., Biochemistry 37, (1998), 10453.

Bacterial methane oxidation is known to be effected by oxygendependent methane monooxygenase enzymes that form methanol as the
stable product. In Methylococcus capsulatus (Bath) this reaction is
catalysed by either a soluble enzyme (sMMO) that contains a diiron
oxo-bridged hydroxylase component or a copper-containing
membrane-associated enzyme (pMMO) in which the nature of the
active site species is presumed to contain copper. The sMMO complex
has been shown to be comprised of three components (hydroxylase,
reductase and regulatory protein) in which NADH2 reacts with the
reductase and 02 and methane interact with the hydroxylase. Recent
studies by small angle X-ray scattering techniques have indicated that
the reductase interaction with the hydroxylase brings about a
significant structural alteration in the latter and is a prelude to the
overall activation process. The opening up of the diineric hydroxylase
permits access of the substrates (02, CH4 and electrons) to the diiron
active site and it appears that the regulatory protein may well serve to
stabilize this particular conformation. In this presentation 1will show
how biophysical studies on the complex have been used to indicate
how methane is bound to the enzyme and activated by a high valent
ferryl species to form the product methanol.

Structure of Fe-only Hydrogenase.

Juan C. Fontecilla-Camps, Yvain Nicolet#, Claudine Piras#,
and Claude E. Hatchikian
# IBS/LCCP CEA-CNRS, 41, Av. des Martyrs, 38027, Grenoble and
BIP UPR 9036-CNRS, 31, Joseph Aiguier, 13402 Marseilles,

The structure of the heterodimeric Fe-only hydrogenase from D.

desulfuricans is reported (1). The so-called H-cluster is composed of a
typical [4Fe4S] cubane bridged to a binuclear active site Fe center
containing putative CO and CN ligands and one bridging 1,3propanedithiol molecule. The conformation of the subunits can be
explained by the evolutionary changes that have transformed monomeric
cytoplasmic enzymes into dimeric periplasmic ones.
Several lines of evidence hint at Fe2, the metal ion from the diiron center
that is not bonded to Cys382, the only protein ligand to the center, as being
the primary hydrogen binding site: 1) Fe2 has an empty coodination site
whereas Fel's coordination sphere is saturated; 2) there is a plausible
proton transfer pathway going from the neighboring Lys237 to the
molecular surface.and 3) there is a hydrophobic channel that runs from the
molecular surface to a point near the vacant coordination site of Fe2. This
channel may provide the substrate with access to the active site.
]?he unrelated active sites of NiFe and Fe-only hydrogenases have several
common features: coordination of diatomic ligands to an Fe ion; a vacant
coordination site on one of the metal ions representing a possible substrate
binding site; a thiolate-bridged binuclear center ; plausible proton and
electron transfer pathways and substrate channels. The diatomic
coordination to Fe ions makes them low-spin and favors low redox states
which may be required for catalysis. The complex EPR signals typical of
Fe-only hydrogenases arise from magnetic interactions between the
[4Fe4S] cluster and the active site diiron center. The paucity of protein
ligands to this center suggests that it was imported from the inorganic
world as an already functional unit.
(1) Nicolet, Y., Piras, C., Legrand, P., Hatchikian, E.C. and FontecillaCamps, J.C (1999) Structure 7, 13-23

Abstracts FEBS'99

s79

17 Extremophiles
Extremophiles and their adaptation to hot environments
K. O. Stetter
Lehrstuhl fiir Mikrobiologie, University o f Regensburg,
Regensburg, Germany

Water-containing terrestrial, subterranean and submarine hightemperature areas harbour a variety o f hyperthermophilic bacteria and
archaea which are able to grow optimally above 80C.
Hyperthermophiles are adapted to their hot environments by their
physiological and nutritional requirements. As a consequence, cell
components like proteins, nucleic acids and membranes have to be
stable and even function best at temperatures around 100C. The
chemolithoautotrophic arehaeon Pyrolobus fumarii is able to grow at
113C and, therefore represents the upper temperature border o f life.
For the first time (vegetative) cultures o f Pyrolobus and its close
relative Pyrodictium are observed to be able to survive autoclaving
(lh; 121C; 2bar).

Insights into the mechanism of protein

stability from studies on glutamate dehydrogenase
D.W. Rice
Krebs Institute, University of Sheffield, Sheffield, $10 2TN, UK

The structures of a number of glutamate dehydrogenases

isolated from species that spar the spectrum of thermal
stability from mesophiles to hyperthermophiles have been
solved by X-ray crystallography in an attempt to
understand the molecular basis of the adaptation of
enzymes to extreme conditions.
The comparison of ~hese
structures has revealed that the hyperthermophilic
enzymes contain striking networks of ion pairs which are
formed by regions of the protein which possess a high
density of charged residues.
Such regions are not found
in the mesophilic enzymes and the number and exten~ of
ionpair formation is much more limited suggesting that
the formation of these networks may well represent a
major stabilising feature.
To test this proposal,
site-directed mutagenesis has been used to attempt to
enhance stability through the introduction of new ion-pair
networks.
Progress on the structure determination of
members of this enzyme family and the results of the
mutagenesis progcamme will be presentefl.

Yip et al. Structure, 3, 1995, 1147

Vetrianl et al. PNA8 95, 1998, 12300

Towards the understanding of the molecular adaptation

of psychrophilie enzymes
Ch. Gerday
Laboratory' of Biochemistry, University of Liege, Belgzum

Psychrophilic organisms adapted to permanently cold habitats

produce enzymes able to efficiently catalyse biochemical
reactions at temperatures often below 0 C These enzymes display
two general properties; they have a high specific activity at low
and moderate temperatures and they have a high thermosensitivity
probably reflecting a more flexible structure [1] In order to
understand the rules governing the molecular adaptations of these
enzymes to cold, 13 enzymes were investigated often up to the
determination o f their 3D structure either by X-ray
crystallography or protein modelling The structures have been
compared to those of homologous mesophilic and thermophilic
enzymes. The adaptation to cold will be illustrated by the analysis
o f the structure of an c~-amylase [2] and a C a 2 - Z n 2- protease [3],
both secreted by Gram" Antarctic bacteria. In the case of the cold
cx-amylase which has been compared to pig pancreatic a-amylase
although the residues involved in substrate binding and catalysis
are strictly conserved, there is a general weakening of the number
and strength o f intramolecular interactions supplemented by
entropic factors leading to a possible increase in the plasticity o f
the molecular edifice. In the case of the Ca2~-Zn2+ protease, the
modification o f some structural parameters gives rise to a decrease
of the general affinity o f the enzyme for Ca 2 and possibly to a
better accessibility o f the catalytic cavity.
[1] Gerday et al., Biochim. Biophys Acta, 1342, (1997), 119
[2] Aghajari et al., Structure, 6, (1998), 1503
[3] Villeret et al., Protein Science, 6, (1997), 2462

Proteins and Membranes from Extreme Halophiles: StructureDynamics-Function Relations depend on Solvent Interactions
G. Zaccai
IBS/CNRS-CEA Jean-Pierre Ebel, 41 rue Jules Horowitz
38027 Grenoble Cedex 1

Proteins and membranes from the extreme halophiles are richly informative models for
the study of solvent effects and interactions.
Two decades ago, it was shown that halophilic proteins bound exceptionally large
amounts of solvent ions in the concentrated salt solutions where they are stable and
soluble [l]. The extent of salt and water binding by the folded protein depends on the
nature of the salt as does the thermodynamics of protein stabilisation [2, 3]. A
solvation-stabilisation model was proposed, in which hydrated salt ions interact
specifically with carboxyl groups on the protein surface, stabilising the fold. Halophilic
adaptation, therefore, would involve a tertiary structure capable of these solvent
interactions. The high resolution crystallographic structure of an extreme halophilic
malate dehydrogenase mutant [4], combined with site-directed mutagenesis [5] has
recently provided detailed information showing that these ideas were basically correct
but that a wider range of interesting solvent interactions is involved, including novel
features such as ion-binding complex salt bridges stabihsing protein subunit
interactions and a large cavity of ordered water molecules at the centre of the enzyme
tetramer.
The solvent environment also has important effects on molecular thermal dynamics that
allow it to be related to biological function. The dependence of dynamics on
temperature and hydration in purple membranes from the extreme halophile,
Halobacterium salinarum, was measured by neutron scattering and strong correlations
were establsihed between the nature of thermal motions and functional aspects such as
much slower conformatinnal changes associated with the light activated proton
pumping activity of the membrane [6, 7].

[1] Eisenberg H et al. Advances mprotem chemistry 43 (1992) 1.

[2] Bonnet8 F et al.. d~ Mol. Btol 244 (1994) 436.
[3] Ebel et al. Biochemistry (1999) in press.
[4] Richard S.B. Th~se Universit~ Joseph Fourier, Grenoble (1998).
[5] Madem D. et al. Eur. d. Biochem 230 (1995).
[61 Rdat V.et al. Proc Natl Aead Sci. (USA) 95 (1998) 4970.
[71 Lehnert U. et al. Biophys d 75 (1998) 1945.

s80

Abstracts FEBS'99

18.1 Molecular mechanisms of cell adhesion

The

E-cadherin/catenin complex and

mouse
embr.~ onic development
]orge Stappert

early

Ma.r-Planck-hl.~titut
tti/ Immunbiologie,
Stubeweg
D-7010S l:rcthurg, Germany

5f.

One of the most basic morphogenetic

events in v e r t e b r a t e
development is the formation of qmple epithelia. In the mouse,
the trophectoderm and the embryonic cctoderm represent t r n e
epithelial cell layer. Using gene targcttlng technology we h a v c
shown that E-cadherin
and catcnln
arc essential
for t h e
formation of these epithelia. Moren~cr. m analyzing g e n e t i c a l l y
altered embryonic stem cells we found thut cadherins i n f l u e n c e
gene expression
and are invo[ved II/ u molecular d i a l o g u e
between the cell surface and the nucleous. Particularly,
[3catenin appears in this respect 1o be a key element. [3-catenin is
u central component of the cadhertn cell adhesion complex a n d
plays an essential role in the Wingle',s/Wut signalling p a t h w a y
We found that the ubiquitin-dependent
proteolysis system is
involved
in
the
regulation
of [3-catenin
turnover
and
nlntugenesis experiments denlonstralcd that substitution of t h e
~erme residues m the glycogen ~ynthase kinase 3[3 (GSK3[3)
phosphorylatioo
consensus
1110111 of
13-catenin
inhibits
ubiqultination
and results nl stablhzatton of the protein. Our
resnlt~
~uggest
that
the
uhiqult Ul-i)roteasome
degradation
pathway may act downstream of G~,K3[~ in the regulation of [3cutemn. We discuss a model where a functional
E-cadherlncatenin complex is not only required for the formation of a n
epithelium hut where in addition cmnponents of the complex ure
invoNed
in the
control
of the
c p i t h e l i a l - m e s e n c h y mul
transition during gastrulatlon lrl k crtebrate development.

Cadherins in the control of epithelial

cell plasticity in morphogenesis
J.P. Thiery, F. Broders, S. Dufour and H. Feracci CNRS UMR
144 and the lnstttut Curie, Parts, France (email: jpthiery@curte.fr)

Epithelial sheets are formed very rapidly during early

embryonic development in a wide variety o f metazoans. These
specialized cell assemblies undergo extensive regional reorganization
leading to the formation and shaping of tissues. Remodelling of
epithelial sheets along with transient or definitive conversion to a
mesenchymal state is particularly spectacular during gastrulation.
These morphogenetic events also occur in many other vertebrate
but most prominently in neural crest, somites, heart and kidney
tissues including the neural tube. The role of classical cadherins has
been studied during the movements of convergence extension in
xenopus embryo gastrulation. Perturbation experiments based on
the use o f an inducible dominant negative mutant of type 1
cadherins have shown the crucial importance of this adhesion
system in notochord formation. The adhesive function o f type 1
cadherin is in part controlled by the homeobox containing
transcription factor otx2 known to interfere with convergence
extension. The role of cadherins type 1 and type 2 has been
addressed in the control of cell motility in an embryonic
environment. The behavior of aggregates of sarcoma 180 ceils
genetically modified to express type I N-cadherin or type 2
cadherin 7 has been analyzed in vitro in response to a fibronectincoated susbtrate and in vivo following their injection in chick
embryo neural crest pathways. Type 1 N-cadherin promotes stable
adhesion of the aggregate while type 2 cadherin is permissive for
migration. These findings are in agrement with preliminary
experiments showing different rates of adhesion between cadherin
type 1 and type 2 in a flow chamber. In vivo, some type 2
cadherins may be preferentially localized in mesenchymal cells
strongly suggesting that they differ significantly in their adhesive
behavior from their closely related type 1 classical cadherin.

E-Cadlldn lind r~'t E . ~ n

i ~ ' ~ / s a' cet,cm' @ im,a ~
M. Mareela, V. No~a, C. Gespachb, E. BnJynee@
aLab. ExpeffmenlalCancero/ogy,Un/v~psityHofplta/,B - ~ Gent,Belg/um
bInsermU482,t ~ ? a l Saint-An&the,75571Paris Cedex 12, France

The E-cadherin/catenincell-cell adhesion and signaling complex is pivotal for

mvasmnof experimental and clinical cancers of epithelial origin [ 1]. Recent experiments
showed that invasion of E-cadherin expressing cancer cells into collagen type I may be
induced by extracellularE-cadherinfragments(sE-CAD)releasedby eclodomainshedding,
as well as by synthetic or recombinant peptides containing the HAV sequence
homologous to the first extracellulardomain of E-cadherin [2]. Release of an 80 kD sECAD is stimulatedby phorboI-12-myristate-13-acetate(TPA) and is inhibited by TIMP-2
overexpression. Comparisonof cell lines with differentchanges in oncogenesand tumorsuppressor genes showed that cancer cells respond to sE-CAD only when they have
reached certain stages of progression, sE-CAD-mediated induction of invasion is
neutralized by tyrosine kinase inhibitors such as herbimycin A and by PI-3 kinase
inhibitors such as wortmannin.Furthermore,sE-CADcauses tyrosinephosphorylation of
[3-catenin and of GSK-3J3and association of GSK-3[~and Shc with the E-cadherin/catenin
complex. The putative relevanceof these experimentalobservations is supported by the
finding of 80-kDa sE-CADand of the remnant40-kDamembrane-associatedfragmentsin
biopsies from humanbreast and colon cancer. It remainsto be elucidated how cancer cells
receive sE-CAD, how this signal is passed downstream of the E-cadherin/catenin
complex and which cellular responses are crucial for the induction of invasion.
Complexity arises from the interaction between the E-cadherin/catenin pathway with
other invasion pathways that may as well act independently. Such pathways include the
non-receptor tyrosme kinase Src and the scatter factor receptor c-MET that both, when
activated, cause invasionconcommitantwith tyrosinephosphorylation of ~3-catenlnand of
p 125FAK. Treatmentwith Wortmanninand transfectlon with dominanl negativeformsof
p110ct pointed to a role for PI3-K in all invasion pathways examined. By contrast,
platelet-activatmg factor [3] and other natural inhibitors of invasion showed some
pathways specificity. Theyare currently used to further unravel the variouspathwaysof
invasion as well as their mutual relationships.
Ill Marcel et al., J. Cell Physiol., 173, (1997) 271; [2] Noe et al., J. Cell Sci., 112,
(1999) 127; [31 Kotelevetset al., J. Biol. Chem, 273, (1998) 14138.

Epidermal cell adhesion and hemidesmosome assembly.

A. Sonnenberg ~, L. Bormdori2, B. Favrez, L. Fontao I, D. Geerts] ,
J. Koster I and M. Nievers t
/ The Netherlands Cancer Institute, Amsterdam. The Netherlands and
e Hopital Cantonal Umversitaire, Geneva, Switzerland..
Hemidesmosomes are multiprotein complexes that mediate adhesion of epithelial
cells to the underlying basement membrane. The transmembrane components of
hemidesmosomes include the integrin tx6134 and BPI80. These proteins are
connected, via the hemidesmosomaI plaque proteins HD 1/plectin and BP230, to
the keratin intermediate filament system. We have investigated the contribution
of various segments of the 134 cytoplasmic domain in the formation of
hemidesmosomes in transient transfection experiments using immortalized
keratinocytes derived from an epidermolysis bullosa patient deficient in the
expression of 134. We found that the expression of wild type 134 restored the
ability of the 134-deficient ceils to form hemidesmosomes, and that distinct
domains of the 134 cytoplasmic tail are required for the localization of
HD1/plectin, BP180 and BP230 in these hemidesmosomes. Using yeast twohybrid and biochemical assays, we confirmed that 134 can interact with each of
these three components. In addition, we found that the COOH terminal portion
of the cytoplasmic domain of 134 is able to associate with a more proximal region
o f the molecule. Although the significance of this potential intramoleeular
folding remains to be established, it may represent a means by which the
interaction of 134 with other elements ofhemidesmosomes is regulated. A current
model for the assembly ofhemidesmosomes has emerged in which the clustering
of c16134 at the basal side of the cells, induced by the binding ofc16134 to ligand
and HD1/plectin, facilitates the formation o f a core, that subsequently serves as
an attachment site for BP 180 and BP230. Additional protein-protein interactions
between BP180 and BP230, HDl/plectin and the 016 subunit, may then help to
stabilize these hemidesmosomes.

Abstracts FEB S' 99

lntegrin signalling in cytoskeleton organization and cell

growth
G. Tarone, M. Brancaccio, S. Degani, S. Guazzone, N.
Mcnini, M. Venturino, L. Morn*, L. Dolce, C. Bozzo* P.
Defilippi, F.Altruda, L. Silango. Dip. di Genetica, Biologia e
Biochimica, Universitfi di Torino (*) Dip. di Scienze
Mediche, Universit/~ A. Avogadro, Italy.
Integrins are major cell surface receptors responsible for transducing signals in
response to cell matrix interaction. Integrin signalling involves activation of
cytosolic tyrosine kinases, monomeric GTPases of the Rho family, MAPK and
transcription factors as STATs. Although the functional consequences of the
activation of these pathways are relatively clear, the molecular mechanisms by
which integrins can activate these pathways is still very poorly defined. Since
signaling requires the integrin cytoplasmic domain, we have searched for
proteins capable to bind this region of the molecule. Using the two hybrid
screening we have isolated two proteins, ICAP and L1L8, which bind to
different regions of the integrin cytoplasmic domain and have distinct
properties. ICAP contains an amino terminal region rich in serine residue
potential targets for PKC and it is involved in the regulation of cell spreading
on fibronectin. L1L8 binds to the membrane proximal region of [31 integrin
cytoplasmic domain in a Ca++ regulated manner and its is expressed
selectively in muscle tissue. These molecules are likely candidate as transducer
of integrin signals. A second aspect we have studied is the ability of integrins
to utilize growth factor receptors as down stream sgnalling molecules. We have
shown that upon cell matrix binding, integrins can induce tyrosine
phosphorylation of the EGF-R in absence of ligands for this receptor. Tyrosine
phosphorylation of EGF-R leads to ERK MAPK activation in response to
integrin stimulation and to survival signals. The matrix-induced activation of
the EGF-R is likely to require physical interaction of integrins and EGF-R at
the cell surface. (Supported by grants form Telethon, AIRC and MURST).

s81

s82

Abstracts FEBS'99

18.2 The cytoskeleton in morphogenesis and signalling

Cytoskeleton cross-talk during cell motility
J. V. Small, I. Kaverina, O. Krylyshkina, K. Rottner
Department of Cell Biology, Institute of Molecular Biology, Austrian
Academy of Sciences, Billrothstrasse 11, Salzburg, 5020, Austria.
Cell crawling entails the co-ordinated creation and turnover of substrate
contact sites that interface with the actin cytoskeleton. The initiation and
maturation of contact sites involves signalling via the Rho family of small G
proteins, whereas their turnover is under the additional influence of the
microtubule eytoskeleton. By exerting relaxing effects on substrate contact
assemblies in a site- and dose-specific manner, microtubules can promote both
protrusion at the front and retraction at the rear, and thereby control cell polarity.

Re-organization of the actin system in cell motility,

endocytosis and cytokinesis

G. Gertsch, K. Barisic, J. Faix. R. Ne.ujahr.

J.-M. Schwartz, J. Niew6tmei', l.Weber
Max-Planck-lnstitutfur Biochemie, D-82152 Martmsried, Germany.

Activities of the actin cytoskeleton depend on the intracellular redistribution of proteins and their assembly into functional complexes at
the leading edge of a cell, at a phagocytic cup, or in the cleavage furrow.
Three actin-binding proteins will be analysed using GFP-fusions:
coronin, a member of the WD40 repeat family of regulatory proteins,
cortexillin, a member of the spectfirdc~-actinin family, and the tail
domain of talin. Their re-distribution is analysed in the highly motile
cells of Dictyostelium, where chemotactic stimulation induces actin reassembly within less than 10 seconds and cytokinesis takes only 2
minutes, Mitotic cell division in Dictyostelium is of interest for two
reasons.
(1) When anchored to a substratum these cells do not need myosin II in
order to make a cleavage furrow [1]. Therefore, the importance of other
proteins is obvious. (2) Two of these proteins, coronin and cortexillin,
sort out during cytokinesis: coronin is accumulating in ruffles at the cell
poles and cortexillin is targeted to the cleavage furrow. Domain analysis
of eortexillin 1 revealed that the infbrmation for translocation to the
furrow resides in a fragment of 93 amino-acid residues comprising a
PIP_, binding motif. This C-terminal fragment is also capable of rescuing
cytokinesis in cortexillin I-null mutants [2]. Mechanisms of protein
translocation during mitotic cell division will be discussed.

[1] Neujahr R, Heizer C, Gerisch G (1997): J Cell Sci 110: 123-137.

[2] Weber I, Gerisch G, Heizer C, Murphy J, Badelt K. Stock A.
Schwartz JM, Faix J (1999) EMBO J 18: I01-109,

Control of actin dynamics in cell motility.

Marie-France Carher*. Fanza Ressad*, Val~ne Laurent*. Thomas
Lolsel*, Coumaran Egile*+, PhilippeSansonetti", Dominique
Pantaloni*. *LEBS, C.N.R.S. Gif-sur-Yvene,France, and "*Unitdde
Pathogdnie microbiennemol6culaire.InstltutPasteur, Paris, France.
Stimulation of eukaryotic cells by growth factors or chemoanractants activates a
signahng cascade of reactions leading to actin polymerization, which drives the
extension of lamellipodia or filopodia. Numerous barbed ends, generated beneath the
plasma membrane, achvely grow, pushing the leading edge forward at rates of 1-20
gin/rain. The growth of barbed ends is fed by the depolymerlzation from pointed
ends at the rear of the lamellipodium. Hence lamellipodium extension results from
the rapid turnover of actin filaments according to a treadmilling mechanism. The
following key mechanistic issues are raised by this actin-based motility process : I)
how are barbed ends generated in response to signaling ; 2) how is the intrinsically
slow treadmilling of actin filaments increased to support the observed fast barbed
end growth : 3) how is the actin assembly machinery connected to the membrane to
elicit movement. Actin-based propulsion of Listeria or Shigella in acellular extracts,
which can be blochemically or immunochemically modified, provides a tool for the
molecular and physical approach of movement. ADF/cofilins are phosphorylationregulated actin dynamizing factors which enhance the turnover of actm filaments,
and increase the rate of Listeria movement, by increasing the rate of
depolymerization from the pointed ends. This results m an increase m the steadystate concentration of ATP-G-actin, which feeds barbed end growth. Profilin acts in
synergy with ADF to increase the turnover of actin filaments over 100-fold. The
Arp2/3 complex multiplies growing filaments by barbed end branching. This effect
of Arp2/3 is activated by N-WASP, a multipartner connector protein that links the
actin cytoskeleton to the signaling pathway. Binding of N-WASP to IcsA (VirG)
expressed at the surface of Shigella allows local activation of Arp2/3 and initmt]on
of actin assembly at the surface of Shigella. The protein VASP binds to the prolinerich central domain of ActA at the surface of Listeria via its EVH1 N-terminal
domain and to F-actm via its EVH2 C-terminal domain. In attaching the actin tall to
the bacterium. VASP organizes directional actin assembly and is responsible for the
generation of propulsive force. In contrast, VASP is not required for the movement
of Shigella. and the necessary attachment of the aetin tail to the bacterium m thls
case is provided by N-WASP itself.

Scar and the Wiskott-Aldrich Syndrome protein (WASp)

regulate actin nucleation through the Arp2/3 complex
L.M. Machesky, H.N. Higgs, L. Blanchoin, R.D. Mullins,
D. Kaiser, R.C. May, A. Secchi, M. Hall, R.H. Insall,
H. Wehland, T.D. Pollard
Department of Biochemistry, Universityof Btrmingham,
Edgbaston,Bwmingham B 15 2 ~ UK
The Arp2/3 complex, a 7-protein complex containing the actin-related
proteins Arp2 and Arp3, may regulate actin polymerization at the leading
edge of a migrating cell and during other cellular activities. Arp2/3
complex localizes to regions of dynamic actin assembly, including
lamellipodial protrusions and nascent phagosomes. In vitro, Arp2/3
complex crosslinks actin filaments, binds to the slow-growing (pointed)
end and nucleates new filaments. Together, these activities appear to
assemble a dendritic network of actin filaments leading to protrusion of
the plasma membrane. We found that the p21-Arc subunit of the Arp2/3
complex binds to (Wiskott-Aldrich Syndrome) WASp family proteins.
This binding regulates the actin nucleating activity of the Arp2/3
complex as well as its localization in cells. WASp family proteins
participate in signalling pathways downstream of receptor tyrosine
kinases, Rho-family GTPases (Cdc42 and Rac) and heterotrimeric Gproteins. WASp-family proteins also contain a binding site for
monomeric actin and a proline-rich region that binds to SH3 domains and
possibly profilin. Overexpression of the Arp2/3 complex binding
fragment of WASp or Scar causes delocalization of the complex in cells
and rearrangement of the actin cytoskeleton. Cells Overexpressing Scar
or WASp fragments that delocalize the Arp2/3 complex do not form
lamellipodia and cannot complete phagocytosis. In vitro, Scar and
fragments containing the actin and Arp2/3 complex binding sites enhance
the nucleation activity of the Arp2/3 complex. These proteins also
inhibit the actin-based motility of the pathogenic bacterium Listeria
monocytogenes, probably via binding to the Arp2/3 complex. We
propose that Scar (and likely other WASp-family proteins) controls actin
polymerization downstream of various signal inputs by modulating the
localization and activity of the Arp2/3 complex.

Abstracts FEBS'99

s83

Beta-catenin/armadiUo in cell adhesion

and signal transduetion
Walter Birehmeier
Max-Delbrueck-Centerfor MolecularMedicine,
Robert-Roessle-StrasselO, D-13125 Berlin, Germany

Beta-catenirgarmadillo is a component of both the cadherin cell adhesion

system and the wnt signaling pathway. Wnt signaling increases the levels of
cytosolic beta-catenin by preventing its ubiquination and degradation via
proteasomes. This allows direct interaction of beta-catenin with transcription
factors of the LEFfI'CF family and modulation of gene expression. The tumor
suppressor gene product APC induces degradation of heta-catenin which is
dependent on phosphorylation by the serine/threonine kinase GSK3beta. APC
and beta-catenin mutations found in human tumors prevent degradation.
Thus, the regulation of stability of beta-catenin is central for wnt signaling in
development and tumor progression.
We have shown that the protein conductin forms a complex with beta-catenin,
the tumor suppressor gene product APC, and GSK3 beta. Conductin induces
beta-catenin degradation, whereas mutants of conductin that are deficient in
complex formation stabilize beta-catenin. Fragments of APC that contain a
conductin-binding domain also block beta-catenin degradation. Thus
conductin and the related protein axin are components of the multiprotein
complex that directs beta-catenin to degradation. Formally, conductin is
located downstream of GSK3 beta and APC but upstream of siamois, LEF-1
and beta-catenin.
References:
Behrens J. et al., Science, 280 (1998) 596.
Meiners S. et al., Oncogene, 16 (1998) 9.
Niemann C. et al., J. Cell Biol., 143 (1998) 533.
Behrens J. et al., Nature, 382 (1996) 638.
Weidner K.M. et al., Nature 384 (1996) 173.
Huelsken J. et al., J. Cell Biol. 127 (1994) 2061.

s84

Abstracts FEBS'99

19.1 Molecular mechanisms in neuronal and muscular diseases

Intraneuronal filamentous deposits
in neurodegenerative diseases
M. Goedert
MRC Laboratory of Molecular Biology,
Hills Road, Cambridge CB2 2QH, laid

Abundant neuritic plaques and neurofibrillary lesions constitute the

defining neuropathological characteristics of Alzheimer's disease. The
paired helical filament (PHF) and the related straight filament (SF) are
the major fibrous components of the neurofibrillary lesions. They
consist of all six brain isoforms of the microtubule-associated protein
tan. The discovery of exonic and intronic mutations in the tan gene in
frontotemporal dementia and parkinsonism linked to chromosome 17
(FTDP-17) has shown that tan dysfunction causes neurodegeneration.
We have grouped known tau mutations into three classes depending on
their effects on tau isoforms composition, tau filament morphologies and
affected cell types. Most missense mutations lead to a reduced ability
of tan to interact with microtubules, whereas intronic mutations and
some missense mutations lead to an overproduction of four-repeat tan
isoforms. Some missense mutations also stimulate heparin-induced
assembly of tan protein. Lewy bodies and Lewy neurites, the defining
neuropathological characteristics of Parkinson's disease and dementia
with Lewy bodies, constitute the second most common filamentous
intraneuronal pathology, after the neurofibrillary lesions of Alzheimer's
disease. The discovery of missense mutations in a-synuclein as a rare
cause of familial Parkinson's disease has led us to the finding that
c~-synuclein is the major component of Lewy body filaments. Moreover,
multiple system atrophy has been shown to be a third
~-synucleinopathy. Recent developments in the field of intraneuronal
filamentous inclusions have thus shown that tau protein and C(-synuclein
account for the inclusions in the vast majority of late-onset dementias.
Genetic studies have shown that thesc proteins are central to the
neurodegenerative process.

Physiological function of APP and its A-amyloid domain

- implication for A l z h e i m e r ' s disease
K. Beyreuther, C. L. Masters"
ZMBH, Univ. Heidelberg, D-69120 Heidelberg, Germany and Dept.
Pathology, Univ. Melbourne, Parkville, Victtoria 3052, Australia
To understand synaptic loss and neurodgegenemtion in Alzheimer's disease
(AD), we have tried to consider what the physiolgocal functions of the
arnyloid precursor protein (APP) and g amyloid peptide (BA4) are. The
latter is a metabolic product of APP and the major subunit of amyloid
plaques which are characteristic of AD. The normal function of APP is not
known, but from studies in transgenic D. melanogaster and primary
neurons, we believe that APP has a function at the synapse in modulation of
cell adhesion where it may be involved in repair and forgetting [1 ].
When we studied the transport of APP down the axons and dendrites, we
discovered that the axonal transport was dependent on the betaA4 domain
of APP [2]. This suggested that the 13A4 domain could function as major
axonal sorting signal o f APP and binding site of a receptor that controls the
recruitment of APP into axonally transported vesicles. Removal of betaA4
occurs at all sorting stations of APP such as at the ER/cisGolgi,
TGN/endosomes as well as at the cell surface [3]. Therefore, this flA4
release may regulate receptor binding. Because both, the apoE e4 allele and
lower estrogen may be associated with higher cholesterol levels in brain and
risk of developing AD, the influence of cholesterol on neuronal BA4
generation was studied [4]. By reducing the cellular cholesterol level of living
hippocampal neurons with lovastatin and methyM3-cyclodextrin, the
formation of secretory and intracellular 13A4 is drastically reduced. This
shows that cholesterol is required for BA4 formation to occur and implies a
link between brain cholesterol, APP transport, BA4 production and AD.
Our finding that 13A4 could function as axonal targeting signal of APP
suggests that BA4 formation regulates APP transport and implies that
accumulation of BA4 in AD may disturb the axonal transport of APP and of
other proteins which use the same transport machinery. Furthermore, both
thephysiological and pathological regulation of APP transport by 13A4
production appears to be linked to cholesterol. From these molecular
tnsights, it i s ' l ~ e l y that effective therapeutic and preventive strategies will
probably be based on combination therapies, such as today's
neurotransmitter replacement combined with a drug to protect against the
above-mentioned toxic effects of gA4 whereas prevention will include brain
cholesterol lowering and estrogen replacement slrategies.
~ ] FoBgreen, A. et al., Proc. Natl. Acad. Sci. US.A, 95, (1998) 13703;
ienari. P. et at., Cold Spring Harbor Quant. Syrup., 61 (1996), 575; [2 t
Hartmann, T. et al., Nature Med., 3, (1997), 1016; [4] Simons, M. et ai.,
Proc. Natl. Acad Sci. US.A, 95, (1998), 6460.

The presenilins and amyloid precursor protein in Alzheimer's

disease: is it processing or signaling that goes wrong ?
B. De Strooper*,W. Annaerti, P. CupersI, D Hartmann2, P Saffig3and R. Kopan~
IFlanders Inslltutefor Btotechnolog~ , CME. K U Leuven, Belgium :.4nat Inxtitut der CAU
KteL JZentrum Biochem und Mol Zellbtologw, Grttingen, Germany aDtI' of DermatologY
and Department of Mol Btology and Pharm Washmfon, USA

Presemlin 1 (PSI) ts a multitransmembraneprotein located in the endoplasmatic

reticulum Missense mutations of PSI are an important cause of familial
Alzhmmer'sdisease. In contrast, homozygotePSI-/- mice dte late in embryogenems and display a severe growth retardation,which is most outspoken in the
caudal region. In the brain a collapse of the subventricularzone, hemorrhagies
and cortical dysplasia featuring leptomeningealfibrosis and overmlgrationof
cortical plate neurons into the marginal zone Is observed, resembling human type
2 lissencephaly. This neuronal mtgratmndisorder ts associated with the
disappearanceof the majority of Cajal-Retziuspioneer neurons from the
developing cortical anlage. The deficient somitogenesistogether with aberrant
Notch immunostainingin the brain, suggest that presenilin 1 modulates the Notch
signaling pathway in agreement with previouslydocumentedgenetic interactions
between Notch and PSI homologues in C elegans. The Notch pathway is
involved in crucial cell fate dectmonsduring developmentand requires hgandinduced cleavage of the Notch receptor for signal transduction.We identify this
cleavage step as the specific biochemicaldeficit caused by the absence of PS 1.
We have previouslydemonstratedthat PSI also facilitates a proteolytic activity
called y-secretasethat cleaves the mtegral membranedomain of Amyloid
Precursor Protein (APP). Since y-secretaseinhibitors block also Notch
processing,we suggest that related protease activities are responmblefor cleavage
withm the predicted transmembranedomains of Notch and APP. While the PS
have been imphcated in many cell biologicalprocesses, we would like to propose
that their primordial function is the (control o0 proteolytic processing of a subset
ofintegraI membraneproteins. Disturbanceof this function could explain the
multiple effects of PSI defictency or PS1 overexpressionin cell culture systems.
Targeting PS 1 function to lower amyloid peptide production in the brain of
Alzheimer+sDisease patients may therefore cause severe side effects.

Mechanism of polygiutamine expansion diseases

J-L Mandel
fnstltut de G~n~tiqueet de Biologte Mol~culaireet Cellulatre(1GB3JC),
CNRS/1NSERM/ULP, 67404 lllkirch Strasbourg, France.
Instable expansions of trinucleotide repeats have been found associated,
since 1991, to 12 neurological diseases. These diseases are often characterised
by some form of anticipation, linked to the tendency of pathological alleles
to further expand, and by biases in the parental origin of mutations leading to
the most severe forms. Triplet diseases can be divided in 4 classes. The first
three correspond to large expansions in non coding sequences, leading to
pleiotropic diseases. The fourth class corresponds to moderate expansions of
CAG repeats coding for polyglutamines, found in 8 purely neurodegenerative
diseases, that includes Huntington's disease and 5 spinocerebellar ataxias.
Polyglutamine expansions cause a gain of toxic property in the target
proteins, with the possible exception of SCA6 These diseases are
characterised by adult-onset neuronal death in selected but different regions
of the nervous system, and by an inverse correlation between length of the
polyglutamine expansion and age of onset. However, the age of onset for
patients carrying the same expansion can vary by as much as 20 years.
The proteins implicated in these diseases show no common features apart
from the polyglutamine tract, and their distribution shows no obvious
correlation with the site of neurodegenerescence. Recent observations in
patients and in mouse models suggest that aggregation of polyglutamine
containing proteins within the nucleus may constitute a common pathological
mechanism. We have characterized a monoclonal antibody (1C2) that
recognizes selectively the mutated proteins in liD and in SCAs 1, 2, 3 and 7
(this has allowed the cloning of the SCA2 and SCA7 genes). The efficiency
of the detection is dependent on polyglutamine length, and thus parallels
disease severity. This indicates that the pathological threshold observed in
patients correspond to a conformation change recognized specifically by this
antibody.
Cell lines and transgenic mice that carry and express a mutated protein
have been constructed in various labs, and some show pathological effects.
They allow an investigation of the disease mechanisms. We have recently
obtained a cellular model where expression of full length mutated huntingtin
protein results in the formation of ubiquitinated nuclear inclusions, that
contain however only the N-terminal region of huntingtin. This model
appears thus to recapitulate major steps observed in patients brain.

Abstracts FEBS'99

Self-assembly of polyglutamine-containing
huntingtin fragments into amyloid-like fibrils
and implications for the pathology
of Huntington's disease
E. Wanker
Max-Planck-lnstitut fftr Molekulare Genetik, lhnestrasse 73,
D-14195 Berlin (Dahlem), Germany.

Huntington's disease (HD) is an autosomal dominant progressive disorder

characterized by a selective neuronal cell death in the cortex and striatum. The
disorder is caused by an elongated CAG/polyglutamine (polyQ) repeat in the
first exon of the HD gene encoding the huntingtin protein of unknown
function. In unaffected individuals the CAG repeat varies from 6 to 39 repeats,
whereas in HD patients between 36 and 180 units have been reported. For the
first time, we have shown in our laboratory that HD and also the inherited
neurodegenerative disorders DRPLA, SBMA, SCAI, SCA3 and SCA7 could
be the result of a toxic amyloid fibrillogenesis similar as Alzheimer's disease.
We demonstrated that the proteolytic cleavage of GST-huntingtin fusion protein
leads to the formation of insoluble high molecular weight protein aggregates
only when the polyQ expansion is in the pathological range. Electron
micrographs of these aggregates revealed a fibrillar or ribbon-like morphology.
Subcellular fractionations and ultrastructural techniques showed the in vivo
presence of these structures in the brain of mice transgenic for the HD mutation
as well as in the brain of HD patients. Recently, we also found that the
formation of amyloid-like huntingtin aggregates in vitro is not only dependent
upon polyQ repeat lenght, but is also critically dependent on protein
concentration and time. Furthermore, the in vitro aggregation of huntingtin can
be seeded by preformed fibrils. Together, these results suggest that amyloid
fibrillogenesis in HD, like in Alzheimer's disease, is a nucleation- dependent
polymerization.

s85

s86

Abstracts FEBS'99

19.2 Molecular mechanisms in infectious diseases

Bacterial virulence factors: the case of Helicobacter pylori
C. Montecucco

CNR Centerfor Btomembranes and Department of Biomedical Sciences

Vta G. Colombo, Patova, Italy
Pathogenic
strains
of
Helicobacter
pylori
are
responsible for the pathogenesis of severe gastroduodenal diseases. This bacterium can resist very
acid environments because its urease creates a nearly
neutral microenvironment. Aided by its spiral-shape
and propelled by its flagella, H. pylori crosses the
mycus layer of the stomach and attaches firmly to the
apical
domain
of the
epithelium
via
specialized
adhesins and via other outer membrane bound proteins,
including a neutrophil activating protein of 15 kDa
(HPNAP) and a vacuolating cytotoxin of 95 kDa (VaeA).
VacA
is
activated
by
low pH
and
thus
becomes
hydrophobic and capable of inserting into the lipid
bilayer. VacA has two major cellular activities. A)
it causes the formation of vacuoles which originate
from the fusion and enlargement of late endosomal and
lysosomal
compartments.
VacA
at
low
pH
makes
transmembrane anion specific channels, which support
the activity of the electrogenic vacuolar ATPase
proton pump. Membrane permeant amines present in food
or generated by the urease swell the vacuoles via an
osmotic effect. Vacuolation causes mis-targeting of
acid hydrolases, inhibition of antigen processing and
eventually lead to cell sufferance and death. B) it
induces a selective increase in the paracellular
route
of
permeability
of
polarized
epithelial
monolayers
to
small
molecules.
The
passage
of
nutrients from the basolateral side to the apically
located
bacteria
supports
their
growth.
A major
damage to the stomach mucosa is due to a strong
recruitment of neutrophils.
HPNAP causes a strong
activation of neutrophil oxygen radicals production
via a pertussis toxin sensitive G protein-coupled
HPNAP
receptor.
It
is
believed
that
neutrophil
induced tissue degradation makes additional nutrients
available
to the bacterium.
Oxygen
radicals
can
generate mutagenic substances which could contribute
to the
increased
risk of
stomach
adenocarcinoma
associated to chronic H. pylori infection.

M o l e c u l a r a n d c e l l u l a r bases of epithelial cell

i n v a s i o n by e n t e r i c p a t h o g e n s
P.J. Sansonetti, Unit6 de Pathogrnie Microbienne
Molrculaire, INSERM U 389, Institut Pasteur.
28 rue da Dr Roux, F - 75724 Paris crdex 15, France

Entry of Shigella into epithelial cells winch are not professional phagocytcs revolves a
complex signalling pathway that elicits a macropinocitic event leading to internalization
of the bacterml body. Upon contact w~ththe cell surface, S. fle.u~ert acttvatc~ a Mxi/Spa
specialized secretory apparatus through which Ipa invasms are secreted, two of which
(IpaB, 62 kDa and IpaC, 42 kDa) form a complex which resorts as a pore In the
eukaryotic cell membrane where It serves two purpo~c~,it permits ~qn3echon,of bacterial
porteins such as IpaA and IpaD into the cytoplasm. It also elicits major rearrangements of
the host cell cytoskeleton, essentially tile polymerization of actin filaments that m'e all
similarly polarized with their positive ends onentcd towards the inner face of the
cytoplasmic membrane. Activation of the cascade of small GTPases (i.e. Cdc42, Racl and
Rho) by the IpaB-IpaC complcx accounts for thcsc evcnts. These actin filaments lbrm
plastin-mediated bundles that support membrane projccttons which achieve bacterial entry.
The tyrosme kinase pp60~~: act as a regulator that amphfies the extension of cellular
projecuons by down-regulating the rho-mcdmtcdsignals. Ezrin, which *s actively rccrmted
in the cellular projections, also plays a major role ill the extension of tho cell surface
projections that engulf the bacteria. Shigella subscquently mduces tile formation of an
adherence plaque at the cell surface In order to promote its entry. The entering bacterium
allows translocation of another Ipa protein, ipaA, which binds amd activates vmculin
thereby completing maturatton of the entry focus by inducing the formation of a dense
actin cup. Once intracellular, tile bacterium lyses its phagocytic vacuole, escapes into the
cytoplasm and starts moving by reducing polar, directed polymerization of actm on its
surface, due to the expression of IosA, a 120 kDa outer membrane protein which becolncs
locahzed at one pole of the mtcroorganlanl, lbllowing cleavage by SopA, a plasmidencoded surface protease. N-WASP appears to bc a key ligand of IcsA for actm
polymerization. In the context of polmlzed cpithehal cells, bacteria then reach the
intermedlatejunctmn and engage their components, particularly the cadherins, to form a
protrusmn which is actively mternahzed by the adjacent cell. Bacteria then lysc the two
membranes, reach the cytoplasmic conlpartment again, and re~,unle actin-driven
movement.

Helicobacterpylori adherence in the human

Neisseria Host Cell Interactions - Adherence,
Signalling, Cell Invasion, Phagocytosis and
Apoptosis
T. F. Meyer

Max-Planck-lnstitutfiir lnfektionsbiologie, Berlin, Germany

The genus Neisseria includes two pathogenic species, Neisseria gonorrhoeae

and Neisseria meningitidis, which primarily infect human mucosal surfaces and
rarely cause systemic infections. Initial binding to the mucosa is mediated by the
pill while expression of the phase-variable Opa outer membrane adhesins leads to
an intimate association with target cells via heparan sulfate proteoglycan
(HSPGs) or CD66 receptor molecules. These Opa-mediated interactions
represent one precondition for the endocytic uptake or the phagocytosis of the
bacteria which involves several distinct signalling pathways, depending on
receptor and cell type. [1,2 ]
One co-factor for gonococcal invasion appears to be PorB, a typical gramnegative bacterial porin which exhibits a variety of striking properties. ProB is
capable of translocating from the bacterial cell surface into target cell membranes
where it functions as a GTP/ATP regulated pore. As a consequence, CA 2. influx
occurs which on one side may enhance invasion perhaps through the activation
of protein kinase C (PKC) and, on the other side, induces apoptosis through the
activation of calpain and cysteine proteinases. Interestingly, PorB structurally
resembles the mitochondrial porins representing common constituents of
eukaryotic cells. Since recent evidences assume a critical role of mitochondria as
central integrators of apoptotic cell death, the infection with pathogenic Neisseria
species may resemble aspects of the interaction of ancestral endosymbionts with
early eukaryotic organisms.J3]
[1] Dehio, C. et al., Trends MicrobioL 6, (1998) 489; [2] Naumann, M. et
al., Current Opinion in Microbiology. 2, (1999) 62; [3] Mtiller. A. etal., EMBO
.L 18 (1999) 339.

gastric mucosa
T. BorOn
Dept. of Odontology,Ume~ University,SE-901 85, Ume~i,Sweden

Helicobacter pylori, has emerged as the causative agent in chronic active

gastritis and acid peptic disease. H. pylori has adapted to the hostile and
acidic environment of the h u m a n stomach. Once established in the host, the
bacteria can persist for the lifetime of the host. The chronic infection has
also been correlated to the development of gastric cancer. H. pylori colonize
the human gastric mucosa by adherence both to the mucous epithelial cells
and to the mucus layer lining the gastric epithelium. The microbial affinity
for specific receptor structures, in combination with unique tissue-specific
distribution o f receptors, will restrict the colonization to a limited number of
hosts and tissues. This process is often referred to as tissue tropism.
We have previously show that the fucosylated histo-blood group antigens
Lewis b and H are the carbohydrate structures that specifically mediate
adherence o f Hi pylori to human gastric epithelial cells [ 1]. The fucosylated
b l o o d g r o u p antigens are also present as natural clearance factors in
secretions such as saliva and h u m a n milk. There is an interesting clinical
relevance o f bacterial attachment mediated by the fucosylated H and Lewis
b blood group antigens since individuals o f blood group O phenotype are in
higher risk to develop acid peptic disease.
For the purification of the blood group antigen binding adhesin, BabA, a
novel technique was developed, Receptor Activity Directed Affinity
T a g g i n g (ReTagging) [2]. We propose that B a b A - m e d i a t e d H. pylon
adherence to gastric epithelial tissue plays a critical role in efficient delivery
of H. pylori virulence factors that damage host tissue directly, and that incite
i n f l a m m a t o r y r e s p o n s e s a n d p r o v o k e a u t o i m m u n e reactions, that
c u m u l a t i v e l y lead to d e v e l o p m e n t o f chronic i n f l a m m a t o r y disease.
U n d e r s t a n d i n g o f the m e c h a n i s m s s u p p o r t i n g the adhesin proteinc a r b o h y d r a t e receptor interactions should aid in the development o f
antimicrobial strategies against Hi pylori infections and associated chronic
inflammatory disease. The presence of biologically conserved motifs and
peptide sequences provides a unique target for vaccine based antimicrobial
strategies.
[1] T. B o r r n et al., Science, 262, (1993) 1892.
[2] D. Ilver et al., Science, 279, (1998) 373.

Abstracts FEBS'99

s87

The Prion Protein in Health and Disease

C.Weissmann, F.Montrasio, A.Raeber, M.Klein $

l.Hegy$, E.Flechsig, T.Riilicke+ and A.Aguzzi$

D.Shmerling,

lnstitut fur Molekularbiologle. $1nstitut fur Neuropathologie and

+Biologtsches Zentrallabor. Universitat. Zilrich
The prion is believed to be devoid of nucleic acid and identical with PrP*, a
modified form of the normal host protein PrP C. The "protein-only" hypothesis
propounds that PrP* multiplies by causing conversion of PrP c into PrP*. Prusiner
proposed that a protease-resistant form of PrP, PrP s, which accumulates in
serapie-infected tissue is PrP*. The physiological function of PrP C is still
unknown. PrP knockout (Prnp/o) mice develop and behave normally. As
predicted by the "protein-only" hypothesis, they are resistant to scrapie and do
not propagate scrapie agent.
Prnp/o mice that express mini-transgenes encoding full-length PrP regain
susceptibility to scrapie. Removal of amino acids 32 to 93 (comprising the five
octarepeats) still enabled susceptibility to scrapie, however incubation time was
prolonged, prion replication diminished and the clinical picture modified. Deletions
to residue i06 no longer sustained development of scrapie. Intriguingly, deletions
to amino acid 121 or 134 led to spontaneous severe ataxia and neuronal death
restricted to the cerebellar granule layer I-2 months after birth. This condition was
not transmissible. The defect was abolished by crossing in a single wild-type PrP
allele. We propose that in wild-type mice, PrP interacts with a conjectural ligand to
elicit a signal which can also result from the interaction of the ligand with a
conjectural PrP-like protein, explaining why ablation of PrP gives no obvious
phenotype. We postulate that truncated PrP can interact with the ligand without
giving rise to a signal and competes efficiently with the PrP-like protein, thus
acting as an inhibitor of the latter.
In mouse scrapie, prions accumulate in the spleen early on. Follicular dendritic
cells accumulate PrP Sc and may thus be the site of prion replication. Mature B
cells, whether or not they express PrP, are essential for prion accumulation in the
spleen. We have determined that infectivity in spleen is mainly associated with B
and T cells and to a lesser extent with the stromal fraction (which contains FDCs);
strikingly no infectivity was found on circulating lymphocytes of the same mice.
Transgenic PrP knockout mice expressing PrP only on B or on T cells do not
accumulate prions after intraperitoneal inoculation. One explanation for all these
findings is that prions are synthesized in FDCs (the maintenance of which is
known to be dependent on mature B cells) and can be transferred to splenic
lymphocytes, which are in intimate contact with FDCs.

Posters

Abstracts FEB S' 99

s91

5.1 RNA splicing and trafficking

(Su/5.1/001)

HLA-DQ genes expression at transcriptional and post

transcriptional levels in heterozygous cell lines.
M. Cesari, J.J. Hoarau, F.Cadet, M. Pabion

(S~5.1~0~

l~boratoire de Biochtmie et G~n~tique Mol~culaire, Universitd de la R~ttnton.

97715 Saint-Dents, France (DOM)

Regulation of HLA DQ genes transcription is a complex phenomenon

because the allelic polymorphism associated to these genes and their
promoters is a putative source of differential allele expression [1]. Both
transcriptional and post transcriptional regulation could account for the
density of the molecules expressed at cells surface and then for the
specificity of immune response. Different methods have been developed to
evaluate the functional consequences of this polymorphism but at the
present time, no universal method allows to measure neither the steady
state level nor the half-life time of mRNAs species of both DQA1 and
DQB 1 polymorphic genes in heterozygous celt lines.
Here, we propose a potent method, based on relative quantification of RTPCR products that analyses the differential expression of all DQAI or
DQB1 alleles combinations [2]. This new relative RT-PCR quantification
method is extremely simple to perform, consists in a limited number of
steps and is very efficient : it is possible to treat 50 mRNA samples within
two days.
This method has been used to analyse the differential expression of HLA
DQB1 0201/0402 alleles in human heterozygous lymphoblastoid B cell
line. Indeed, nucleotidic sequences of proximal Upstream Regulatory
Region of these alleles exhibit significant differences. DQB1 0402
promoter is able to mediate a transcription strength twice more efficient
than 0201 one. In addition, 0402 mRNA steady state level is also governed
by a remarkable post-transcriptional regulation. Indeed, an important part
(20%) of 0402 primary transcript is derived by alternative splicing into a
short mRNA translated into a non-functional protein. Despite their variable
sequences and lengths, no difference between DQB1 0201, and 0402
mRNAs half-lives was observed in B lymphoblastoid cells.
[1] l~eatyJ.S. et al, Mol Cell Biol., 15, (1995),4771.

Molecular genetics of severe junctional epidermolysis

bullosa with spontaneous amelioration. Y. Gachea, S.
Chavanas ~, C. Bodemerb, C. Prost c, J. Uittod, J.P. Ortonne~, G.
Meneguzzia. lnserm U385, Nice; bNecker, "St Louis Hospitals, Paris,
France; dJefferson Medical College, Philadelphia, PA, USA.

General improvement of the epithelial lesions with aging has been

reported in some patients suffering from junctional epidermolysis bullosa
(JEB). We studied a patient presenting a rapid evolution from severe to mild
PA-JEB associated with reduced expression of integrin ot6134. Genetic
analysis of the gene (IGTB4) encoding integrin [34 revealed compound
heterozygosity for a point mutation in exon 31 underlying severe PA-JEB,
and a point mutation (3986-19T->A) located in the lariat branehpoint
consensus sequence of intron 31. By performing expression of ITGB4
minigenes in vitro, and RT-PCR on mRNA isolated from the patient's
keratinocytes, we demonstrated that under the influence of epigenetic cell
factors, the intronic defect drives synthesis of either wild-type or abnormal
134 mRNAs. In another patient with severe JEB associated with absent
expression of laminin 133, genetic analysis of gene LAMB3 revealed
heterozygosity for the nonsense mutation R635X in exon 14, and a two-base
deletion (1587delAG) in exon 13 resulting in expression of mRNAs with an
in-frame exon skipping of exon 14. With age, a strong reduction of the
blistering tendency correlated with enhanced expression of laminin-5 and
improvement in number and structuration of hemidesmosomes. At the age of
five, RT-PCR amplification of total RNA purified from skin biopsies
demonstrated upregulation of the internally-deleted [33 mRNAs. Biochemical
analysis of the patient's keratinocytes demonstrated synthesis of functional
laminin-5 molecules with a shortened 130-kDa [33 chain missing the Cterminal EGF-like repeat of domain IlI, and the two cysteines of domain II
thought to stabilize the heterotrimer. Our results demonstrate that cellular
factors can reverse the effect of splice site mutations in genes associated
with severe EB. Identification of the mechanisms underlying reversion of the
clinical phenotype is expected to pave the way to new therapeutic
approaches of subsets of these disabling disorders.

[2] CesariM. et al., lmmunogenettcs, (acceptedfor pubhcation).

(Su15.1/003)Rrp8p and Riolp new factors involved in rRNA

processing in Saccharomycescerevisia~
J.P.Gdugne, E.Vanrobays, C. Bousquet-Antonelli, Y. Henry,
M. Caizergues-Ferrer
Laboratoire de btologie molbculaire Eucarvote,'CA~RS, 118 route de
Narbonne, 31062 Toulousve, France.

GAR1 encodes an essential snoRNP protein required in different

aspects of the rRNAs processing pathway in S.cerevisiae: 18S RNA
production and rRNAs pseudourydilation [ 1].
Using synthetic lethal screens with different GAR1 mutant alleles, we
uncovered mutations in potential functional partners of Garlp.
Besides mutations in R O K I ( a putative RNA helicase [2]), G R C 5
(which encodes the ribosomal protein L10) and SNR30, we obtained
mutations in two other functionaly uncharacterized ORFs: YDR83w
(RRP8), YOR119c (RIO1).
R R P 8 encodes a non essential protein with putative methylase motifs.
RIO1 is an essential gene of unknown function [3].
In order to characterize a potential involvement of these proteins in the
biogenesis of the ribosomes we
(1) analysed their cellular localisation
(2) studied the rRNA processing in cells depleted of these
proteins
(3) attempted to identify functional partners either via
genetic methods (synthetic lethal screen) or biochemical approaches:
coiramunoprecipitation with tagged Rrp8p or Rio lp.
[1] Bousquet-AntoneUi et al. EMBO J. 16 (1997) 4770
[2] Venema J. et al.Mol.Cel.BioI. 17 (1997) 3398
[3] Angemayr M. et al. J.Biol.Chem. 272 (1997) 31630

(811/5.1/004)

Targeting of metaliothionein (MT) mRNA is necessary

for its protein Ioealisation and function
M. Levadoux a, P. Mahona, H. Wallace b, J. Beattiea, J. Hesketha
Rowett Research Institute, Aberdeen, AB21 9SB, Scotland, UK
b University of Aberdeen, Aberdeen, AB25 2ZD, Scotland, UK

In eucaryotic cells, some mRNAsare localised in specific cytoplasmic region,

and/or associated with the cytoskeleton. This is due to targeting signals withir
the Yuntranslated region (YUTR) [1]. mRNA localisation provides
mechanism to synthesise proteins close to their site of function, for example.
perinuclear mRNA loealisation could facilitate subsequent import of proteir
into the nucleus. These phenomena may be very important for eel
organisation and development. MTs are a family of low-molecular-mas,
proteins, characterised by their binding capacity for transition metals [2]. The)
have a major role in metal metabolism and may also protect DNA againsl
oxidants [3]. MT has been reported to be found in the nucleus during S-phas~
of the cell-cycle. We transfected CHO cells, which have a low constitutive
level of MT expression, with either the full MT-gene (MTMT) or with M~[
5'UTR and coding region linked to the 3'UTR of glutathione peroxidas~
(MTGSH). Immunoeytochemistry and in situ hybridisation showed both th~
MT mRNA and protein were localised in the perinuclear cytoplasm in the
MTMT cells whereas no distinct localisation was found in the MTGSH cells
Cell fractionation showed that the mR.NA was associated with th~
cytoskeleton in MTMT cells but not in MTGSH cells. The cells were ther
synchronised in S-phase by serum depletion/repletion. After serum repletior
MT was found in the nucleus of MTMT cells but not in the MTGSH cells
These data suggest that perinuclear localisation of MT-I mRNA and it,
association with the cytoskeleton is necessary for MT protein localisation:
particularly for the shuttling of MT protein into the nucleus during S-phase
Functional studies demonstrated that the extent of DNA damage was lower ir
the MTMT than the MTGSH, showing that a loss of MT protein localisatior
led to a reduced protection of the cell against DNA damage. We propos~
therefore, that perinuclear localisation of mRNAs coding for MT is necessar3
for subsequent transport and targeting of proteins into the nucleus and that th~
localisation of the protein within the cell is important for its function. Thi,,
phenomenon could be relevant to a wide range of mammalian nucleaJ
proteins.
[1] Hesketh, J.E., Exp. Cell Kes., 225 (n2), (1996) 219.
[2] Harner, DH., Annu Rev. Biochem., 55, (1986) 913.

s92

(Su/5.1/005)

Abstracts FEBS'99

KIS, a protein kinase with an RNA Recognition Motif.

A. Maucuer, V. Manceau, S, Ozon, A. Sobel

(Su/5.1/006)

INSERM U440, ]FM, 17 rue du For d Mouhn. 75005 Parts, France

We are studying a novel kinase which possesses a characteristic RNA

Recognition Motif (RRM). We initially identified this kinase as a stathmin
partner in the two-hybrid system and thus called it KIS for "Kinase
Interagissant avec la Stathmine" [1]. Stathrnin is a small cytoplasmic
ubiquitous phosphoprotein that is enriched in the nervous system. It is a
major phosphorylation substrate in response to diverse cell stimulations and
we proposed that it acts as a general regulator interacting with multiple
target proteins.
KIS is formed by the juxtaposition of an N-terminal kinase domain that is
not strongly related to any previously characterized one and a C-terminal
RRM containing domain [2]. This unique structure leads us to speculate that
KIS acts as a regulator of gene expression through phosphorylation and
regulation of RNA associated factors. Interestingly the RRM domain is
highly homologous (40% aa identity) with the C-terminal RRM of the
splicing factor U2af, which suggests a functional homology. Our analysis of
KIS expression indicates a particular implication in the developing as well
as the adult nervous system. Furthermore, when over-expressed in cultured
cells, KIS accumulates in the nucleus which might be relevant for its
proposed implication in controlling the metabolism of RNAs.
In order to analyse the catalytic activity of KIS we produced it in bacteria.
recombinant KIS autophosphorylates and phosphorylates stathrnin
on serine residues. It phosphorylates also other classical substrates such as
MBP and synapsin. A systematic approach for identifying KIS substrates in
brain fractions indicates that KIS phosphorylates a few specific substrates.
In vitro, we observe that KIS activity is repressed by histones and the Cterminal domain is necessary to have full autophosphorylation activity.
Phosphorylation might also participate in regulating K I S activity.
In vitro,

Identification of KIS substrates and regulations will shed light on its

implication in the control of protein expression.

UmagasserG, Hear,armM, R ~
H and Berg~P
lnstiUe forBkanxikalAgog Researchof theAttsaim
,~atm'ty of Scias:es

Complex alternative splicing of the GH-V gene in the human testis

The human (h) growth hormone variant (GH-V) gene is expressed during
pregnancy in the syncytiotrophoblast and as shown recently in the normal
human testis, In addition to the classical transcript, encoding for the 22 K major
form, also intron D retaining processed mRNAs (GH-V2) have been described
in both tissues. In the present study we analyzed testicular GH-V RNA
alternative splicing patterns, a major source for GH-variability. We observed
three types of GH-V derived mRNAs by RT-PCR amplification of GH/PL
mRNA, subsequent cloning into appropriate vectors, vector amplification,
restriction-endonuclease map-analysis and double-strand sequencing of GH-V
clones. Apart from the conventional splice-product, encoding classical hGH-V
(22 K, 191 amino acids) and intron D retaining mRNA GH-V2 (230 aa) we
detected an additional GH-V mRNA variant GH-VA4 utilizing a competitive
splice-donor site 4 bp 5' of the conventional exon 4/intron D splice-donor site,
but retaining the genuine intron D/exon 5 splice acceptor site. This mRNA
encodes a putative 25 K protein of 219 aa in length, having the first 124 aa and
thus, two and a half structural alpha-helices in common with hGH-V, hGH-VA4
has lost the N-glycosylation site at Ash 140 of hGH-V, but aquires a novel site
at position 148 as well as a cystein-rich domain in the 65 carboxyl-terminal aa,
potentially involved in multiple disulfid-bridge formation. Tissue specificity
and possible functions for testicular physiology remain to be investigated.
Acknowledgement: This work was supported by the Austrian NB (#6120)

[1J Maucuer at al., Proc. Natl. Acad. Sci. U.S,A., 92, (1995) 3100,
[2~ Maucuer etal., J. Biol. Chem., 272, (1997) 23151.

(Su/5.1/007)

Identification of an alternative spliced variant of human

Hypoxia-Inducible-Factor HIF-Ict.
E. Gothie, D.E. Richard, E. Berra, G. Pages and J. Pouyss6gur
Centre de BtocltHme

C.VRS- [/MR6543 - 06100 Ntce- Franc e

Mammahan cells are able to sense oxygen and regulate a number of genes
in response to hypoxia (EPO, VEGF,...). The transcription factor HIF-1
was identified as an important key component of the hypoxia sensing
system. HIF-1 is a heterodimer of two members of the basic helix-loophelix transcription factor superfamily containing a PAS (Per-ARNT-SIM)
domain : HIF-Ic~ and HIF-II3/ARNT. Recently, an increasing number of
closely related proteins were found (HIF-2a, HIF-3ct, ARNT2, ARNT3).
The several putative dimers could be implicated in multiple signaling
pathways.
During the cloning of the already described H I F - l a subunit, we isolated an
additional eDNA clone. PCR analysis and sequencing reveals that this
clone lacks 127 bp ofexon 14 compared to wild type HIF-Ict.
The rnRNA of this small form is expressed in several human cells lines
(293, HeLa, HepG2), human skin, but apparently not in rodents.
Western blot analysis of the putative form (HiP-let TM) shows a lesser
molecular weight than wild type H I F - l a s~-(~.
When compared in transient transfection assays, both recombinant proteins
HIF-10. TM and H I F - l a 826 were shown to dimerize with HIF-I I3/ARNT and
to activate the VEGF promoter upon hypoxia. However, the shorter
HIF-16t isoform was 3-fold less active than H I F - l a 826, a result consistent
with the lack of the C-terminal transactivation domain. As expected, this
small isoform competes with the endogenous as well as the transfected
wild type HIFI~.
One of the crucial question now is to evaluate whether this spliced variant
protein is expressed in human cells, and if yes, to determine the ratio
3~
[HIF-1ff.736/ HIF-Iff. 7 ], half life, regulation and exact role of this protein
in hypoxic stress.

(Su/5.1/008)

Terminal Stem: A highly Degenerate Motif that Directs

Nuclear Export of Pol III Transcripts in Many Eucaryotes.
C. Gwizdek a, E. Bertrand b, J-C. Lefebvre a A. Doglio a
a Lctboratoire de Vtlwlogie. Facult~ de Mddecine de Nice, France. b lnstitut de
Gdn~tiqlw Mol~cuh+tire de Monpellier. France.

The sequence elements determining the nuclear export of RNA are generally
not well characterized. These elements are however crucial since the
exported RNAs have to be selected from a large population of nuclear RNA.
We have characterized the export determinants of the adenovirus VA1
RNA, which is produced by RNA polymerase III. We found that the
terminal stem of the VAI R N A is necessary and sufficient for its nuclear
export in mammalian cells, yeast and Xenopus oocytes. This stem has the
RNA 5' end bases paired with its 3' end and leaves only Y terminal Us
unpaired. With a detail mutagenesis of this stem, we have shown that some
specific features are required to make it competent for export. Specifically,
the stem should start with the RNA 5' end, should be longer than 12
nucleotides and should have the RNA 3' end following the stem, between 3
to 8 bases away. Remarkably, pollIl RNA of artificial sequences that
present a such terminal stem are efficiently exported as well. This suggest
that any RNA that can fold according to these rules will be efficiently
exported from the nucleus to the cytoplasm+ regardless of its primary
sequence.
In conclusion, we have discovered a highly degenerate motif, the terminal
stem motif, that promotes efficient export of pol III RNA, in many different
eucaryotes. This discovery is of direct practical interest, since the terminal
stem motif can he used to efficiently deliver therapeutic RNA such as
ribozymes, antisenses or aptamers, to the cell cytoplasm.
Key words: nuclear export, pol III transcripts, terminal stem+

Abstracts FEBS'99

(Su/5.1/009)

AROM: a new gene encoded by the opposite strand of

the MCH gene.
L. Borsu, F. Presse, J.L. Nahon. I.P.M.C. - CNRS UPR 41l, 660
route de lucioles, Sophia-Antipolis, 06560 Valbonne, France.

The melanin-concentrating hormone (MCH) is a cyclic peptide synthesizec

predominantly in the lateral hypothalamus of mammals. It is involved in the
regulation of the stress response, food-intake behavior and reproduction. MCIm R N A is i n d u c e d f o l l o w i n g NGF and lithium t r e a t m e n t s ol
pheochromocytoma PC12 cells. In resting cells, the MCH cDNA prob~
hybridizes to several RNA species of 1.6, 3.5 and 4.0 kb while the mature
MCH mRNA (1 kb) is absent. Here, we investigated the molecular structur~
and regulation of these MCH-related RNAs. Northern-blot data indicated tha
these MCH RNA species overlapped the exon-intron of the MCH gene as wel
as the 5' and 3' flanking regions. Sense probes yielded a pattern similar to tha
observed with a double-strand MCH cDNA suggesting that all RNA form:
were in an antisense orientation with respect to MCH mRNA. Combined RT
PCR and Race-PCR investigations revealed the existence of multiple splice
RNA variants, some of these sharing putative exon domains. These RNAs arc
transcribed from a new gene called AROM for Antisense RNA Overlappin~
MCH gene. Sequence analysis of human and mouse genomic clones isolate~
from bacterial artificial chromosome libraries indicated that the AROM gent
contained at least eleven exons and spanned about 36 kb. Several long open
reading frames (ORF) could be predicted from the deduced amino acic
sequence of the largest RNA species (3.5 and 4.0 kb) while the short unsplice~
RNAs (1.6 kb) were devoid of a coding region. The longest ORF~
corresponded to putative proteins named p64, p54 and p50 according to thei
molecular mass. All exhibited sequence identities with a short region o
bacterial helicases but did not share known functional domains, suggestin~
that they formed part of a new protein family. Western blot analysis performec
with antibodies directed against either the N- or C-terminus of p64 indicatec
that AROM proteins are expressed in PC12 cells and various rat organs. Ir
PC12 cells and developing organs, the amounts of MCH and AROM RNA~
were found to be inversely related. Collectively, our findings suggest that the
AROM gene is coordinately expressed with the MCH gene and that i~
producing two types of ant~sense RNAs, one type encoding proteins whose
functions are presently unknown and the second type without ORF that ma 3
be involved in post-transcriptional regulations of MCH expression.

s93

s94

Abstracts FEBS'99

6.1 Structure and function of chromatin

(Su/6.1/010)

EUKARYAL NUCLEOSOME AND HIGH RESOLUTION CRYSTAL

STRUCTURES OF ARCHAEAL HISTONES
A.M. Babu~, K. Decanniere~, K. Sandmanb,2 J.N. Reeve~, U. Hememanna

(Su/6.1/Oll)

"Forchungsg~ppe Krlstallograph~e. MDC pur Molekulare Mediz~n. D13092 Berbn. Germany.

~Oepartraent of Mwrobtology, The Ohto State Umverstty. Columbu~ Ohw 43210. USA

A protein designated HMf (Histone M e t h a n o t h e r m u s f e r v i d u s ) is a h~stone

isolated from the hyperthermophile M. fervidus, a methanogenic archaeon that
grows optimally at 83C. It contains two homologous, small (-7.5 kDa)
polypeptides, HMfA and HMfB. Archaeal histones bind double stranded DNA in
vitro, forming structures that resemble eukaryal nucleosomes[ 1,2]. High resolution
crystal structures of archaeal histones have been determined. Comparative studies
of crystal structures of archaeal histones with the crystal structure of the eukaryal
nucleosome core particle[3] are presented.
Sequence alignment based on structures of histone fold regions of archaeal
histones and core histones suggests that they acquired a common three dimensional
structure in spite of low sequence similarity. The rms deviations of structurally
equivalent superimposed C~ atoms varies from 1.1 to 1.6 A. for histone monomers
and from 1.3 to 1.5 A, for histone dimers. Based on the H3-H4 tetramer as present
in the nucleosome, HMfA and HMfB homotetramers were modelled and binding
properties of the archaeal tetramer models were studied. The main site of predicted
interaction with DNA was the toop region between tx~ and t~3 of the protein. The
loops of the archaeal tetramers are modelled to bind to DNA similar to the
corresponding loops of the H3-H4 tetramer. The shape and charge distribution at
the DNA binding surfaces of archaeal histones resembles core histones. DNA
binding is mainly through hydrophobic interactions. Most of the residues revolved
are conserved in the archaeal histones at similar positions as in the H3-H4
tetramer. High affinity binding of the protein to negatively charged runs or groups
is observed in the HMfA crystal structures (i.e., sulfate group and chloride ion).
Strikingly, the sulfate group binds to the HMfA homodimer at a position exactly
equivalent to the binding site of a DNA backbone phosphodiester group on the
surface of H3-H4 suggesting a closely similar mode of histone-DNA interaction in
the nucleosome and the archaeal nucleosome-like particle.

Fhe nuclear uRrastructure of apical meristematic cells nl

plants shoot growing at low positive temperatures,i
Baranoxa E.N., l-~dyKov ~,.. ~t..411-Ru.~s~aInsn rim. ol
lgrt~ tdtlo'al BlOlet'/l?lf*lllgX', 12 "L~OMoscow. Rttss~a

Changes in nuclear s raacttnes (cba'omatin. nudeolus, extra-nucleola~

RN~P products) were ~tudied in shoot apical meristematic cells ot
winter wheal (Trificur'~ aestivum), winter 13"c (Secalc cereale), :dfalfa
(Medicag,~ sativa) se~.llings under a rnolonged (up to 60 da3sl
influence of Im~ ~eml>elatures above zero ( -2 ._-l"f").
In cell nuclei ~e observed helerochromatin loosening, nficropuil
formalton, a marked neixgork ol RNP-fibfilles and grannies in nmnber
of nuclear I~odies. "Ihese nucleoli si~atiticantb exceeded in size fllose
ol the control seedlings frozen at -?2 ~C and contahled a developed
granular component which totaled a net-~xork-like structure of th~
ribonudeoprotein origin.
I hesc changes in nuclear structures reflect ~he activation of speciiic
lznlclinllS tmdel" |lie inflD.ence ()f low temperatures above zero. the
aclix,tfion bring as,ociated ~ith adaptation w ~etnperalur and
cmali/aliop,

References:
[1] Sandman K, Pereira SL and Reeve JN (1998) CMLS, 54, 1350-1364
12l Reeve JN, Sandman K and Daniels CJ (1997) Cell, 89:999-1002
[3] Luger K, M/ider AW, Richmond RK, Sargent DF and Richmond TJ (1997)
Nature, 389:251-260.
(Su/6.1/012)

Affinity modification of eucariotic chromatin with

reactive derivatives of oli~onacleotide t~d(AC)6
L. Bozhenok. E. Khazina, A Vladimlrova, N.Kobets.,
E. Chernolovskaya., T. Godovikova, V. Vlassov, D Knorre
:Vovo.~tblr~k ln~tltute o/lloorgam~

('henn~tr~'. ,Vovowbtrsk.

Ru.~ta

Affim~' modtficatmn of euka~'otic cells chromatin with d e m a t w e s

of ohgonucleotide pd(AC),,, bearing alkylating and photoreactwe groups
at the 5"-end has been investigated. These reagents targeted to (TGL
repeats form complexes with complementau' DNA sequences m
chromatln and react with DNA and chromatm proteins [1]. Both
compounds with photoreactl~c p-azidoanilinc and 2-mtro-5-azidbenzoxl
group react sequences-specifically via fbnnatlon of the double-stranded
complexes with single-stranded parts of chromatin DNA More efficient
modification of proteins was achieved by the reagent bearing pazidoaniline group [2].
Increase in spermme and sperm~dine concentration that arc known to
facdltate B-DNA to Z - D N A transltmn of (TG), repeats resulted in
stimulatmn of the affinity modification of DNA and chromatme proteins
near (TG). DNA repeats. VJsualizatmn of modificatmn sites b,,
fluorescence mtcroscopy detected the increase of total nuclear signal and
distribution changes of specific fluorescence signals when polyamines
are added. ]'he proteins rno&fied in conditmns which facilitates the B-Z
transition, are symflar ~o the proteins modified m natwe condfl~ons.
These data suggests that q-Z transition can be one of the possible reasons
for single-strand DNA regions accessible for modification existence m
chromatin [2]
Reaction with pd(AC)~, derivative bearing p-azldoamhnc
photoreactive group was used for detection of changes in chromatin
structure Comparison of proteins that reacted m the nucleus from
synchronized cells in S and GI/S phases of cell cycle have demonstrated
differences m the sets of proteins modified [2]
[ I ] Chernotovskaya E L e t al, FEt3S Lett, 303. ( 1992), 269
[2] Bozhenok L N e t al, FEBS Lett., 440. 11998), 38

(Su/6.11013)

Transcriptional inhibitors activate the HIV-1 promoter

c. Cass6, F. Gmnnom, V.T.Nguyen, M.F. Dubols and O. Bensaude
Laboratoire de G~n~tique Mol~culaire,
Ecole Normale Sup6rteure,
46 rue d'Ulm, 75230 Parts Cedex 05, FRANCE.

SUMMARY
Actinomycin
D a n d t x - a m a n i t i n are c o m m o n l y
u s e d to
inhibit transcription.
U n e x p e c t e d l y h o w e v e r , it c o m e s out
that the transcription
o f the h u m a n
immunodeficiency
v i r u s ( H I V - 1 ) l o n g t e r m i n a l r e p e a t s ( L T R ) is a c t i v a t e d in
h u m a n a n d m u r i n e c e l l s e x p o s e d to t h e s e d r u g s w h e r e a s
the
Rous
sarcoma
virus
(RSV)
LTR,
the
human
cytomegalovirus
immediate
e a r l y g e n e ( C M V ) a n d the
HSP70 promoters
are r e p r e s s e d .
T h i s a c t i v a t i o n is a
c h a r a c t e r i s t i c o f the H I V L T R but i n d e p e n d e n t of the N F ~ B
and
TAR
sequences.
In c o i n c i d e n c e ,
the
average
p h o s p h o r y l a t i o n o f the C - t e r m i n a l d o m a i n ( C T D ) f r o m the
l a r g e s t s u b u n i t o f R N A p o l y m e r a s e II is e n h a n c e d in c e l l s
t r e a t e d w i t h a c t i n o m y c i n D and o~-amanitin.
Both the H I V 1 LTR activation
a n d the b u l k C T D p h o s p h o r y l a t i o n
enhancement
are
prevented
by
several
CTD
kinase
inhibitors,
including
5,6-dichloro-1
-8-Dribofuranosylbenzimidazole
(DRB).
T h e e f f i c a c i e s o f the
various compounds
to b l o c k C T D p h o s p h o r y l a t i o n
and
transcription
in
vivo
c o r r e l a t e w i t h t h e i r c a p a c i t i e s to
i n h i b i t the C D K 9 / P I T A L R E
k i n a s e in v i t r o .
H e n c e , this
kinase
is l i k e l y
to c o n t r i b u t e
to the a v e r a g e
CTD
phosphorylation
i n v i v o and to the a c t i v a t i o n of the HIV-1
L T R i n d u c e d by a c t i n o m y c i n D.

Abstracts FEBS'99

(S~6.1~1~

ltistone deacetylase 1 can repress transcription by

binding to the transcription factor Spl
A. Doetzlhofer, H gotheneder, M. Koranda, G. Lagger,
V Kurtev, E. Wintersberger and C. Seiser

s95

(Su/6.1/015)

Amplification of subteiomeric Y'-repeat DNA in hdfl

and hdfl tell mutants ofS. cerevisiae
b
B Fellerhoff ~, F. Eckardt-Schupp ~, and A. A. Friedl ~'
~GSF-institute of Radiobiology, 85758 .Veuherberg, Germany,
bRadioblologtcal Institute, Lu~vlg Ataximtlians University,
80336 AIuntch, Germany

lnst. of 3/lolecular Biology, Universtty ofVtenna, Austrta

HDFI (yKU70), coding for the yeast homolog of the human DNA-PK

Reversible acetylation of hist.ones and corresponding changes of ehromatin

structure are substantial elements of regulation of gene expression. We
investigated if histone d,eaeetylase (HDAC) activity is necessary for the GO
specific transcriptional repression of the growth regulated murine thymidine
kina.,;e (TK) gene It was previously shown that the TK expression is
regullated by E2F and Spl cooperativly binding to the promoter [1] We
find that the rnurirte TK promoter can be activated in GO by the specific
HDAC inhibitor Trichostatin A through its Spl site
Coimmunoprecipitation experiments demonstrate a physical interaction
between Spl and HDAC1. The interaction is direct and requires the
carboxyterminal domain of Sp 1 The expression of the transcription factor
E2F-I strongly reduces HDAC1 binding to Spl and Spl mediated
transcriptional repression. Our data suggest that Spl dependent regulation
of the T E gene involves transcriptional repression via HDAC1. Finally we
prese.nt a model how the TK promoter is regulated at the GO/S transition.
[1] Karlseder et ai. (1996) Mol. Cell. Biol. 16:1659-67

(Su/6.1/016)

Immunological approaches to the study of the human

centromere antigen CENP-A.
J. Figueroa. M. Valdivm
Departanlento de B~oqufimca ) Biolog{a Molecu{ar, Facultad de Cienctas.
UnA ersidad de Cddtz, 11510 Puerto Real, Cddtz, Spain,

We have used an anti-CENP-A peptide serum [1] to affinity purified

the CENP-A kinetochore antigen from human placenta nuclear protein
extracts. Microsequencing of the purified protein band from SDS gels
have demonstrated the feasibility of the purification scheme for a low
abundant human centromere protein. Formaldehyde-crosslinked
placenta nuclei were used to tmmunoprecipitate a human centromere
DNA-protein complex containing CENP-A and ot satellite DNA
among other components to be characterized. Microinjeetion
experiments were used to study the role of CENP-A during the cell
cycle progression. Affinity-purified anti-CENP-A antibodies injected
into the nucleus during the early-rephcation stages of the cell cycle,
caused cells to arrest in mterphase before mitosis. These cells showed
highly condensed small nuclei, a granular cytoplasm with a loss of
their division capability. On the other hand, microinjection of
nocodazole-blocked HeLa cells in mitosis, showed typical punctuate
staining pattern of CENP-A for centromeres during different stages of
mitosis and apparently normal cell division. This was corroborated by
time-lapse imaging microscopy analysis of mid-interphase-injected
cells revealing that they undergo mitosis and divide properly.
However, a significant delay throughout the progression of mitotic
stages was observed. These results suggest that CENP-A Is involved
predominantly m an essential interphase event at the centromere
before mitosis. This may include chromatin assembly at the
kinetochore co-ordinate with late replication of satellite DNA to form
an active centromere. The availability of affinity purified CENP-A
could serve to initiate studies on centromenc chromatin assembly "in
vitro".
[1] : Valdivia, M.M. et al. FEBS Lett 1998 Jan 23:422(1):5-9

subunit KuT0, is known to be involved in the DNA double-strand break

repair by non-homologous end joining. Mutants of this gene have shortened
telomeres and are sensitive towards elevated temperature. In subcultivation
experiments we observed that the length of the telomeric repeat tract is
shortened by about 200 bp during the first 30 generations aiter inactivation
of HDf7 and remaining stable furtheron. The plating efficiency of the mutant
at 30 remained unaffected by the telomere shortening for more than 120
generations. When incubated at 37 , however, hdfl mutants exhibited a
dramatically reduced plating efficiency Analysis of temperature resistant
survivors showed that all of them had amplified and redistributed their
subtelomeric Y' DNA in a manner reminiscent of the alternative
chromosome end stabilization observed in mutants deficient for the
telomerase pathway. This finding suggests that thermosensitivity in hdfl
mutants is related to some temperature-induced alteration at their telomeres.
tell mutants, whose telomere length is similar to that of hdfl mutants, were
not found to be temperature sensitive, and subclones grown at 37 did not
show Y' amplification, thus showing that short telomeres pet" se do not cause
thermosensitivity. The reduction of the telomere length is additive in hdfl
tell double mutants, thus indicating that both genes act in separate pathways
affecting telomere length. Interestingly, in hdfl tell double mutants strong
amplification of Y' DNA was already evident after growth at 30 , thus
showing that inactivation of the TELl-dependent pathway deteriorates the
hdfl phenotype.
We propose that telomere function in hdfl mutants is readily lost upon
introduction of further, telomere-associated alterations, and that it can be
restored by amplification of Y' DNA

(Su/6.1/O17)

Prothymosin a modulates the interaction of

histone H! with hromatin
Z Karetsou.R. Sandaltzopoulos*.O. Tsolas, P.B. Becker*. T, Papamarcaki
Umversitv ofloanmna. Greece, EMBL, Hetdelberg, Germany

Prothymosin u (ProTc0 is amongst the most abundant proteins in a

mammalian nucleus at amounts matching those of core histones. Yet, the
function of this conserved, small (12.5 kDa) and acidic protein remains
elusive. In our search for its cellular partners, we have demonstrated an
interaction between ProTa and linker histone H1 which is dependent on
concentration and temperature [1]. Further evidence for the physiological
relevance of this interaction was obtained by immunoisolation of a histone
H 1-ProTGt complex fi'om crude cell extracts [2].
We have analysed the effects of ProTa on the interaction of histone HI with
chromatin using a cell-free system for chromatin reconstitution under
physiological conditions on bead-immobilised DNA [3]. ProTct failed to
interact stably with chromatin in the presence or absence of HI. If HI is
titrated into a chromatin assembly reaction, the nucleosome repeat length
(NRL) is increased to the physiological value of approximately 200 bp, but
further addition of H 1 results in even wider nucleosome spacing with NRLs
reaching 220 bp. Remarkably. the presence of ProTu during chromatin
assembly did not aft~ct the incorporation of sufficient HI to increase the
NRL to the physiological value of roughly 200 bp, instead, it prevented the
association of excess HI. In a complementary experiment. ProTc~ was able
to detach a fraction of H l from chromatin, if excess HI (over the amount
required to establish the physiological spacing) bad been incorporated. By
contrast, the amount of Hi required to increase the NRL to 200 bp resisted
extraction by ProTu. Importantly. despite the lower affinity of HI lbr naked
DNA compared to chromatin, ProTGt was not able to release H1 from naked
DNA. These results suggest the presence of at least two different interaction
modes of ill with chromatin that can be distinguished by their sensitivity to
ProTet extraction. The properties of ProTct suggest a role in fine-tuning the
stoichiometry and/or mode of interaction of H 1 in chromatin.
References
1. Papamarcaki, T., Tsolas, O., FEBS Lett. 345 (1994), 71
2. Karetsou, Z., et al., Nucleic Acids Res. 26 (1998), 3 l 11
3. Sandaltzopoulos, R., et al., EMBO J. 13 (1994), 373

s96

(Su/6.1/018)

Abstracts FEBS'99

Role of the histone N-terminal tails in nueleosome

dynamics: In vitro analysis.
V. Morales, F. Fabre, H Richard-Foy and A. Hamiche

(Su/6.1/020)

(Su/6.1/020)

Divergent promoters - identical regulation: chromatin

structure as unifying theme? Thymidine kinase as example
M. Sauer, E Wintersberger
Inst. o f 34olecular Biology, Umv. o f Vienna, Dr Bohr-Gasse 9/2,
1030 Vtenna, Austria

The gene of cytoplasmic thymidine kinase (TK), an enzyme necessary for

the fine regulation of the pool ofthymidine precursors, is conserved between
hamster, human mouse and rat. Expression is strictly growth regulated in all
of these species on a variety of levels but especially by transcriptional
regulation. Most of the upstream sequence is conserved between these
species, but a closer look reveals a short stretch with remarkable differences.
Strikingly, the elements identified as functional for the promoters are located
within this divergent region and they are not Conserved between these
species. The detailed mechanism of transcriptional growth regulation seems
therefore to be different
In the murine promoter single binding sites for Spl and E2F are responsible
,

for the growth regulated expression

of the gene 12 . Expression
of the rat TK
gene is solely regulated by Spl. Both promoters lack a TATA box. The
human TK promoter contains a TATA box, binding sites for E2F and Sp 1, as
well as two CCAAT boxes. All of them seem to contribute to the regulation
of the promote?. The hamster promoter finally includes a CCAAT box, a
TATA box and binding sites for Spl but no E2F binding site (Smensen,
P.W., pers. comm.).
A model which could explain the similar regulation of these divergent
promoters, involving reversible chromatin modification, will be presented.

[1] Ogris, E ~ al., J. Virol. 67, (1993) 1765

[2] l~arlseder, J. et al. Mol. Cell. Biol. 16 (1996), 1659
[3] Tommasi, S. et al. J. Biol. Chem. 272 (1997), 30483

function
Univ.

Regulation
in vivo.
of

Texas

of ehromatin-mediated
L. Rastelli and S.
M.

D.

Anderson

Cancer

Center, 1515 Holcombe Blvd., Box 316, Houston, TX

77030; s-mail: majumder@mdanderson.org

LB?vfFI1BCG.CNRS, 118 route de Narbonne 31062 Toulouse France

It has recently been proposed that the histone (H3-H4)2 tetramer undergoes
structural changes, which allow the particle to accommodate both negatively
and positively constrained DNA [1]. We have demonstrated in vitro, using a
single nucleoprotein particle assembled on a topologically constrained DNA
minicircle [2], that adducts on the single histone H3 cysteine, which have no
effect on the structure of the nucleosome, limit the structural transitions that
the (H3-H4)2 tetrameric nucleoprotein particle can undergo [3] We have
used the same approach to investigate the role of the historic tails in the
dynamics of the nucleosome. We have either cleaved the tails by treating the
particles with clostripain or have deleted them by mutating the genes and
expressing the histones in E. coil Removal of the histone tails did not affect
the structure of the nucleosome. In contrast, in the absence of H2A-H2B
removal of histones H3 and H4 tails dramatically affected the structure of
the tetrameric nucleoprotein particle such that the DNA now adopted a
relaxed conformation. The effect of histone tail acetylation was also
investigated, either using hyperacetylated histones isolated from cells
treated with butyrate or trichostatin A, or histones treated in vitro with
recombinant histone acetyltransferases. Hyperacetylation of histone H3 and
H4 tails does not affect the structure of the nucleosome. In contrast it
induced significant change in the structure of the tetrameric nucleoprotein
particle. These results provide new insights on the possible role of histone
acetylation/deacetylation in transcription control.
[1] Hamiche et al., Proc. Natl. Acad. Sci. USA, 93, (1996), 7588.
[2] Goulet et al. Nucleic Acids Res., 15, (1987), 2803.
[3] Hamiche et al., J. Biol. Chem, 273, (1998), 9261.

enhancer
Majumder.

In mammals, enhancers are believed to stimulate transcription

from RNA polymerase I1 promoters primarily by relieving
their chromatin-mediated repression. Interactions responsible
for enhancer function are developmentally acquired. Factors
responsible for this repression are not present in the paternal
pronuclei of 1-cell embryos, m a k i n g them i m p e r v i o u s to
e n h a n c e r function. However, a l t h o u g h s u c h chromatinmediated repression is observed in oocytes, maternal pronuclei
of 1-cell embryos, and the 2-cell embryos, the enhancer
function first appears in 2-cell embryos, a stage in development
that c o r r e s p o n d s to the b e g i n n i n g of major zygotic gene
expression (ZGE). The lack of enhancer function prior to 2-cell
stage is also not d u e to the absence of specific activation
proteins, but appears to be due to the absence of an essential
coactivator activity. The coactivator activity first appear in 2cell embryos, and is required for enhancers driven by different
classes of transcription factors. The absence of the coactivator
activity and the corresponding enhancer function appears to be
u n i q u e to oocytes and 1-cell embryos, s u g g e s t i n g that it
provides a safeguard against p r e m a t u r e activation of genes
prior to ZGE. Finally, microinjection of purified chromatin
c o m p o n e n t s in m o u s e preimplantation embryos reveal that
histone H1 is not essential for chromatin-mediated repression
and subsequent relief of the repression by enhancer function.
Majumder et al., (1993) EMBO J. 12, 1131-1140; Majumder et
al. (1997) EMBO I. 16, 1721-31; Lawinger et al. (1999) J. Biol.
Chem. 274, 8002-11.

(Su/6.1/021)

Setl & Mec3 interact in DNA repair and telomere functions

V.SchramkeI ,Y.Cordal,M.LongheseZ,V.Brevet3,E.Gilson3&V.G61il
I LISM CNRS 31,chemin J Aiguier 13402 Marsedle France
2 Universita degh Studt dt Mdano Via Celorta 26 20133 Milano haly
3 LBCM ENS 46, allde d'ltahe 69364 Lyon France

The protein Setl from Saccharomyces cerevisiae has been identified by the
presence of a specific domain, the SET domain, common to many chromatin
associated proteins. It has been shown, that deletion of Setlp alleviates
telomeric proximal effects (TPE) (1). We found, that Setlp physically
interacts with Mec3p through its SET domain (2). Mec3p is a checkpoint
protein required for delay of the cell cycle after DNA damage. Our results
show, that the deletion of SET1 increases the viability after DNA damage of
mec3 mutants but also of other checkpoint mutants (radg, radl7, rad24), in a
process mostly independent of Rad53p, a signalling kinase involved in
checkpoint control. Nevertheless, SET1 deletions do not restore any of the
three DNA-damage checkpoint responses regulated by Mec3p. In addition,
we found that, contrary to the deletion of SETI, deletion of MEC3 enhances
TPE and even attenuates the silencing defect of a set1 mutant. Moreover,
restoration of TPE in a set1 mutant by overexpression of the isolated SET
domain requires Mec3p. Finally, unlike set1 mutants which show slight
telomere shortening, mec3 mutants have elongated telomeres, suggesting that
Mec3p negatively regulates telomere length. Our results suggest that Setl and
Mec3 have antagonistic effects on two processes regulated by chromatin
structure: repression of telomere-proximal transcription and telomere length
maintenance. Recently, it has been shown that SET domain proteins can
interact with phosphatases and antiphosphatases and that this interaction is
involved in the regulation of cell differentiation (3). By analogy, Set1 and
Mec3 may exert their antagonistic functions through the phosphorylation of
until now undetermined, common targets.

(1) Nislow C. et al. MoL Biol. Cell,, 8, (1997), p. 2421

(2) Corda Y., Schramke V. et al. Nature Genet., 21, (1999), p.204
(3) Cui X. et al.,Nature Genet., 18, (1998), p.331

Abstracts FEBS'99

(Su/6.1/022)

Molecular characterization of mouse histone

deacetylase 1
C. Seiser, J. Taplick, G. Lagger and V. Kurtev
Institute of Molecular Biology, Vienna Biocenter, Vienna, Austria

Acetylation of conserved lysine residues of core histones is believed

to modulate the interaction between DNA and the N-terminal histone
tails. Histone deacetylation leads to chromatin condensation and
reduced accessibility of specific DNA sequences for high molecular
weight protein complexes like the RNA polymerases. Recently it was
shown that several eukaryotic transcription factors are able to recruit
histone deacetylases (HDACs) to specific promoters. Previously we
have identified mouse histone deacetylase 1 as interleukin 2inducible gene in T ceils [1]. Here, we analyzed the domains of
HDAC1 that are important for nuclear localization of the protein and
for the binding of different HDAC 1-interacting proteins.
[I]

Bartl, S., Taplick, J., Lagger, G., Khier,H., Kuchler,K. and

Seiser, C. (1997) Mol. Cell. Biol. 17:5033-5043

(Su/6.1/024)

Change in nuclear texture and DNase I sensitivity in

mulfidrug-resistant human ovarian IGROVI cells
S }'atoll]t, ('. /'rente.~aux..I.l.htfer
f )ltt(; Ilel)Ll\ !i.i2063, I!'R53, t,'f;R l)harmaoe, 5]/oO)~elms t;)'ance

We showed previously Ihat muitidrug-resistant cancer cells displayed nuclear

texture changes as assessed by image analysis [1] In this work, two tumoral
cell line ;ariants were studied" the human ovarian carcinoma celt line
IGROV1 and the muhidrug-resistant subline OVI-VCR selected with
vincfistine. We confirmed by immunocytochemistry, RT-PCR~ and Southern
blot that OVI-VCR cells overexpress P-glycoprotein and its corresponding
mRNA without mdrl gene DNA amplification. Cell smears of these two celt
popuIatmns ~sere stained wtth Feulgen method and analysed b3 image
cytometry. As compared to drag-sensitive cells. OVt-VCR display a chromatin
global decondensation as assessed by textural features analysis. Several studies
suggest lhat transcriptionally or potentially actise genes exist in an altered
conformatim~ in nuclear interphase chromatin In order to correlate this
decondensation w~th ahewations m chromatin stnaclure, DNase I was used as a
probe to preferentially digest potentially active genes in chromatin Genomic
I)NA was extracted from both celt lines nuclei after treatement wtth various
concentrations of DNase I. OVI-VCR cells DNA displayed an increased
(about 5 fold) DNase 1 sensitivity, suggesting an increased chromatin
accessibility To investigate further the presence of putative DNase 1
hypersensitive sites IHS) in the vicinity of the mdrl gene promoter~ genomic
DNA from both cell lines was analysed by Southern blot alter digestion with
DNase 1 and restriction enzymes (Hind ill, Barn HI, EcoR]~ and Kpn[).
According to the mdrl probe (l 3kb) and enzymes used, no change in DNase
HS sites could be visualised
[I] Dufer el al, Int J, Cancer. 60,(1995), 108
This work was supported by Comltes I)epartementaux de la Haute Marne et de
I'Aube de la 15gue Fran~;aise contre le Cancer

s97

(Su/6.1/023)

Dynamics of Chromatin Domain Organization in

Xenopus Early Development
Yegor Vassetzky, Alan Hair, and Marcel M6chali
Genome Dynamics and Development, Institut de Ggngtique
Humaine, CNRS, 141, rue de la Cardonille, 34396 Montpellier

A dynamic change in the organization of different gene domains

transcribed by RNA polymerases I, II, or III occurs during the progression
from quiescent (pre-MBT) to active (post-MBT) embryos during Xenopus
development. Both for the rDNA domain, c-myc, and the somatic 5S gene,
a transition from random to site-specific anchorage to the nuclear matrix
occurs when chromatin domains become active. In parallel with this
specification of the nuclear matrix attachment sites, the average size of DNA
loop domains containing the 5S gene increases from 5 to 20 Kbp during the
mid-blastula transition.
LM-PCR-mediated genomic footprinting in the 5S domain during
development revealed that a subpopulation of the genes became specifically
attached to the nuclear matrix and contains the hallmarks of a determined
chromatin after the mid-blastula transition. In contrast, the same analysis
performed on the total 5S gene population does not permit to reveal a
specific organization.
These data emphasize the role of nuclear architecture in regulated
gene expression during development, and also provide a new method to
reveal determined chromosomal territories. The mechanisms underlying
such specification and their involvement in the establishment of active
chromosomal domains during the determination of cell lines in the embryo
will be discussed in detail.

s98

Abstracts FEBS'99

9.1 Molecular mechanisms of secretion

(Su/9.1/025)

An uncorrqJetitivemechanism for Ihe inhibition of Sec7 domain.

catalyzed nucleotide exchange on ARF1 by B~.feldin A
B An~'a~', APoyrod~, S Rc/~eaua J Adg~b,J Oaerfi~, (1,Jacksonb

(Su/9.1/026)

alPMC-CNRS, 660 route des Luctoles, 06560 Valbonne, France. bSBGM,

CEA/Saclay, 91191 and CLEBS-CNRS, 91198 Gif-sur- Yvette, France

Sec7 domains are all helical domains of 200 amino acids, which
catalyze GDP to GTP exchange on ARF, a small GTP-binding protein
involved in vesicular trafficking. Sec7 domains feature a hydrophobic groove,
which is the binding site for ARF, and an essentml glutamate residue which
destabilizes GDP and Mg 2 on ARFI. The exchange activity of some
members of the Sec7 family, such as the yeast proteins Geal/2p is inhibited
by the fungal metabolite Brefeldin A (BFA). Other members of this family,
such as the mammalian protein ARNO, are BFA resistant. In yeast, a genetic
approach has shown that the BFA sensitivity depends on some residues of the
Sec7 domain [1, see also the abstract of Peyroche et al.]. These residues
cluster in a small patch, which overlaps the binding site for ARF. For
instance, replacing the adjacent residues F190-AI91 in the hydrophobic
groove of ARNO-Sec7 by the cognate residues of Gealp (an Y-S pair),
renders ARNO-Sec7 sensitive to BFA in vitro and in vivo.
The BFA sensitivity of the F190Y-A 191S mutated form of ARNOSec7 was studied in vitro, using [A17]ARF1-GDP, a soluble truncated form
of ARF1, as a substrate. Preincubation of BFA for few seconds with both
[F 190Y-A 191S]ARNO-Sec7 and [A17]ARF I-GDP was reqmred for
inhibition. Furthermore, increasing the [A17]ARFI-GDP concentration,
increased BFA inhibition of SecT-catalyzed nucleotide exchange. Thus,
despate the fact that the Y-S pair of See7 domains belongs to the binding site
for ARF, BFA does not act as a competitive inhibitor. Further analysis of the
kinetics of GDP/GTP exchange at various [A17]ARFI-GDP and BFA
concentrations shows that BFA acts as an uncompetitive inhibitor, freezing
[A17]ARFI and the Sec7 domain in an abortive complex, m which GDP/GTP
exchange is dramatically slown down. Gel-filtration analysis using [3H]GDP
labeled [A17]ARFI-GDP shows that this abortive complex contains GDP. We
suggest that B F A is trapped at the interface between ARF1-GDP and the Sec7
domain, and hinders the conformational change in the switch regions of
ARF1, that accompanies the release of GDP.
[ 1] Peyroche et al. Molecular Cell, in press (issue of March 1999)

(Su/9.1/027)

Membrane fusion machinery is involved in post transGolgi cleavage of Semliki Forest Virus precursor p62
A.M. Band, J M~tta, L K~_ri~iinen, E Kuismanen
Dtvision of Btochemtstrv, Umversity of Helsinkl, Finland

Many biologically active proteins, peptide hormones and viral proteins are
initially synthesized as large inactive precursors which are activated by
proteolytic cleavage. Semliki Forest Virus (SFV) spike glycoprotein precursor
p62 is cleaved to E2 and E3 by the membrane associated calcium dependent
serine endoprotease furin. When BHK 21 cells are infected by SFV, and p62
precursor is accumulated to trans-Golgi network (TGN) at 20C there is no
cleavage of p62. By increasing the temperature to 37C the transport block is
released and cleavage occurs. We have previously demonstrated that exit from
TGN is needed for the cleavage of p62 and that there is communication
between early endosomes and this post TGN compartment [ 1]. We have now
investigated the possible involvement of membrane fusion machinery
in the cleavage of p62 by using streptolysin O (SLO)-permeabilized BHK 21
cells. The lost cytosol was replaced by using rat brain cytosolic extract
N-ethylmaleimide (NEM)-sensitive fusion protein (NSF) is inactivated by
alkylating agent NEM. Treating the cells and the cytosol with 1 mM NEM
caused the inhibition of the cleavage of p62 (control 26.9_+2.7%; I mM NEM
2.5_+1.3%, mean _+SE, n=3-4 observations). A recombinant NSF stimulated
the cleavage of p62 in the SLO-permeabilized cells (control 26.6_+0.7%,
I~M NSF 37.0_+ 1 0%, mean_+SE, n=4 observations, p<0 01 versus contol,
t-test) Soluble NSF attachment protein (SNAP) binds and activates NSF. We
used c~SNAP (L294A) mutant, which binds but does not activate NSF. ~SNAP
mutant inhibited the cleavage of p62 in SLO-permeabilized BI-[K 21 cells
(control 26.3_+0.7%; 200 lJg/ml c~SNAP mutant 5 1_+09%, mean_+SE,
n=3 observations) Rabs are the small GTPases which are implicated
in the regulation of the membrane fusion. Rab GDP-dissociation inhibitor
(GDI) binds the GDP-bound, inactive form of Rab An excess of recombinant
GDI caused inhibition of the cleavage of p62 in the SLO-permeabilized BHK
21 cells (control 100%, 5 ~M GDI 48.9_+5 7%, mean_+SE,n=3 experiments).
These results suggest that membrane fusion machinery is involved in
the cleavage of p62 in the post TGN site.
References: [1] Sariola et al., J.Cell Sei., 108, (1995) 2465.

FUSION BETWEEN SECRETORY VESICLES AND

INSIDE-OUT PLASMA MEMBRANE VESICLES
L Arrastua, A. F. Quincoces, U Ugalde
Biochemtst~ Unit, Facul~ of Chemistry, Umverst~. of the Basque
Country, P.O.BOX 1072, E-20080 San Sebastian, Spare

We have focused our effort in the design of a in vitro system to

study the fusion mechanism between secretory vesicles and inside-out
plasma membrane vesicles from Saccharomyces cerevisiae.
We have localised components of the fusion machinery in our
subcellular fractions, i.e., Snc lp and Sso lp on secretory vesicles and Sso
lp on inside-out plasma membrane vesicles [1] Other cytosolic
components, like See 18p and Sec 17p, have also been detected in these
fractions.
The fusion reaction is followed by the increase in fluorescence
given by the dilution of the lipidic fluorescent marker Octadeeyl
Rhodamine B [2] which has been incorporated previously in the secretory
vesicles membrane.
Our process is temperature and cation dependent and is
independent of externally added ATP. This process is inhibited by specific
antibodies against v / t- SNAREs and by trypsin. Taken all these results
together we postulate that our process is a fusion process between
secretory vesicles and inside-out plasma membrane vesicles.

[1] Pfeffer S.R., Annu. Dev. Cell Dev. Biol., 12, (1996), 441
[2] Hoekstra et al., Biochemistry, 23, (1984), 5675.

(Su/9.1/028)

Characterization of glycosylation of a vesicular

monoamine transporter
C.Bedet, J.P. Henry, B. Gasnier
CNRS UPR 1929-Biologle de la Sdcr~tion /IBPC
13 rue P et M Curte, 75005 Paris, France

Catecholamines and serotonin are loaded into secretory vesicles by a

H+/monoamine antiport catalyzed by a Vesicular Monoamine Transporter
(VMAT). The current topological model of VMAT predicts 12
transmembrane domains (TM) with a large, N-glycosylated, intravesicular
'loop' connecting TM1 and TM2. To investigate the role of Nglycosylations, the three consensus sites N-X-S/T present in the first loop of
the bovine VMAT2 isoform were suppressed, individually or
simultaneously, by mutation of the asparagine codon to glutamine. The
glycosylation state of the mutants expressed in COS-7 cells was analyzed by
Western Blotting and their activity was determined by ligand binding and
substrate uptake assays. Partial mutagenesis revealed that each of the 3 sites
was actually glycosylated and a treatment with glycopeptidase F
demonstrated the absence of N-glycosylation in the triple mutant. Identical
affinities for ligand and substrate were observed for the wild-type and triple
mutant, showing that N-glycosylation is not necessary for VMAT activity.
However, these analysis revealed that the triple mutant was much less
expressed, suggesting that N-glycosylation is critical for the stability or the
rate of synthesis of VMAT. A careful analysis of the single and double
mutants revealed site-specific differences in electrophoretic mobility,
suggesting different processing of the three N-glycans. To investigate such
differences, the mutants were treated with neuraminidase. Surprisingly, the
triple mutant was sensitive to the enzyme, suggesting that VMAT might be Oglycosylated. Th~s hypothesis raises new questions about the role of
glycosylation in VMAT.

Abstracts

s99

FEBS'99

(Su/9.1/029)

Secretion of laecase from the wood-degrading fungus

Trametes versicolor in yeast expression systems

(SuP).I/030)

P. Cassland, S. Larsson, L.J. J6nsson

Department of Biochemistry, Physiology and Nutrition

The Norwegian School of Veterina O, Sctence, Oslo, Norway

Applied Mtcrobtology, Lurid Universlty/Lund lnstttute of Technology,

P.O Box 124, SE-22l O0 Lund, Sweden

The copper-containing blue oxidase laccase catalyses the reduction of molecular

oxygen to water and the one-electron oxidation of organic substrates, primarily
phenolics, to radicals. Laccases are produced by many plants and fungi. The
white-rot basidiomycete Trametes versicolor secretes several isoforms of
laccase, presumably involved in the degradation of the aromatic plant polymer
lignin. Laccase is of practical interest for use in enzymatic processes in the
conversion of hgnocellulosics. Potential applications include the catalysis of
lignin degradation in the pulp and paper manufacture and detoxafication of
phenolic fermentation mhibitors in the conversion of lignocelluloslcs to fuel
ethanol [1]. Heterologous expression of laccase in yeasts is of interest for
producing recombinant laccase and for construction of inhibitor-resistant yeast
strains. Laccase-encoding cDNAs were isolated from Z versicolor and used
for expression in the yeasts Pichia pastoris [2] and Saccharomyces cerevisiae .
Active recombinant laccase was secreted both by P. pastoris, using the AOXI
promoter for expression, and by S. cerevisiae, using the PGK promoter.
Factors affecting the level of active secreted laccase included choice of cDNA,
choice of host strain, the use of the original laccase secretion signal or of the S.
cerevisiae alpha-factor secretion signal and cultivation conditions such as pH,
aeration and temperature. Recent results indicate that simultaneous
overexpression of proteins in the secretory pathway is an alternative approach
to increase the level of active heterologous laccase.
[l] J6nsson LJ et al., Appl Microbiol Biotecbnol, 49, (1998) 691
[2] JOnsson LJ et al., Curr Genet, 32, (1997) 425

(Su/O.1/031)

Stabilization of the SNARE complex by GTP-bound Rab3a

C. Desnos, E. Lejeune, O. Martinez, M.P. Chich. J.P. Henry
and F. Darchen

The rapid growth of pigs in the perinatal period puts a heavy demand on their
hematopoietic system. At birth, the hormone regulating erythropoiesis,
erythropoietin (EPO), is barely detectable in plasma. After birth, the activity
increases greatly the first 1-2 days, wbereafter it drops rapidly [1]. These
changes in Epo activity are not easily explained on basis of current concepts
on regulation of Epo secretion.
in piglets not supplemented with iron, a serious anemia develops
during the first 1-2 weeks after birth. InJection of iron (180 mg
subcutaneously) in two weeks oId piglets results in a marked increase in Epo
activity [2]. A similar response to iron has been observed in mice. We have
now demonstrated that the response to iron is dependent on the hemoglobin
(Hb) status of the animal. In two weeks old piglets with Hb below 60 g/l, the
Epo activity in plasma on an average increased 5-fold during the first 24 h
after injection, whereas the corresponding changes in iron treated piglets
with Hb above 60 g/l and in untreated controls were insignificant.
In adults, there exists a correlation between Hb and the Epo activity
in plasma. In neonatal mice and humans, significant correlations between Hb
and plasma Epo has not been found. Such observations have led to the idea
that other factors than tissue availibllity of oxygen might be important in the
regulation of Epo in the early postnatal period. In the two week old piglets
examined in the present study, however, a high correlation between log
plasma Epo and Hb was found (r2--0.75, n=50, p<0.001 ).
Bleeding of prenatal piglets (-5 days) greatly increased the level of
EPO mRNA in liver, while the kidneys responded moderately. These
findings are in contrast to observations in adult humans and mice where the
kidneys are responsible for the EPO response to an anemic stress. Data on
the relative contribution of the liver and kidneys to the EPO activity in
normal and bled perinatal piglets will be presented.
References:
1. Sjaastad, O.V. et al. Acta Vet. Scand. 33 (1992) 249.
2. Sjaastad, O.V. et al. Acta Vet. Scand. 37 (1996) I33.
(Su/0.1/032)

[1] Johannes et al., J. Cell Sci., 109 (1996) 2875-2884

Identification of critical motives in the proneurotensin for its processing and regulated secretion
S.FELICIANGELI. P.KITABGI, and J-N BIDARD lnstaut de
Pharntacologw Moldcuhure et Cellulat~ e, CNRS, Valbotme, France.

CNRS UPR 1929, IBPC, 13 rue P et M. Curw. 75005 Paris, Frame

Rab3 is a monomeric GTP-binding protein implicated in the control of

regulated exocytosis in neuroendocrine cells. It was previously reported
that clostridlal neurotoxins (TeTx and BotA) cleave their targets (VAMP
and SNAP-25) less efficiently after injection of a GTPase-deficient Rab3
protein (Rab3Q81L) in cholinergic Aplysia neurons, suggesting that
Rab3-GTP stabilized the SNARE complex [1]. In order to further
investigate this point, clonal PC12 cell lines stably expressing different
Rab3a mutated proteins were established. Since Rab3a mutated protein
was c-myc tagged, it was easily distinguished from the endogenous Rab3a
by immunofluoresence or western-blot. In PC12 cells expressing
Rab3aQ81L, a 50% inhibition of monoamine release was observed
compared to control cells, in agreement with transient expression
experiments. Assembled SNARE complexes were biochemically detected
by SDS-PAGE and sucrose gradient. We observed two major SDSresistant complexes (60 kDa and 100 kDa) recognized specifically by antisyntaxin, anti-SNAP-25 and anti-VAMP antibodies. The amount of
complex increased after N-ethylmaleimide or NGF treatment. In resting
conditions, a slight decrease of the amount of complex was observed in
Rab3aQ81L-expressing cells compared to control cells. However, upon
stimulation of the secretory activity (2 minutes in high KC1), the amount
of complex decreased in control cells but remained inchanged in
Rab3aQ81L-expressing cells. No physical interaction between Rab3a and
the components of the SNARE complex could be detected by coimmunoprecipitation. These results suggest that GTP-bound Rab3a
stabilizes the SNARE complex by preventing its dissociation during
exocytosis. Therefore, Rab3 seems to play a role downstream of the
complex assembly.

Studies on erythropoietin in perinatal pigs

R.B. David, I. Harbitz, A.K. Blom, H.R. H0gfisen,
T. Framstad, O.V. Sjaastad

The mechanisms by which prohormone precursors are sorted to the

regulated secretory pathway (RSP) m neuroendocnne cells remain unclear,
despite all previous investigations. Here, we attempted to identify the sorting
signal(s) within the common neurotensm (NT)/neuromedln N (NN)
precursor (proNT/NN), the structure of which is depicted below.
proNT/NN

I
I

c
pro-region

KR KR RRKR

IIIIIIIIV/AINI I

n__.l I
U~l
NN NT tail

Several point and deletmn proNT/NN mutants were constructed,

transiently expressed an the endocrine insulinoma Rip-tag cell line, and the
processing and secretion of the precursor mutants was examined. Our data
show that the N-terminal proregion, the disulfide bond within it and the Cterminal tail played no role in the sorting and processing of proNT/NN in the
RSP. In contrast, a mutant precursor deleted after the NN sequence was not
processed, did not exhibit regulated secretion, and was constitutively released
at a high rate. Mutation or deletion of one or several of the four dibasic
sequences found in the C-terminal region of proNT/NN led to the following
results: As long as at least one dibasic site was not mutated, the precursor
was processed and its maturation products were correctly sorted to the RSP.
In contrast, removal of all the dibasics prevented sorting to the RSP.
However, constitutive secretion was not enhanced m this case. We conclude
that the KR-neurotensin motif is essential to target proNT/NN to the RSP and
that dibasic sequence(s) within the C-terminal region of proNT/NN are
reqmred for the processing and storage of the precursor in regulated secretory
vesicles.

s 100

(Su/9.1/033)

Abstracts F E B S ' 9 9

A role for a PDZ protein in the early secretory pathway for

the targeting of proTGF-c~ to the cell surface
J. Fernfindez-Larrea, A. Mer[os-Su~irez,J. Urefia, M Borrell,
J. Baselga, J. Arnbas

(Su/9.1/034)

I,aboratort de Re, erda Onl ol~gtca, Hospttal General Unlversttart Uall

d'llebron, Ps.e I'all d'lh, bron It9-129, Barcelona 08035, 5~Jam

The correct localization of proTGF-a, a prototypical type I trans-membrane

cell surface growth factor, has been found to rely in a strikingly simple
structural determinant: the presence of a very C-terminal cytoplasmic valine.
proTGF-c~ C-terminal mutants are not transported to the cell surface but are
retained in an early secretory compartment, probably the ER [1]. Using a
two-hybrid screen, we identified two TACIPs (proTGF-A lpha _Cytoplasmic
domain_Interacting P_roteins), referred to as 1 and 18, putatively involved in
the targeting of proTGF- a to the cell surface. Both TACIPs show a lack of
interaction with some proTGF-c~ C-terminal mutants that do not reach the
cell surface, and were identified as the PDZ proteins alpha AI Syntrophin
and Syntenin/mda-9 [~], respectively. Using a panel of proTGF-a point
mutants, we determined that the specificity of the protein that possibly
mediates the transport of proTGF-a coincides only with that of
Syntenin/mda-9/TACIP18. Furthermore, we show that the cytoplasmic
domain of Syndecan-2, another transmembrane molecule known to bind
TACIP18, supports the correct trafficking of proTGF- a/Syndecan chimeras.
This result is likely due to the capability of the cytoplasmic tail of Syndecan
to bind TACIP18 since point mutations that disrupt this interaction also
disrupt the normal trafficking of proTGF- a/Syndecan chimeras. Coherent
with a role in or near the ER, TACIP 18 was found primarily in an intracellular
compartment that co-localizes with ER markers and interacts specifically
with immature, intracellular forms of proTGF- a and not with mature, cell
surface proTGF- a. In addition, expression of TACIP18 anti-sense cDNA
results in the reduction of cell surface proTGF-a, whereas over-expression of
TACIP18 is associated with and increment of mature plasma membrane
proTGF- a These results indicate that the interaction of the PDZ protein
TACIP18 with proTGF-c~ in the early secretory pathway is necessary for
the correct targeting of the latter to the cell surface [3].
[1] Brdey et al., Mol Biol Cell, 8. (1997) 1619. Urefia et al., J Cell Sci (1999) in
press. [2] Ahn et al., J Biol Chem 271 (1996) 2724. Lin. J. J., et al., Gene 207,
(1998) 105. [3] Fernandez-Larrea et al., Mol Cell (1999) In press.
(Su/9.1/035)

Identification of retention signal of ER membrane protein.

V. Gomord, C. Richer-Potier, C. Bonneau, L. Faye
LTI-CNRS ESA 6037, IFRMP 23, Universit~ de Rouen,
76821 Mont St Aignan, France. vgomord@crihanj~r

In order to remain in the E R , and therefore to be diverted from the bulk

flow of secreted proteins, soluble or membrane bound ER-resident
p~oteins require specific retention or retrieval signals. Signals and
mechanisms responsible for soluble protein retention in the ER of plant
cells have been extensively studied in our laboratory (Gomord et al.,
1997 and Poster of Pagny et al.: "ER retention mechanisms of proteins in
plant cells").
Recent evidences indicate that a carboxy-terminal di-lysine or a Nterminal di-arginine cytoplasmic domain maintain type I or type II
transmembrane proteins respectively in the ER of mammalian cells.
Calnexin is an abondant ER type I membrane protein and a well known
ER chaperone that transiently binds nascent glycoproteins. In order to
delineate the portion of Arabidopsis calnexin (AC) primary sequence that
is responsible for the ER retention of this protein, we have first fused a
reporter protein with the transmembrane domain and different version of
shortened cytoplasmic tails of AC. When expressed in tobacco cells,
these fusion proteins remain in the ER membrane, independently of the
length of their cytoplasmic tail. In contrast, a fusion protein containing
the cytoplasmic tail of AC and the transmembrane domain of the
tonoplast intfinsec protein (alpha-TIP) is not retained in the ER. These
studies suggest that the transmembrane domain plays an important role in
the retention of calnexin in the plant ER.
Gomord et al., 1997, Plant J., 11,313-325.

C h a r a c t e r i z a t i o n of a vesicular inhibitory a m i n o acid

transporter
A. Dumoulin ~, P. Rostainga, C. Bedet b, S. Ltvi ~, M.F.
Isambert b, J.P. Henry b, A. Triller~, B. Gasnierb
alNSERM U 497, hCNRS UPR 1929, 75005 Paris, France

A majority of mammalian central nervous system neurons secrete amino

acids as neurotransmitters. This process requires their accumulation into
synaptic vesicles, which is mediated by an amino acid transporter driven by
the vesicular H+-ATPase. Recently, the identification of the Caenorhabditis
elegans unc-47gene by in vivo [1] or in sthco positional cloning [2] allowed
the cDNA cloning of a mammalian y-amino butyric acid (GABA) transporter
presumed to be located in the synaptic vesicle membrane. In addition, our in
situ hybridization data in rat brain suggested that it might also take up glycine
and thus represent a general Vesicular Inhibitory Amino Acid Transporter
(VIAAT; ref. 2). We have now investigated the localization of VIAAT in
neurons by raising a polyclonal antibody against its N-terminal domain
(amino acids 2-127, mouse sequence).
Western Blot analysis revealed a strong 58-kDa immunoreactive band in
homogenates of VIAAT-transfected COS-7 cells and of diverse regions of the
rat central nervous system. Light microscope immunocytochemistry in
primary cultures or tissue sections of the spinal cord revealed that VIAAT was
localized in a subset (63-65%) of synaptophysin-immunoreactive terminal
boutons. Among the VIAAT-positive terminals around motoneuronal somata,
33% were immunoreactive for GAD65, a marker of GABAergic presynaptic
endings. Labelling was also found massively apposed to clusters positive for
glycine receptor or for its associated protein gephyrin. In the cerebellar cortex,
VIAAT labelling was found at typical GABAergic terminal boutons. At the
ultrastmctural level, VIAAT irnmunoreactivity was restricted to presynaptic
boutons exhibiting classical inhibitory features and, within the boutons,
concentrated over synaptic vesicle clusters. Pre-ernbedding detection of
VIAAT followed by post-embedding detection of GABA or glycine on serial
sections of the spinal cord or cerebellar cortex indicated that VIAAT was
detected in glycine-, GABA- or GABA- and glycine-immunoreactive boutons.
Taken together, these data further support the view of a common vesicular
transporter for these two inhibitory amino acids, which would be responsible
for their corelease from the same synaptic vesicle at some synapses. An
additional, unexpected finding was the existence of a few glycineimmunoreactive terminals devoid of VIAAT, which raises the question of a
distinct vesicular inhibitory amino acid transporter in some cells [3].
1- Mclntire et at., Nature, 389, (1997) 870.
2- Sagn6 et al., FEBS Lett., 417, (1997) 177.
3- Dumoulin et al., J. Cell Sci., (1999) in press.
(Su/9.1/036)

Expression during bell pepper early fruit development of

genes encoding proteins involved in vesicle fusion events
G. Houln~, J. Proust, M.L. Schantz, S. Akrim, R. Schantz
lnstttut de Biologie Mol~culaire des Plantes/CNRS-Universit~ Louis Pasteur
12, rue du G~n~ral Zimrner, 67084 Strasbourg Cedex, France

The architecture and the final size or shape of an organ or the

whole adult plant is largely determined by the linked phenomena which
takes place at the cytokinesis, a major step of cell division. Early fruit
development has been choosen as a model for the study of cytokinesis.
Moreover, it is one of the few developmental processes that involves
activation and sudden stop of cell divisions. In plants, cell division
requires a centrifuge extension of a cell plate which grows and merges
with a predetermined region of the parental plasma membrane. For a
better understanding of the mechanisms controlling the formation of the
cell plate, we have analyzed several proteins known to be associated with
vesicle fusion, a basal process of the plate forming :
a-Annexins
interact in a calcium-dependent manner with
membrane phospholipids. Two genes, Sp32 and Sp38, have been
characterized in bell pepper. The expression of Sp32 is modulated during
fruit development and peaks during early stages. On the other hand,
immunolocalization of this annexin in intercellular junctions of TBY2
cells, forming a ring structure under the plama membrane, suggests
involvement of annexins in the process of cell divisions.
b- C A F C P is an AAA protein homologous to yeast Secl8p or
human NSF. These cytosolic proteins assemble into a 20S membraneassociated protein complex with 0t-SNAP (soluble proteins) and v/tSNAREs (membrane receptors). This complex is involved in the
docking/fusion step of all cellular vesicle fusion events. During bell
pepper fruit development cafcp is constitutively expressed, the specificity
of the fusion event in a peculiar secretory pathway could be given by the
associated v/t-SNAREs. Recently, Lukowitz et al [1] has characterized in
A. thaliana a v/t-SNARE (named K N O L L E ) specific to cytokinesis. We
have isolated and characterized the bell pepper counterpart of knolle. The
gene is monogenic and specifically expressed in the early stages of fruit
development. These stages are characteristic of mitotic activities as shown
by the expression pattern of the histone and cyclin B genes.
c- Another AAA protein, CAFP, has been analysed and the results
are presented on the poster of Akrim et al.
[1] Lukowitz et al., Cell, 84, (1996) 61.

Abstracts FEBS'99

(Su/9.1/037)

s 101

A tobacco syrtl3ax.i~ w i t h a key role in hormonal

control of guard cell ion channels

B. L e ~ n ,

(Su/9.11038)

D. Geelen, F.J. Ouintero~ and M.R. Blatt

~__.o~
or

,f~

. and

S. Pagny, A~ Cabanes, P. Lerouge, L Faye and V. Gomord

~s

Syotaxins and r e l a t e d SNARE complex p r o t e i n s p l a y c e n t r a l

roles in the machinery of intraeellular vesicle trafficking,
~esicle fusion and secretion. We describe a novel syntaxin
(t-SNARE) homolog from tobacco, Nt-Syrl, that mediates in
signalling evoked by the plant water-stress hormone abscisic
acid (ABA). At the cellular level, ABA is best characterized
by its action in regulating K + and el- channels at the plasma
membrane of stomatal guard cells, leading to stomatal
closure that reduces transpirational water loss from the
leaf. The Nt-SYRI gene was identified in an heterologous
expression-cloning screen for ABA-receptor elements in
Xenopus Zaevis oocytcs. Nt-SYPI encodes a membrane-anchored
protein that is associated with the plasma membrane.
Northern as well as western analysis indicate upregulation
Of Nt-Syrl by ABA and drought stress. Loading Nieotiana
guard cells with Botulinum C toxin, a syntaxin specific
protease, prevents ABA action in controlling K* and CIcharmels. Manmlalian syntaxin proteins have been shown to
interact with a whole range of interactnrs involved in
slgnalling and vesicle trafficking and Nt~Syrl contains
three predicted coiled-coil domains. If Nt-Syrl functions
within a complex, we reasoned that a soluble, C-truncated

fragment of Nt-Syrl i n c l u d i n g the p u t a t i v e p r o t e i n - p r o t e i n

i n t e r a c t i n g ~omains can compete with t h e n a t i v e Nt~Syrl
f o r p r o t e i n p a r t n e r s . Indeed, upon loading guard c e l l s
with Sp2, v o l t a g e clamp r e c o r d i n g s showed a complete l o s s
of ABA s e n s i t i v i t y f o r the K~ and CI- c h a n n e l s . These
r e s u l t s imply t h a t Nt-Syrl i s a key element in an ABAsignalling cascade and implicate its interaction with
other elements in a signalling protein complex.

(Su/9.1/039) Switching BFA sensilivily in vivo and in vitro. A

Peyro=/~"~,B.
An~rmy, S. Robhezu, J. ~.k~r* md C. JaScson*.*SI~GM CEA/Sc~.
91191 G~sl~YvetteCo:kx IPMC CNRS. 06560Vdtx*um

ER retention mechanisms of proteins in plant cells

Laborato,re LTI CNRS ESA 6037, IFRMP 23, Universitd de Rouen, 76821
Mont Saint Atgnan, France

The current experiments were designed to characterize the

contribution of recycling in the retention mechanisms of proteins in the ER
of plant cells.
Our goal was to compare the structure of glycans N-linked to a model
glycoprotein: the carrot invertase (Inv) when it is either transported to the
cell-wall or retained in the ER. The structural analysis of these glycans will
give a good indication as to whether or not ER resident proteins recycle
between ER and the Golgi in plants.
In an attempt to retain the recombinant thvertase in the ER of tobacco
cells, we fused the HDEL peptide sequence at the C-terminus of the protein.
Furthermore, to facilitate the immunodetection of the recombinant invertase,
we have fused the FLAG epitope (9 amino acids) at the C-terminal end of
the recombinant glycoprotein.These different forms t)f the same recombinant
glycoprotein (InvFLAG and InvFLAGHDEL) have been expressed in
tobacco cells (BY2) and a screening of transformed cells was performed
with antibodies directed against the invertase and FLAG sequences.
Results were obtained concerning the immunolocalization and the Nglycan processing of the recombinant invertase in the secretory pathway. We
have also purified InvFLAG and InvFLAGHDEL for a detailed structural
analysis of their glycans and these structures were compared to the
oligosaccharides N-linked to ER resident glycoproteins such as calreticulins.
Our results strongly suggest that ER retention of InvFLAGHDEL only
depends on its very active (HDEL dependant) recycling from the Golgi
apparatus, where N-glycans are maturated back to the ER. Contrastingly, the
structure of glycans N-linked to plant calreticulins are not in favor of a
strong contribution of recycling in the residency of natural reficuloplasmins
in the plant ER.

(Su/9.1/040)

An in vitro model for the study of ceruinplasmin synthesis

and secretion regulation.
P.Pisu, DBellovino, MCarbonaro and SGaetani
lstituto ,\'az~o~mle della Nutnztone, 13a Ardeatina 546, 00178 Roma, Italia

ADP-ribosylation factors (ARFs) are small GTP-binding protems

implicated in a number of different protein transport steps, mcluding
ER-golgi and mtra-golgi transport. ARF cycles between inactive GDPbound and active, membrane-associated GTP-bound forms. We have
identified a protein (Gea 1p) from S. cerevisiae that is a component of a
complex possessing Brefeldin A (BFA)-sensitive ARF GDP/GTP
exchange activity [1]. Brcfeldin A (BFA) is a hydrophobic compound
that has dramatic effects on the structure and function of intraeellular
organelles, particularly the Golgi apparatus. Gealp possesses a domam
in common with Sec7p, a protein necessary for intra-golgi transport.
We show that the major in vivo targets of BFA in the secretory
pathway of budding yeast are the three members of the See7 domain
family of ARF exchange factors in this organism: Gealp and Gea2p
(functionally interchangeable) and Sec7p. The Sec7 domain ~s also
found in the human protein ARNO. Purified ARNO has potent ARF
guanine nucleotide exchange activity. The ARNO See7 domain alone
has ARF GDP/GTP exchange activity. However, this activity is
completely insensitive to BFA [2].We provide evidence that the See7
domain plays an essential role in BFA sensitivity using chimeric
proteins in which the Sec7 domains of Gealp and Sec7p were replaced
by that of ARNO. Specific residues within the See7 domam are
important for BFA inhibition of ARF exchange activity, since
mutations m these residues of Gealp (sensitive to BFA) and of ARNO
(resistant to BFA) reverse the sensitivity of each to BFA in vivo and in
vitro. We demonstrate that the target of BFA inhibiuon of ARF
exchange activity is an ARF-GDP-Sec7 domain protein complex, and
that BFA acts to stabthze this complex to a greater extent for a BFAsensitive See7 domain than for a resistant one.
[ 1] Peyroche et al., Nature, 384,(1996),479
[2] Chardin et al., Nature, 384,(1996),481

Ceruloplasmin is a bleu-copper oxidase glyeoprotein present in the sera of all

vertebrate species and synthesized mainly by the hepatocytes Ceruloplasmin is
an acute-phase protein and its serum concentration increases during infection,
inflammation and tissue injury. Although the biosynthesis and secretion of
ceruloplasmin has been extensively investigated, the mechanisms and the
regulation of its synthesis and secretion has not been well elucidated The
studies have been performed using animals, primary cell cultures and
hepatocarcinoma cell lines However experiments that utihze animals and
primary cells derived from their livers are expensive, time consuming and the
results are not always reproducible, and hepatocarcinoma celts are not well
regulated It has been shown that the untransformed stable cell line MMH (Met
Murine Hepatocytes), generated from liver explants of transgenic mice
expressing a constitutively active truncated form of a human hepatocyte growth
factor receptor (cyto-met), represents an innovative tool for in vitro studies of
liver function In particular the cells have been used successfully for the study
of retinol-regulated retinol-binding protein secretion Here, we have utilized a
line of MMH celts, the D3 cells, isolated from the liver of transgenic mice 3
days after birth, to investigate the mechanisms of ceruloplasmin synthesis and
secreuon, the factors involved and the cell compartment where copper is
incorporated. Preliminary experiments have been performed by metabolical
labelling of D3 cells with 3~S-Methionine-cysteine, ceruloplasmin
immunoprecipitation in cell lysate and culture medium by specific antibody, to
check if D3 cells synthesize and secrete the protein Several agents were
utilized to increase ceruloplasmin synthesis, in order to make the model more
convenient for the detection of factors involved in the aquisition of the folding
proper for secretion The results have demonstrated that D3 cells represent a
suitable model for the study of the regulation of ceruloplasmin secretion

s102

Abstracts FEBS'99

(Su/9.1/041) MUTATIONAL ANALYSIS O F MUNC-18-2

Kirsi Riento ~, Maria Kauppi 1, Katja Kivinen-', Sirkka
Ker~nen ~', and Vesa M. Olkkonen ~

(Su/9.U042) Regulation of protein transit in colonic cells HT29

d.P.Tenu, R.Auger, P.Robin, G.VIal, B,Camler
M.N,Ra) mond, B.Rossignol
c;~:RS U,IIR ~619, Bdt, 430, Untt'ervtld Paris X/. c/1405 Orsav. France

National Public Health Institute, Mannerheimintie 166, 00300 Helstnki,

Finland and ~VTI;, P.O.Box 1500, 02044 VTK, Ftnland

Munc-18-2 is a mammalian Secl-related protein that is predominantly

expressed in the epithelial cells of various tissues. In several epithelia
Mune-18-2 localizes at the apical plasma membranes, and forms a
complex with the t - S N A R E syntaxin 3. In the present study we made a
panel of Munc-18-2 mutant forms in order to gain novel insight to the
mode of action of Secl proteins. A set of mutations was designed based
on the sequence comparisons within the Sec 1 protein family, In addition,
functionally interesting mutations corresponding to those characterized m
other Secl-related proteins were generated in Munc-18-2. The in vivo
experiments were carried out m Caco-2 cells using the Semliki Forest
virus expression system. The mutants display a spectrum of syntaxin 3
binding capacities, the in vitro and in vivo binding being in good
agreement. The ability to bind syntaxin 3 correlates well with the ability
of the Munc-18-2 protein variants to reduce the quantity of SNAP23 in
immunoprecipitated syntaxin 3 complexes. Interestingly, mutants unable
to bind syntaxin 3 display significant association with cellular
membranes, indicating that interactions with other components play a
role in this membrane association. The functional effects of wild-type
Munc-18-2 and the mutants on the apical vesicle trafficking in Caco-2
cells are under investigation. The present data can he used as a basis for
the reinterpretation of the results reported previously on Secl proteins.

The regulation of mtracellular protein trafficking, unhke the regulation of

secretory" granule exocytosis has not been extensix el,, studied.To study th~s
phenomenon, colorer epithelial cells HT29-c119A are a g3d model, the3 arc
polarized, the3 secrete proteins among which (, 1-antitrypsm represents about
90%
(as deterrelned by immunoblott~ng, lmmunoprecJp~tat~on of
radiolabelled proteins and ELISA methods) and the 3 can be stimulated x ia
different types of receptors. Secretion occurs vra a const~tutix e pathss ay, st)
that the rate of secretion dircctly reflects the rate of protein transit.
The rate of apical protein transit can be regulated by different mechanisms. It
Is enhanced by phorbol esters ( PdBu and PMA),by thc chohnergic receptor
agonist carbachol and, as shown recently b 3 Jdhng et al [1], by agents
~shmh increase the cAMP level: forskohn and x asoacm e intestinal peptlde
(VIP). It ~s lowered by ethanol which d~erts the phosphohpase D (PLD)catalyzed production of phosphatidtc acid (PA) to phosphat~dylethanol (PEt).
Effects on protein secretmn of carbachol and PdBu, on one hand, of
carbachol and forskolin, on the other hand, are ad&tix e. It was shown,
using the " 20C block", that a TGN or a post-TGN step of the transit was
the target of the regulator3' events.
PdBu and carbachol act v~a different signalhng pathways. The fact that
bismdoylmalmm~de inhibits the PdBu-st~mulated transphosphat~d31ation
arm it3' of PLD to the same degree as it mhiNts protein release triggered by
Pdf3u suggests that phorbol esters regulate protem traffic partly ~ ia the actton
of a PKC-act~vated PLD. By contrast, carbachol stimulauon does not lead to
PLD activation but rather reduces a production of PA vm the acttvat~on of the
phosphomos~tol-phospholipase C (PI-PLC), and possibly also the
phosphocholine-phospholipase C (PC-PLC) The mechanism by" uhich
protein transit ~s regulated by agents mcreaslng the cAMP level ~s
independent of a PA production. In conclusion, ~ e sho*~ed that m HT29cll9A cells, the regulatmn of apical protein transit by' phorbol esters and
carbachol and actv~anon of P L D and PLC, which lyoth lead to an increase of
the PA le~ el, are closely related e~ ents.
[11 Jilhng et al., J.B~ol.Chem. 271. (1996) 4381.

(Su/9.11043) Regulation of protein transit in colonic cells HT29

J.P.Tenu, R.Anger, P.Robin, G.Vml, B.Camrer
M.N.Raymond, B.RossJgnol
CNRS U.1IR 8619. Bdt. 430. Umverrlld Parts X]. ~1405 :)tsar. l"rance

(s~9.1~aa)

Expression and morphogenetic role of a multigene family

coding secretory granule proteins in Paramecium.
L. Vayssi~, A. Galvani, N. Garreau, L. Sperling
Centre de g~nOtlque moI&ulalre. CNR~ 91 ] 98 Gtf sur Yvette. France

P a r a m e c i u m is a unicellular eucaryote that possesses a regulated secretory

The regulation of intracellular protein trafficking, unhke the regulauon of

secretory granule exocytos~s has not been extensi~ el 3 stu&ed.To study thJs
phenomenon, colorer eplthehal cells HT29-c119A are a good model : the 3 are
polarized, the', secrete proteins among which <t 1-antitrypsm represents about
90%
(as d e t e r m i n e d by lmmunoblotting, lmmunopreclpltation of
radlolabelledprotelnsand ELISA methods) andthey can be stlmulated via
different types of receptors. Secretion occurs via a constitutive path',~ ay, so
that the rate of secretion directly reflects the rate of protein transit.
The rate of aptcal protein transit can be regulated by dfflerent mechanisms, It
is enhanced by phorbol esters ( PdBu and PMA),by the chohnerDc receptor
agonist carbachol and, as sho':,n recentl3 by Jilhng et al [1], by agents
which increase the cAMP level: forskohn and ~asoacm e intestinal peptide
(VIP). It is lo'aered by ethanol which diverts the phospholipase D (PLD)catalyzed production of phosphatidlc acid (PA) to phosphatldylethanol (PEt).
Effects on protein secretion of carbachol and PdBu, on one hand, of
carbachol and forskohn, on the other hand, are additive. It was shown,
using the " 20C block", that a TGN or a post-TGN step of the tranmt was
the target of the regulatory events.
PdBu and carbachol act via different signalhng pathways. The fact that
Nsindoylmalmmide inhibits the PdBu-stlmulated transphosphat~dvlation
actl,~lt3,'of PLD to the same degree as it inhibits protein release tnggered by
PdBu suggests that phorbol esters regulate protein traffic part/,,' ~ra the action
o f a PKC-actlvated PLD. By contrast, carbachol stlreulahon does not lead to
PLD actp. ahon but rather reduces a pr~xluctlon of PA vla the activation of the
phosphomos~tol-phospholipase C (PI-PLC), and possibly also the
phosphochohne-phosphohpase C (PC-PLC). The mechanism by ~hich
protein transit is regulated by agents increasing the cAMP level is
independent of a PA producuon. In conclusion, u e showed that m HT29c119A cells, the regulation of apical protein transit by phorbol esters and
carbachol and act~vatmn of PLD and PLC, u hlch both lead to an increase of
the PA level, are closely related ex ents.
[1] Jdling et ai., J.BiohChem. 27 1, (1996) 4381.

function involving the biogenesis of elaborate secretory granules, the

trichocysts, and their regulated exocytosis in response to appropriate
extraeellular stimuli. In order to build up the crystalline secretory granule
contents, P a r a m e c i u m uses a multigene family organised in sub-families
that encode precursor proteins. Within each subfamily, 4 to 8 co-expressed
genes code for nearly identical proteins (1). 1- Functional slanificance of
such a family for.the.elaboration of t _ ~ ' s t a l l i n e structure. Subfamilyspecific antibodies used in electron microscopic immunolocalization
experiments revealed distinct and non overlapping patterns of decoration of
the trichocyst crystalline matrix, supporting the idea that the different
polypeptides are not interchangeable during the assembly of the secretory
granule matrix, and have different morphogenetic roles. 2- Functional
analysis of mutants created by gene sil~:'ncing. Exploiting the possibility to
inactivate genes in Paramecium using a homology-dependent gene silencing
phenomenon (2), we have specifically silenced different subfamilies of TMP
genes, and then created secretory mutants with distinct phenotypes. The
analysis of these mutants provided a second, strong argument that the
different TMP genes have different functions in trichocyst biogenesis. 3Effects of regulated exocytosis on the expresston of Paramecium secretory
protein ~enes. Rapid and synchronous exocytosis of all of the =1000
trichocysts per cell can be triggered by vital secretagogues such as
aminoethyldextran (AED) without harming the ceils. Using this exocytotic
trigger, we found that the transcription of TMP genes undergoes rapid,
transient and co-ordinate activation in response to massive exocytosis,
leading to an increase in the pool of TMP mRNA. Genetic dissection using
a collection of exocytosis-deficient mutants and the mutants created by gene
silencing indicate that the gene activation is regulated by signals generated
at the plasma membrane, according to the state of occupation of pre-formed
exocytotic sites.
(I) Madeddu, L. et al., 1995. Mol. Biol. Cell, 6, (1995) 649 ; (2) Ruiz, F. et
al., Mol Biol Cell, 9, (1998) 931.

Abstracts FEBS'99

s103

10.1 Functions of glycosylation

(Su/10.1/045)

Glycosylation analyses of the Clostridium tyrobutyricum

bacterial flagellin
L. Bedouet, F. Arnold, P. Batina, F. Talbot, P. L6cher,
P. Le Chevalier and G. Robreau

(Su/10.1/046)

IPBS CNRS, 205 Route de Narbonne, 31077 Toulouse, France

U.B.O., LUMAQ. IUT de Qutmper, 29334 Qutmper, France.

The anaerobic Gram positive, spore forming bacterium Clostridium

tyrobutyricum is the organism responsible for the late spoiling of
Emmental or similar cheeses by the production of gas and butyric
acid. In order to count C. tyrobutyricum spores in milk after
membrane filtration, murine 21E7-B12 monoclonal antibody was
produced. Immunoelectron microscopy demonstrated that the
21E7-B12 Moab recognized a surface-exposed epitope on the flagellar
filaments. After flagellar extraction, the purified flagellin showed an
apparent molecular mass of 46 kDa and was labelled with
digoxigenin-hydrazide after mild periodate oxidation. Non-reductive
13-elimination experiment with diethylamine showed that flagellin is a
O-glycosylprotein, the resultant MW is 45 000. Furthermore the
21E7-B12 Moab binding was lost. Amino acid analysis of the
glycopeptides obtained after pronase digestion of flagellin indicated
that O-glycosylation probably occured via the hydroxyl group of
serine. Otherwise, 13-elimination partly deglycosylated flagellin, thus
revealing additionnal linkages probably via asparagine or tyrosine.
Competitive ELISA between flagellin and carbohydrates ligands for
21E7-B12 Moab showed that maltopentaose was the best of tested
competitors. Thus glycans removed from flagellin by 13-elimination
may contain linear maltodextrin. Moreover, amino acid sequence of
flagellin contains 11 potential N-glycosylation sites and one sequence
rich in serine which could be modified byO-glycosylation.

Subeeilular localization of PAPS Synthetases, the enzymes

responsible for sulfate activation in mammals
S. Besset, F. Amalric, J.P. Girard

Sulfation is a major modification of many molecules in animals, including

mucins, proteoglycans, glycolipids, hormones, neurotransmitters, drugs and
xenobiotics. To serve as a substrate for cytosotic or Golgi-resident
sulfotransferases, sulfate must first be activated by conversion into a highenergy donor, 3'Phospho Adenosine 5'PhosphoSulfate (PAPS). Two PAPS
Synthetases (PAPSS), bifunctional ATP Sulfurylase / APS Kinase enzymes
sufficient for PAPS synthesis, have been identified in mouse [ 1] and human
[2, 3]. Major physiological processes are controled by PAPS availability, as
evidenced by murine brachymorphism and human spondyloepimetaphyseal
dysplasia, two orthologous diseases caused by profound undersulfation of
cartilage proteoglycans due to mutations in the PAPSS 2 gene [1, 3].
In this study, we show that human PAPSS1 unexpectedly accumulates in
the nucleus of mammalian cells. Mutagenesis studies show that the APS
Kinase domain is both necessary and sufficient for nuclear targeting of the
enzyme, while the deletion of a short catalytically dispensable sequence at
the amino-terminus of the APS Kinase domain completely abolishes nuclear
accumulation of PAPSS 1. In vtvo evidence that PAPS Synthetases are fully
functional when targeted to the nucleus is provided by showing that human
PAPSS 1 expressed in yeast strains deficient in APS Kinase or ATP
Sulfurylase activities localizes to the nucleus and concommitantly
complement the methionine auxotrophy of both strains.
Interestingly, we found that PAPSS 2, which exhibits m vitro PAPS
synthesis activities similar to those of PAPSS 1, localizes to the cytoplasm
of mammalian cells, but accumulates mostly in the nucleus when coexpressed with PAPSS 1. Altogether, these results suggest that PAPSS 1
regulates the subcellular localization of PAPSS 2 and indicate that a
sulfation pathway might exist in the nucleus of eukaryotic cells.
[1] Kurima et al., Proc. Natl. Acad Sci. USA, 95, (1998) 8681.
[2] Girard et al., FASEB J, 12, (1998) 603.
[3] ul Haque et al., Nat Genet, 20, (1998) 157.

(Su/lO.1/o48)

Role of N-glycosylation and cysteines

in human angiotensinogen.
J. Cderier, A-P. Gimenez-Roqueplo, P. Corvol and X.
Jeunemaltre. 1NSERM U36. Colldge de France, Paris, France.

Human angiotensinogen, the specific substrate of renin, is a

heterogeneous glycoprotein constitutively secreted by the liver. During
pregnancy, a 2:2 covalent complex between angiotensinogen and proform
of eosinophil major basic protein (proMBP) have been identified.
Angiotensinogen is involved in blood pressure regulation and M235T
polymorphism has been associated with essential and pregnancy-induced
hypertension. We have performed a systematic site-directed mutagenesis
study of the four putative asparagine-linked glycosylation sites (Asn-XSer/Thr) which has demonstrated that all the sites are actually Nglycosylated. The suppression of the Asn 14 glycosylation site led to 5
times lower Km and 10 times lower kcat. The N-deglycosylated mutant
(N-4) exhibited a single form at 48 kDa on SDS-PAGE. Interestingly, the
N-4 mutant had a higher catalytic effiency (kcat/Km = 5.0 versus 1.6 IxM"
l.s-l) than the wild-type protein and could be of value for X-ray
cristallography studies. Human angiotensinogen contains four cysteines.
We have demonstrated the existence of a disulfide bond between CysTMand
Cys 135 by mass spectrometric determination after trypsic digestion. The
complex anglotensinogen-proMBP was produced in vitro. Using several
eysteine mutants of angiotensinogen cDNA, we have demonstrated that
Cys232 is crucial for complex formation. The 235M variant generates a
higher quantity of complex than the 235T angiotensinogen. Human renin
hydrolysis of complex angiotensinogen-proMBP was slower than the
monomeric angiotensinogen without influence of the M235T genotype. All
these data suggested that N-glycosylation of human angiotensinogen is
responsible of its heterogeneity and that angiotensinogen-proMBP
complex could be a storage form of angiotensinogen during pregnancy.

s104

(Su/I0.II049)

Abstracts FEBS'99

Expression of fecosylated glycoconjugates by cells from

patients with acute myelocytic leukemias
C. Dera.pbPe', C. Piechio b, M. Roscini b, C. Zamponi b, C.
Emiliani , M. Aubery', M.J. FogliettP and M. Bernard a
bUniversita di Perugia, Italy - aUniversitg Paris V France

Numerous studies have demonstrated that many human tumours express

fucosylated glycoconjugates that are absent in corresponding normal
tissues (see, for example Hakomori, Adv. Cancer Res., 1989, 52, 257331). In this point of view, we studied fucosylation of glycoconjugates
from leukemic cells of patients with different acute myelocytic leukemias
(AML O, 1, 2 and 3). Fueosylation of the HL 60 cell line (corresponding to
AML 3 type) was also studied, since we previously observed (Dosbaa et
al.. Carbohydr. Res., 1992, 236. 259-265) that these cells exhibited some
similarities in expression of the ct-L-fucosidase. These HL 60 ceils are
able to differentiate into granulocyte-like cells by treatment with
dimethylsulfoxide. After treatment these cells exhibited an expression
profile for c~-L-fucosidase comparable to that of normal granulocytes.
Cells were metabolically labeled with tritiated mannose and lysed.
Glycoproteins were intensively digested with Pronase and resulting
glycopeptides were analyzed by affinity chromatography on immobilized
lectin columns of Lens culinaris and Ulex Europeus which have affinity
for core and peripheral fucose residues, respectively. We observed that
leukemic cells from patients express high levels of glycoproteins carrying
peripheral fucose residues. A large diversity was observed in the affinity
of the AML glycoproteins for the Lens culinaris lectin: AML 0 and AML
2 express high levels (about 80%) of glycoproteins, when AML 1 and
AML 3 express lower levels (12 to 22 %) of such glycoproteins carrying
core fueose residue. It is interessant to note that HL 60 cells express low
level of glyeoproteins with core fucose residue, like AML 3 cells. Their
treatment with dimethylsulfoxide (and also with all-tram retinoic acid
which induces the same type of differentiation) results in a dramatic
increase in the level of peripheral fucose residues (9 times more) and a
decrease of core fucose residues (an half). Possibility of relationships
between fucosylation of the cell glyeoproteins and activities of c~-Lfucosidases and fueosyltransferases are discussed.

(Su/10.1/051) H O R M O N E INDUCIBLE GLYCOSYLATION OF STAY5

C. Gewirmera, B. Groner"
aGeorg-Speyer-Haus.Institutefor Biomedical Research,
PauI-Ehrlich-Str.42-44, 60596 Frankfurt, Germany
The STAY (signal transducer and activator of transcription) proteins
are a family of transcription factors involved in cytokine, growth
factor and hormone signal transduction. Cytokine binding to their
receptors leads to the tyrosine phosphorylation of associated
cytoplasmic protein tyrosine kinases which belong to the JAK (Janus
kinase) family. JAKs phosphorylate STAY proteins which then
dimerize, translocate to the nucleus and bind to specific DNA
sequences of responsive gene promoters. Secondary modifications
regulate protein activation and interaction with cofactors. We
investigated the possible functions of different secondary
modifications to the STAY proteins. Tyrosine phosphorylation at
residue 694 is the crucial event in the STAY5 activation process.
Phosphorylation on serine and threonine residues has also been
observed [1, 2]. A novel form of glycosylation has been discovered
in which single N-acetylglucosamine monosaccharides are Oglycosydically linked to serine or threonine moieties (O-GIcNAc).
The potential functions for O-GIcNAc include nuclear transport,
protection from proteolysis, blocking sites of phosphorylation on
serine and threonine and regulation of protein function [3].
We used galactosyltransferase assays and immune precipitations
with succinyl wheat germ agglutinin lectin directed against Naeetylglucosamine to show that STAY5 is O-linked glycosylated in
HC11 cells upon treatment of the cells with lactogenic hormones.
This glycosylation event coincides with translocation of STAT5a
from the cytoplasm to the nucleus and indicates a additional
regulatory pathway of STAY5.
[1] Groner et al., Curr. Opin. Genet. Dev. 5 (1995): 587
[2] Gouilleux et al., EMBO J. 13 (1994): 4361
[3] Haltiwanger et al., JBC 267 (1992): 9005

(Su/10.1/050)

Effect of glycosylation of 13-casein on its

foam and emulsion properties
J. Dziuba, M. Darewicz, Z. Dziuba
Department of Food Biochemistry, OUA T 10-957 Olsztyn, Poland

Bovine 13-casein (13-CN) is a flexible, approximately random coil protein. It

can be used for its functional properties, such as surface-active, foam- and
emulsion-forming and -stabilising properties. A number of attempts have
been made to improve functional properties of milk proteins by chemical
modifications. Chemical modifications are useful tools in the study of
structure-function relationship of proteins. One of such modification,
glycosylation is reported in this abstract. The present research work was
carried out to determine the effect of covalent binding of glucose molecules
to ]3-CN on its foam and emulsion properties. Glycosylation was carried out
according to the method by Nessar and Furth [1]. The foaming and
emulsifying properties of intact and glycosylated 13-CN were studied at pH
6.7 and 1=75 mmol/L [2]. Foam properties were studied by measuring the
volume of foam which was produced during whipping. Emulsion-forming
ability and instability of the emulsions were investigated by measuring
particle size distribution and the decrease of the turbidity at 500 nm,
respectively. The mass spectrometry results of glycosylated 13-CN suggested
that no more than nine glucose molecules would bind to Lys residues. Foam
forming properties of glycosylated I3-CN were better as compared with
intact one. Glycosylated 13-CN produced slightly smaller emulsion droplets
than an intact one. An increase in emulsion stabilising properties of
modified sample was observed. A prerequiste of proteins to form emulsions
is their adsorption onto the oil/water interface. Therefore, the secondary
structure of intact and glycosylated 13-CN, both in solution and adsorbed
onto a hydrophobic teflon/water interface, were studied as well as by farultra violet circular dichroism [3]. It appeared that secondary structure was
induced aider adsorption onto the interface. Based on the information
obtained in this study a model with the hypothesis for structure-function
relationship of intact and glycosylated 13-CN will be presented.
[1] Nessar, A., Furth, A. Anal. Biochem., 192 (1991) 119.
[2] Darewicz, M., Dziuba, J., Caessens, P., Gruppen, H. (submitted).
[3] Jongh, H., Goormaghtigh, E, Killian, A. Biochemistry, 33 (1994)
14521.
(Su/10.1/052)
Regulation of al,3-galactosyltransferase expression in
murine F9 cells
D.H Jozmsse~, C.G. van der Linden~,J C.M Baggen~, D.F.R. Muris~,DH.
",'anden Eunden~,N.L Shaperb, JM Shaper~
"Dept. of Medical Chemlstt T. l/rtje Umversltelt. Amsterdam. Netherlamls
bOncolog3' Center. Tire Johns Hopkins Um~er~iO' School of Medicine.
Balttmore. USA

The enzyme UDP-GahGal~ 1,4GlcNAc-R c~l,3-gaIactosyltransferase (c3GAIT) transfers c~-galactose in a 1,3-linkage to the sugar chains of
glycoproteins and glycolipids. The resulting terminal element has the
structure Galc~I,3Gal-R. The enzyme c~3-GalT occurs in a variety of
mammalian species, but is absent in humans, apes, and Old World monkeys,
and therefore the latter species are unable to produce the Galccl,3Gal
disaecharide. This structure on endothelial cells is the major target for natural
xenoreactive antibodies during the hyperacute rejection of pig organs
transplanted to primates.
In the mouse, the gene encoding c~3-GalT is expressed in most tissues and
cell types, with the exception of male germ cells and hepatocytes. Expression
of ~x3-GalT is upregulated in murine F9 embryonal carcinoma cells treated
with retinoic acid. To investigate the regulation of c~3-GalT gene expression,
we have isolated several kb of upstream genomic sequence. Sequences
upstream of the transcriptional start site were able to drive the expression of a
luciferase reporter gene transfccted into F9 cells and COS cells. By deletion
analysis we have identified a minimal fragment able to mediate a 5-6-fold
stimulation of luciferasc gene transcription in F9 cells treated with retinoic
acid. In this fragment two putative transcription factor binding elements were
identified by data base search, one of which was bearing a weak resemblance
to a DR2 direct repeat of RAREs. EMSA analysis and site-directed
mutagenesls studies were employed to determine if any of the elements thus
identified were directly involved in retinoic acid stimulation of c~3-GalT
expression, and to investigate which factors might be involved.
Supported by Human Frontiers Science Program Grant RG 414-94 M, and
EC Biotechnology grant BIO4-CT97-2242.
References:
D.H. Jozlasseetal. (1989)J. Biol. Chem. 264, 14290-14297
D.H. Jozlasseetal (1992) J. Blol Chem. 267, 5534-5541
S.K. Cho et al (1996) J Biol Chem. 2"71,3238-3246

Abstracts FEBS'99

(Su/10.1/053)

Improvement of erythrocyte storage conditions in blood

banks importance of deleukocytation.

s105

(Su/10.1/054)

D. Bratosina, S. Leszcz'ynsbkyb,O Fontalne~ J. Descampsb

J-J. Huart, J. MontreuiP.
lnstlattM de Btochlmie al Aeademiet Romcme, P.O. Box 2-2. Re-78200 Bucurestt;
b Etabhssement Rdgional de TransfusionSanguine. BP 2018, F-59012IJlle Cedes;
Untverstt~ des Sciences et Technologtes de Lille. Laboratotrede Chimte
Bwlog~que, UMR I l l du CNRS. F-59655 Vdleneuve d'Ascq Cedex.
The in vivo phagocytosis of red blood cells (RBC) by maerophages, occuring 120
days after birth of the cell, is mainly dependent on two signals: (i) The exposure of

[3-galactosyl residues in terminal position, due to desialylation of RBC membrane

glycoconjugates and (to The exposure of phosphafidylserine (PS) residues in the
cell membrane outer leaflet which triggers the phagocytosis.
In order to explain by which molecular and cellular mechanism, 30 % and 70 % of
transfused RBC disappear from the circulation after 1 and 3 days respectively, we
have hypothefized that the in vitro senescence of RBC could be due to an identical
mechanism of desialylatien- phosphatidylserine exposure.
The knowledge of the mechanism of the in vitro senescence of RBC is of the
highest importance since it could lead to improvemeuts of the storage conditions in
blood banks by increasing the time of viability of stored RBC (less than 3 weeks at
the present time). [n this way, polytransfused people could be protected from
infectious diseases due to the immune-depressive action of iron overload in
macrophage. For the study, the RBC stored during 4 weeks in SAGM medium
were prepared using an in line filtration set "LEUCOFLEX LST-1 - Reference
FKF 6260 P" from MACOPHARMA (Tourcoing, France). On the basis of the
following results, it is clear that deleukocytation of blood before storage in blood
banks protects the erythrocytes from desialylation and phosphatidylserine exposure
in the outer leaflet of cell membrane and, consequently, from a rapid clearance of
transfused erythrocytes: (i) Determination of RBC binding site number of lectins
specific for sialic acid and 13-galactosyl residues shows that an important
desialylation of RBC membrane glycoconjugates occurs during the storage of
blood in the present conditions of storage. In opposite, the number of [3-galactosyl
residues, which are the signals for RBC capture, increases. RBC membrane
desialylation is confirmed by applying the colorimetric method of Warren. (i 0 In
parallel, PS residues which are signals for RBC phagocytosis, are exposed in the
outer leaflet of RBC membrane. (iiO Consequently, in vitro erythrophagocytosis
progressively increases. Ov) All of these harmful effects are due to the highly
active leukocyte neuraminidase and are avoided by the deleukocytation of blood
before storage in blood banks. In conclusion, blood destined to transfusion must be
deleukocytized. In this regard, deleukocytation of blood before storage in blood
banks is mandatory in France from the 1st of April 1998.

(Su/10.1/055)

Mass spectrometric analysis of post-translational

modifications of Factor IX
J.M. Schmitter 1, M Belghazi ~, N. Bihorcauz, E, Nony 2, 0
Just2, S. Chtourou2 I lnstitut Europden de Chimte B~ologie,
33402 Talence, France 2 L.FB., 91958 Les Ulis, France

Human plasma-derived factor IX (FIX) is a 415 amino acid long

glycoprotein. Relationships between specific post-translational
modifications and in vivo functional efficiency have been reported To
improve viral safety o f F A C T E U R IX-LFB ~" plasma-derived
concentrate intended for hemophilia B treatment, a 15 nm filtration has
been implemented in the production process, as an additional virus
elimination step. Structural characterization o f this product (~IX iSN)
was performed including identification o f the complex post-translational
modifications o f the protein. For this study FIX has been isolated b y
size exclusion chromatography and further digested by specific
endoproteases to cleave between the two potential N-glycosylation
sites. The resulting peptides were separated by reversed-phase liquid
chromatography and characterized by lectin blot analysis and matrixassisted laser desorption ionization mass spectrometry (MALDI-MS).
Using these techniques, the complete sequence o f FIX 15N has been
verified For post-translational modifications study, peptide maps have
been systematically compared to those obtained after N and Oendoglycosidases treatments to remove the corresponding glycans
Molecular masses o f glycopeptides and lectin Not data indicated that
Ash I57 and Asn 167 are predominantly occupied by sialylated
tetraantennary, fucosylated glycans. Set 61 and 63, Thr 159, 169 and
172 are modified with classical O-glycans. After N-deglycosylation, a
new peak at 2542 Da was detected and assigned to the [Thr163Glu185] unmodified pep(ire, in agreement with a partial glycosylation
of Thr169 and 172. M A L D I - M S analysis also revealed Asp 64
hydroxylation, Tyr 155 sulfa(ion and Ser 158 phosphorylation which
play a crucial role in the clearance of FIX. The structural integrity of
this 15 nm filtered FIX is consistent with its efficacy for hemophilia B
treatment.

INVOLVEMENT OF GANGLIOSIDESIN HIGH-AFFINITY UPTAKE

OF DOPAMINE BY RAT STRIATAL SYNAP'rOSOMES. G I'AGt=
I. BARRIER.[r tl[IGtJ[~F. C 1AI I,INT;AIJand .I PORTOUKALIAN

Gangliosides are gl.veosphingolipidsfound in high coneentratmu in neuronal membranes

and lvavc been suggested to be functionnall) mvolxed in s)naptlc transmlssmn On the
other hand. the.~ promote CNS recover3, from mjuD. especially both m behavioral and
ncurochemlcal temls ~utlun the dan(aged dopomme (DA) s)stem

Ho~ever. the

mechanisms underl) nlg are still unknoxxn Tbe aim of the present studs ~as to examine the
role ofgangliosldes in the high-affintl3 DA uplakc m m( striatal s)naptosomes hlcubanon
of s)naptosomcs for 40 nun at 37C xxlth neurammidase of ['~brto ('holerae (NVch). an
el~3 me wluch spiltts off smhc acid from pob smloganghosldes. Ieaviog GM I intact, caused
a 40% loss of lipid-bound siahc acid and major modifications of ganghostdes pattern.
Hox~evcr. 11o modification of DA uplake was observed I1 suggested that earlchment of
s3naptic membranes in GMI is sufficient to roannam the efficacy of the DA uptake In the
seeood set of e\ponments we ins estigated the effects of exogenonsb added GM I After lh
of incubation at 37C ~ith 1.2 x 10"M. 1.2 x 10"M and 3 x 10+M of GMI. 0.023.
tl 214 and t) 804 mnol of GM 1 x~ere stabl3 assoctated to s) naptosomes, respoctwel). At
1 2 ", ltl "M. tins association modified the actwlt~ of the neuronal dopaoulle transporter
(TNDA). since a decrease m both Vnlax and Km (about "~5%)was observed, reflecting a
reduction of the ilnnlber of uptake sues and an increase of the affioit), respoctlxcl?, Wheo
s3naptosemes \sere incubated xuth a 100 fold lo~xer concentration (I.2 x 10~MI. the
reductmn of Vmax was Io~xer thao that observed ;~lth 1.2 x It)~'M GMI (17% xs 35%.
rcspoctlxel)) We postulated that a part of assoemted-GMI is anached to membrane
proteins, l~mtcularl) to TNDA and hindered DA binding to us transporter Oi the other
Itand. a souilar decrease of Kill was obtained at these me eoncenlratlons o[" GMI used.
These findings suggested that an e\tremcly Io~* but crmcal level of asseclated-GMI to
s) naptlc plasma membrane is able to influence the acti~it3. of TNDA

(Su/0.1/056)

Role of complex asparagine-linked oligosaecharides

in the e x p r e s s i o n of a functional thyrotropin receptor
S. Siffroi~, S. Costagliolab, A. Girand", P. Banga, J.L. Franc~
u 38 1NSERM, Facult~ de m~decine, Marseille, France; b IRIBHN,
Universit~ libre de BruxeUes, Belgique, c Division of Medicine, King's
College of Medicine, London, UK.

The thyrotropin receptor (TSHR) plays a key role in the thyroid

regulation. It is a member of the family of G protein-coupled receptor. T S H R
undergoes a series of posttranslational modifications including N-glyeosylation
and proteolytic cleavage with formation of subunits A and B at the plasma
membrane level.
In order to evaluate the functional role of complex asparagine-linked
oligosaccharides of this receptor, a C H O cell line and a K562 cell line expressing
the T S H R were treated by deoxymannojirimycine (dMM), an inhibitor of
mannosidase 1 that leads exclusively to the formation of high mannose type
structures. Treatment of cells with dMM for 16h led to a decrease by 55 % in the
number of T S H binding sites at the cell surface. Flow cytometry using a
polyclonal antibody (R9) or a monoclonal antibody (BA8) directed against the
extracelluiar part of the protein (A subunit) showed this inhibition was not due
to a decrease in the number of T S H R expressed at the cell surface. However the
recognition of T S H R by a monoclonal antibody (3G4) directed against a peptide
localized near the C terminal part of the A subunit was decreased by 50 %. It is
known that after the cleavage between the A and the B subunit, the C terminal
part of the A subanit (called: C peptide) can be removed by protease(s) at the
cell surface level.
On immunoblotting, after deglycosylation using pepfide N-glyeanase F,
the B subunit was visualized as a doublet (36 and 41 kDa). In the control cells,
the species of higher molecular weight was more abundant while after dMM
treatment the band of lower molecular weight was more intense. The difference
in molecular weight between the two peptides is compatible with the removal of
the C peptide.
In conclusion the results show that inhibition of complex type structure
formation leads to (i) an incapacity for T H S R to bind T S H without affecting its
intracellular transport, (ii) an increase of T S H R susceptibility to pro(eases which
remove the C peptide. We then hypothesized that removal of the C peptide from
the A subunit leads to the formation of a non functional TSHR.

"6661 'p~.aaqns '~uoOeld '-1~~ "D'N u~q~L [ g]

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Abstracts FEBS'99

s107

13.1 Neurotransmitter receptors

(Su/13.1/058)

Characterization of the receptor for the neurotransmitter and

the addictive drug T-hydroxybutyrate (GHB)
C. Andriamampandry,V. Kemmel, V. Hechler, S. Gobaille, C.
Schmidt, C. D. Cash and M. Maitre.

(Su/13.1/059)

The fish MCH receptor: a biochemical approach

B. Cardinaud a, T. Suplya, S. Ricois a, J. Duhault b, J.A.
Boutin b, J.-L. Nahona
a IPMC/CNRS, 06560 Valbonne. b ldRS, 91000 Suresnes, France

CNRS, UPR 416, 11 rue Humann, 67000 Strasbourg, France

Gamma-hydroxybutyrate (GHB) is an endogenous brain

neuromodulator which is synthesised and released by specific
neuronal pathways in brain. It is also a drug used in therapeutics or
auto-administered for psychological benefits. Several years ago, the
existence of high affinity binding sites for this substance on
neuronal membranes has been demonstrated. These sites bind only
GHB, synthetic structural analogues and some benzamide
neuroleptics, including (-) sulpiride. Autoradiographic mapping
showed the distribution of these sites only in the rostral part of the
brain (cortex, hippocampus, thalamus, striatum, olfactory tracts and
dopaminergic nuclei). Peripheral organs and glial cells are devoid
of binding sites. The stimulation of GHB receptors expressed in
differentiated NCB-20 neurons appeared to be linked to a reduction
of Ca2 channel activities with a delay of 30-60 s, suggesting that
GHB receptor belongs to the G-protein receptor family.
Furthermore, GTP or its analogues and ADP-ribosylation by
pertussis toxin reduced the affinity of GHB binding, indicating that
the G proteins implicated in GHB receptor coupling are oftbe G, or
Go family. After solubilization, these receptors were purified by
affinity chromatography and preparative electrophoresis. A protein
band of 54 Kda was purified and sequenced to define specific
primers for PCR. A eDNA coding for a protein of 56,146 Da was
obtained with 7 transmembrane regions and 35-40% homology
with GABAB and mGLUR1 receptors. After transfection in CHO
cells, a significant high affinity binding of [3H]-GHB was
observed. This binding is currently being investigated with regard
to its kinetic properties and its pharmacological profile.

(Su/13.1/060)

Role o f Gi p r o t e i n s in d o p a m i n e D2 receptorm e d i a t e d M A P k i n a s e activation

E.J. Lee, E.Y. Choi, K.W. Park, J.H. Baik
Laboratoryof MolecularBiololly Medical ResearchCenter
College of Medicine, YonseiUniverst-'ty.Seou 120-752,SouthKorea.

Two isoforms of dopamine D2 receptor, D2L (long) and D2S (short), differ
by the insertion of 29 amino acids specific to D2L within the putative third
intracellular loop of the receptor, which appears to be important in the
selectivity for G-protein coupling. We have generated D2L- and D2Sexpressing Chinese hamster ovary (CHO) cells, and regulation of the
mitogen-activated protein kinase (MAPK) pathway was examined in these
ceils. D2L and D2S both mediated a rapid and transient activation of MAPK
with dominant activation of p42-kDa MAPK. Pcrtussis toxin treatment
completely abrogated the stimulation of MAPK mediated by D2L and D2S,
demonstrating that both receptors couple to pertussis toxin-sensitive G
proteins in this signaling. The stimulation of MAPK mediated by both D2L
and D2S receptor was markedly attenuated by coexpression of the Cterminus of 13-adrenergic receptor kinase ([3ARKct), which selectively
inhibits G[3"t-mediated signal transduction. Further analysis of D2L- and
D2S-mediated MAPK activation demonstrated that protein kinase C and

tyrosine phosphorylations are involved in these signaling pathways, and this

in a differential manner. By using cell lines which stably overexpress G ~xi
subunits or cell lines with stably transfected antisense Gcd subunits, we are
currently studying the role of specific G proteins and their interaction with
other mediator in D2L- and D2S-mediated MAPK activation. These findings
suggest that subtype-specific regulation of MAPK pathway exists by two
isoforms of D2 receptor, probably dictated by their differential G-protein
coupling. (Supported by the GER Grants GE 96-132 and GE 97-115 from
the Korean MOE and a grant from the KOSEF 981-0505-030-2)

Melanin-concentrating hormone (MCH) is a cyclic peptide which acts as a

skin-paling factor in teleost fishes and regulates a broad array of functions
in mammals. In particular, MCH is a regulator of food-intake behavior in
rodents. The mechanisms involved in MCH action and the nature of its
cellular receptor(s) are still unknown.
To date, the fish melanophore remains the only cellular model possessing a
functional MCH receptor. We developped a rapid and sensitive bioassay
using the skin of the eel Anguilla anguilla, and allowing to measure the
MCH-induced melanosomes aggregation. The rat/human MCH was as
potent as the salmon MCH in this test (ECs0:1.0 and 1.6 nM,
respectively).
Deletions and mutations of the rat/human MCH allowed to define the
amino acids involved in the ligand-receptor interactions. A structure
confering 100% of the activity, and being smaller and less hydrophobic
that the native peptide, is at present used as a backbone in the elaboration
of a new radio-ligand.
The eel skin assay was also used in pharmacological and biochemical
studies, in order to characterize the intracellular signalling pathways
involved in the MCH-induced response. For this purpose, we performed
second messengers measurements (AMPc, GMPc, 1P3) and pharmacology
experiments (pertussis and cholera toxins, staurosporine, phorbol esters,
IBMX, wortmannin, etc.). MCH decreased the intracellular concentration
of cAMP, but this effect was not dependant upon the activation of a PTX
sensitive G-protein (i.e.Gi/Go). GMPc and IP3 concentrations were also
modified. Western immunoblotting of cellular extracts (Anti c-los, antiphospho-tyrosine,
anti-MAP kinase antibodies), and binding of
[35S]GTPgS on membrane preparations were also conducted.
Our results allowed to precise the nature of the fish melanophore MCH
receptor and its intracellular signalling pathways.

(Su/13.1/061)

Identification of novel G protein-coupled receptor kinase

4 isoforms and expression in spermatozoa
T.T. Chuang, E. Christodoulou, C.A. Scorer
Cellular Biochemistry Umt, Glaxo Wellcome Medwines Rsearch Centre,
Gunnels Wood Road, Stevenage, Hertforshire SG1 2NY, U.K

G protein coupled receptor kinases (GRKs) are serine/threonine kinases that

phosphorylate G protein-coupled receptors to initiate homologous
desensitization. Of the six mammalian GRK subtypes, four GRK4 splice
variants, named c~, 13,,/, and 5, have been cloned from human testis RNA.
They differ in whether they contain one or both of two inserts, encoded by
exons II and XV. Northern blot analysis revealed that the testis is the major
site of GRK4 expression. Western blot and immunocytochemistry have shown
that only GRK43' is expressed at the protein level in the testis, specifically in
the spermatozoa (1).
In the present study, we have found that in addition to exons II and XV, exon
XIII can also be alternatively spliced, giving rise to four additional isoforms
of GRK4. We propose that the novel isoforms be designated
~, ~, rl, and 0, corresponding respectively to ~, 13, ~/ and 5 lacking the insert
encoded by exon XIII. A two-step PCR experiment was performed to
establish which of the 8 isoforms were expressed as mRNA in human testis.
The first step used primers that were specific for either the presence or
absence of exon I1, and for the presence or absence of exon XV. In this way
we were able to amplify specifically each of the four known isoforms
(GRK4c~, 13, y and 5). The products of this first round of PCR were then
diluted and a second PCR was performed using primers flanking exon XIII.
In this second round amplification, the size of the fragment generated was
diagnostic of the presence or absence of exon XIII. Using this approach, all
eight forms of GRK4 could be amplified from human testis eDNA, but none
were amplified from control eDNA (from human substantia nigra).
Western Blot analysis of human spermatozoa membrane-associated proteins
confirmed the presence of only one GRK4 isoform. However, contrary to the
previous finding (1), the c~ subtype appears to be the endogenously expressed
GRK4 isoform. This conclusion was based on co-migration studies and
immunoblotting with antibodies specific for different GRK4 isoforms.
(1) Sallese, M. et al. J. Biol. Chem. 272, (1997) 10188

s 108

(Su/13.1/062)

Abstracts F E B S ' 9 9

Epstein-Barr virus-derived vectors

for inducible gene expression in mammalian cells
K. Van Craenenbroeck, P. Vanhoenacker, G. Haegeman

(Su/13.1/063)

Department of Molecular Bsology, Flanders lnstituw for Btotechnology Umverstty of Gent, K L Ledeganckstraat 35. 9000 Gent, Belgtum

Regulated expression of a foreign gene in mammalian cells is often desirable,

especially when constitutive expression of the protein is toxic to the cell. To
this end, we have investigated the use of episomal vectors in combination
with the type l interferon-inducible Mx promoter for high, stable and
inducible expression of the reporter gene product, chloramphenicol
acetyltransferase (CAT), and the human serotonin receptor 5-HTm, a protein
of pharmaceutical interest. Mammalian cells provide the most authentic
background for the expression of G-protein-coupled receptors, as they
comprise all the components necessary for the interaction with G-proteins and
for downstream signaling.
Here, we report on the usefulness of the vector p220.2, an Epstein-Barr virus
(EBV)-derived episomal system. EBV is a herpes virus with a large doublestranded DNA genome that infects human B-lymphocytes and some epithelial
cells; it establishes itself in the nucleus in a latent state as a circular,
muhicopy and extrachromosomally replicating plasmid. It has been well
established that plasmids bearing the EBV replication origin (oraP) can be
stably maintained in human cells if EBNA-1, the only viral protein necessary
for episomal maintenance, is expressed [I ]. The stability of the vector and the
efficient segregation into daughter cells is of great importance for its utility in
stable gene expression.
Using CAT as a reporter, we tested the usefulness of the EBV vector in
combination with the inducible Mx-promoter to achieve regulated gene
expression. Moreover, we also used this episomal vector for stable and
inducible expression of the h5-HTIB receptor. To this end. we inserted an
expression cassette MxCAT or Mx5-HTIs,in the same position m p220.2, but
in two orientations with regard to the oriP sequence. After stable transfection
of these vectors into the human cell lines MG63, HeLaH21 and 293, as well
as the monkey cell line Vero, we found that they were stably maintained and
that no DNA rearrangements occurred. Concerning the expression levels after
induction of the Mx promoter, we consistently found that in all cell lines
tested an orientation-dependent expression was apparent by which a higher
expression was obtained when the transcriptional direction of the expression
cassette was in the same orientation as oriP. The expression levels obtained
remained stable for several months.

(Su/13.1/064)

A soluble form of the head-activator binding protein

W. Hampe, I. Franke, S. Hoffmeister-Ullerich, I. HermansBorgmeyer, J. Urny, B. Riedel, J. Lintzel, H.C. Schaller
Zentrum fiir Molekulare Neurobiologie, lnstitut fiir Entwtckhmgsneurobiologie, Hamburg, Germany; hampe@uke.uni-hamburg.de

The neuropeptide head activator (HA) is conserved from coelenterates to

mammals as is its growth-factor function in the development of the nervous
system. HA is secreted by nerve cells bound to a large carrier complex. It acts
on a membrane-bound receptor which triggers a G-protein coupled signaling
cascade. HA enhances proliferation of neuronal precursor cells, survival of
neurons in culture, and neurite outgrowth, in hydra HA is important for head
specific determination and differentiation processes,
Photoaffinity labeling using a radiolabeled HA analogue indicated a molecular
weight of 200 kDa for the hydra HA binding protein HAB. Using sequence
information from the affinity purified protein, its eDNA was isolated. Sequence
analysis revealed HAB to belong to a novel class of receptors containing one
transmembrane and a small intracellular domain. The amino-terminal
extracellular part consists of a module found in the yeast sorting protein VPS 10
which is followed by repeats typical for the LDL-receptor family and two
fibronectin type III domains. Using a specific polyclonal antiserum a protein of
200 kDa was detected. It was not only found in hydra membranes, hut also in a
soluble form. Site specific proteolysis indicates that the soluble protein is Cteminally truncated, missing the transmembrane domain. Part of the membranebound HAB gets soluble after salt- or pH changes indicating that it is only
peripherally attached. Soluble HAB is released from regenerating hydra in
similar kinetics as the HA peptide.
A mouse homologue with the same combination and order of domains showed
highest expression in specific neurons of the adult brain. Early in brain
development mRNA was detected by in situ hybridization in cortical neurons of
the telencephalon, whereas it was absent from other structures of the forebrain.
This unique expression pattern is consistent with a neurotrophic function of
HA in certain areas of the developing nervous system. Specific antisera indicate
the existence of a soluble form also for the mouse homologue in addition to the
membrane-bound form. Future studies have to show whether HAB is secreted
together with HA or whether it acts as a (co)receptor for HA on target cells.

Homer is a targeting protein for metabotropic glutamate

receptors in neurons
L. Fagni', F. Ango', J.P. Pin I, P.F. Wodey b and J. Bockaerta
"CNRS, 141 Rue de la Cardonille, 34094 Montpellier Cedex 5, France
bJohns Hopkins University, Baltimore, MiD 21205, USA

Homer 1a is a protein that specifically binds to group I metabotropic glutamate

receptors (mGluRs), but its role is still unknown. We investigated whether this
protein could control the targeting of one of these recepors, mGtuR5a, in
cultured mouse cerebellar granule cells. One day after plating, these neurons
were transfected with vectors expressing mGlugSa and/or Homer la tagged at
their N-terminus with HA and e-myc epitopes, respectivley. One week after
transfection, neurons were labeled with specific antibodies raised against the
tags.
When Homer la or mGluRSa were transfeeted alone, Homer la was expressed
in neurites and cell bodies, while mGluR5 was expressed in cell bodies only.
When mGluR5 and Homer la were cotransfected, both Homer la and
mGluR5a were expressed in cell bodies and neurites. After deletion of the Cterminus of mGluRSa which contains the interaction site with Homer la, or
point mutation in these C-terminus region that suppressed interaction of
mGluR5a with Homer la, mGluR5a was found in cell bodies and was no longer
targeted to neurites, even in the presence of transfected Homer la.
Homer l a is the product of an early gene that is induced by elevated neuronal
activity resulting in epileptic seizures or long-term synaptic plasticity. We
depolarized our cultured neurons by 15-30 rain incubation in the presence of
high concentrations of K channel blockers (TEA and 4-AP) or ionotropic
glutamate receptor agonists (N-methyl-D-aspartate + kaninate). These
treatments induced expression of Homer l a in both cell bodies and neurites, as
objectivated by immunolabeling experiments. In the same neurons, expression
oftransfected mGluR5a followed expression of induced endogenous Homer l a
in both cell bodies and neurites.
These results suggest that the inducible Homer la protein is a key signal for
the targeting of mGluR5a to the neuritic compartment in mammalian central
neurons.

(Su/13.1/065)

Fully functional serotonergic or catecholaminergic cells

derived from a neuroepithelial cell line.- O. Kellermanna,
S. Mouillet-Richard~, C. Tournois a'b, S. Lorica, V. MuteF, JM.
Launay b - CNRS URA 1960, Institut Pasteur, 75724 Paris, ~H6pital
Lariboisidre, 75010 Paris (FR), ~Hoffmann-La-RocheAG, Basel (CH)

The F9-derived 1 C l l cell line behaves as a bipotential neuroectodermal

progenitor. In the absence of extracellular signal, 1C11 ceils exhibit a stable
epithelial morphology, express nestin, a marker of neuroepithelial cells, and
genes involved in neuroectodermal cell fate such as Notch and its ligands.
Upon appropriate induction, 1C11 precursor cells convert into fully
functional, either serotonergic (1Cl1") or catecholaminergic (1Cl1"*)
neurons. The two differentiation pathways are mutually exclusive and recruit
nearly 100% of the cells. Along either program, the time-dependent
acquisition of neurotransmitter-associated functions is accompanied by the
onset of neurite extensions and of neuronal markers (N-CAM,
synaptophysin, yy-enolase and neurofilament). 1Cl1" cells acquire a
functional 5-HT transporter and thereby a complete serotonergic phenotype
within 4 days, while 5-HTIB/D , 5-HT2B and 5-HT2A receptors are
sequentially induced. The 1Cl1"* catecholaminergic phenotype is complete
within 12 days and includes an ctm-adrenoceptor and a functional
norepinephrine transporter. The serotonergic and adrenergic receptors
specifically induced along each pathway autoregulate the implementation of
the respective phenotype. 1 C l l thus behaves as a neuroepithelial cell line
with a dual bioaminergic fate. The 100% phenotypic conversion of the 1 C l l
clone in vitro and the receptor-mediated regulation of each program have
implications for dissecting the interplay between genetic and epigenetic
factors involved in the control of serotonergic or catecholaminergic
differentiation.

Abstracts FEBS'99

(Su/13.1/066)

Signaling pathways of the serotonin-2B receptor

s109

(Su/13.1/067)

J.-M. Launay ~, P. Manivet a, C. Toumoisa, S. MouilletRichardb, S. Loricb, L. Maroteaux c, O. Kellermann b - "Service

S. Le Croma, J-D. Vincenta, H.B. Niznik b, P. Vernier a

a Institut A~redFessard, CNRS UPR 2212, 91198 Gifsur Yvette, France
Clarke lnstttute of Psychiat~, Toronto, Canada

de Biochimie, H6pital Lariboisidre, 75010 Paris, ~CNRS URA 1960.

Institut Pasteur, 75724 Paris, "IGBMC, 67404 lllkirch, France

So far, the in vivo signaling processes coupled to serotonin-2B (5-HT2B)

receptors have not yet been identified. A serotonergic cell line enabled us to
characterize four distinct transduction pathways coupled to the 5-HT2B
receptor. 1Cll* serotonergic cells result from the induction of 1Cll
neuroepithelial precursor cells by dibutyryl cAMP. At day 4 of
differentiation, 1Cll* cells express a complete serotonergic phenotype
including 5-HT1B/D, 5-HT2A, and 5-HT2B receptors. 5-HT1B/D and 5HT2B receptors are already functional as soon as day 2 of differentiation,
while 5-HT2A receptors become expressed at day 4.
5-HT2B receptors present on 1Cll*d2 cells appear to be coupled to (i)
phospholipase C13 activity through Gll, (ii) the ras/MAPK pathway, (iii) a
cytosolic phospholipase A2-mediated release of arachidonic acid (AA)
through G12. Crosstalk experiments revealed that activation of the 5-HT2B
receptor inhibits the 5-HT1B/D receptor function via a cyclooxygenasedependent AA metabolite. Finally, 5-HT2B receptors expressed by 1C1 l ' d 2
cells are coupled to both NOS-I (neuronal) and NOS-2 (, inducible ,,)
activities. The C-terminal domain of the 5-HT2B receptor, which contains a
type I PDZ target motif, is critical for its functional coupling to NOS-1 and 2. Besides, the 5-HT2B receptor/NOS-2 coupling also requires G13. These
findings highlight novel G-protein coupled receptor-dependent regulations of
NO production.
The 1C11 cell line thus allows the crosstalks between the multiple pathways
and the targets of the 5-HT2B receptor to be studied in an integrated
serotonergic context.

(Su/13.1/068) Molecular pharmacology ofvasopressin receptors.

B. Mouillac a, N. Cotte a, S. Phalipou ~, D. Morin ~, T. Durroux~,
M.N. Balestre a, R. Seyerb, M. Hibert~, C. Barberis '~.
"INSERM U469, nCNRS UPR9023, 141 rlw de la cardonille. 34094 ~4ontppellier, France, 'CNRS ERS655, 74 rolae du rhin. 67401 lllhtJ ~h. France.

Striking progress has been recently made m the knowledge of the structure
and the function ofvasopressm (AVP) and oxytocm (OT) receptors [I]. "l-he
AVP Via receptor subtype is one of the first ncuropeptide r=.-c?!,~r for
',vhic]l a three-dm~ensional m o d e l o f interaction \~ lib !ty: rtaI.ura[ 13oi125o:!c has

been proposed and expernnentally validated. :\VP binding l~kas been

demonstrated to occur in a pocket formed by the rmg-hke arrangement of the
seven transmembrane domains, in a position equivalent to that described for
cationic neurotransmitters. Moreover, a residue responsible for AVP and OT
receptor subtype selectivity has been identified m the first extracellular loop.
Localization of binding sites for other agonists and antagonists has now been
investigated by a combination of molecular modelling, site-directed
mu~genesis [2] and photoafflnity labelling [3] studies. V2 antagonists, which
haq~been shown to be partial agonists on a constitutively active human V2
receptor mutant, have their species-selective binding controlled by two
residues located in the second extracelhilar loop and the seventh
transmembrane domain, respectively. Via-selective cyclic and linear peptide
antagonists, as well as nonpeptide antagonists, all interact with a central
transmembrane binding site significantly overlapping that of the natural
hormone AVP. Functional properties of each ligand could be determined by a
unique set of interactions in the Via receptor. Our current view of the
functional architecture of AVP and OT receptors will be summarized and
critically discussed.
[ l ] Barberis C. et al., J. Endocrinol., 156, (1998) 223.
[2] Cotte N. et al., J. Biol. Chem., 273, (1998) 29462
[3] Phalipou S. et al., J. Biol. Chem., 272, (1997) 26536.

Comparative regulation of DI dopamine receptors

subtypes in vertebrates

In vertebrates, dopamine behaves as a modulatory neurotransmitter by

acting on two classes (D 1 and D2) of G proteins-coupled receptors. Each of
these classes include three subtypes emerging after two gene duplications
steps which occurred early in vertebrates evolution. Expression of D1
receptors subtypes (D1A, D1B and DIC) in heterologous cells led us to
delineate homologous functional characteristics, derived or conserved for
each D1 receptor vertebrate subtypes. Here, our experiments focused on
desensitization, one of the fundamental mechanisms of receptor regulation.
Quantification of desensitization consequences on intracelhilar
messengers was performed by cAMP accumulation assay. D1A receptor
desensitized strongly and quickly. DIB receptor hold high constitutive
activity and a desensitization profile close to D1A but with a weaker
amplitude, similar as what is observed in human. D1C receptor, a subtype
which does not exist in mammals, was not able to undergo desensitization
and conserved a high basal activity in COS cells. Moreover, fusion between
each of the D1 receptors subtypes from eel and Green Fluorescent Protein
allowed us to analyse by confocal microscopy the different morphological
phenomenon of desensitization (uncoupling and endocytosis). Nevertheless,
the use of transient transfection and the lack of total disappearance of
receptor from the made membrane quantification very difficult.
By using this two complementary approaches, we should now be able
to investigate the multiplicity of intracellular pathways of D1 receptors
regulation and to fully describe the conserved cellular functions typical of
each receptors subtypes.

(Su/13.1/069)

Modulation of Somatostatin Receptors, Somatostatin

Content and Gi Proteins in Rat Brain by Substance P.
L. Puobla, M. Griera, M.C. Boyano-Ad~inez,R.M.IzquierdoClaros, A. Ocafia, S. de Frutos, E. Arilla. Dept. of Biochemistry
and Mol. Biol., Alcald University, Alcald de Henares, Madrid, Spain.

The peptide Substance P (SP), a member of the taehyldnin family, and

the tetradecapeptide somatostatin (SS) arewidely spreadthroughout the central
nervous system where they play a role as neurotransmitters and
neuromodulators. A colocalization of both neuropeptides has been
demonstrated in several rat brain areas. Moreover, SP receptors have been
localized in rat cortical and hippocampal somatostatinergic cells. The aim of our
study was to investigate whether SP could modulate SS signalling pathways in
the rat frontoparietal cortex. The results show that acute (24 h) but not chronic
(7 days) administration of SP (50, 250 or 500 gg/kg,i.p) induces an increase in
the density of SS receptors (650 + 26, 688 30 and 634 26 fmol/mg protein
vs. 526 16 fmol/mg protein, p<0.01, p<0.01, p<0.05 respectively) in rat
frontoparietal cortical membranes as compared to controls. SS-like
immunoreactive content (SSLI) was likewise increased in the frontoparietai
cortex after acute administration of either 250 or 500 gtg/kg of SP (32.32.9
and 25.12.1 ng SS/mg protein vs 18.01.6 ng SS/mg protein, p<0.001 and
p<0.05, respectively) whereas the lowest dose (50 gg/kg) did not induce any
significant effect. Western blot analysis of the frontoparietal cortical cq and oh
subunits of inhibitory Gi proteins demonstrated a significant decrease in Gkx~
levels in frontoparietal cortical membranes from SP (250 gtg/kg)-treated rats as
compared to controls. Since SS receptors are coupled to the AC system via Gi
proteins, we also examined basal and FK-stimulated AC activity as well as SSmediated inhibition of the enzyme. No significant differences were detected in
either basal or FK-stimulated AC activity in SP (250 gg/kg)-treated rats. SSmediated inhibition of basal and FK-stimulated AC activity, however, was
significantly decreased in SP (250 gtg/kg)-treated rats. These results
demonstrate the existence of a relationship between the SP and the SSergic
system which may be involved in the regulation of CNS functions such as
memory and learning, nociception and the sleep-wakefulness cycle,

s 110

(Su/13.1/070)

Abstracts FEBS'99

The 14-3-3 ~ protein interacts with the third intracellular

loop of different c~ adrenergic receptor subtypes
L. Prrzeau*, J. Richman, S. Edwards and L. Limbird
Dept Pharmacology, Vanderbilt Uni Med Ctr, Nashville, TN 37232-6600,
USA *now at CCIPE, rue de la Cardonille, 34094 Montpellier, France

It has been shown that ix2 ARs are localized to and function on the
basolateral surface in polarized renal epithelial cells; targeting to this surface
involves regions within the transmembrane domains, whereas the third
cytoplasmic (3i) loop is critical for surface retention of the receptors [1]. In
order to identify proteins that may contribute to this retention mechanism, in
vitro translated [35S]Met-labeled genl0 fusion proteins with the 3i loops of
the cx2A AR (V217-A377), ~2B AR (K210-W354) and ff.2C AR (R248V363) were used as ligands in gel overlay assays. The [35S]Met-3i loops of
the t2B and c2C ARs identify a protein doublet of -30 kDa in cytosolic
extracts derived from MDCK cells or pig brain cortex; the ct2A3i loop
interacts with the same proteins, but to a lesser extent and requiting a longer
incubation time. Purification of these proteins by sequential DEAE and size
exclusion chromatography revealed, via protein microsequencing, that they
are the ~ isoform of 14-3-3 proteins. These interactions are also obtained
with native ~2 AR subtypes assessed using a solid phase binding assay with
[35S]Met-14-3-3~ as a probe and solubilized x2 AR as the target. In
addition, interactions with native receptors are diminished if not eliminated in
structures where the 3i loop has been deleted. Attenuation of these
interactions in the presence ofa phosphorylated Raf-1 peptide corresponding
to the 14-3-3 binding domain (residues 251-266) but not by the nonphosphorylated peptide is evidence for the functional specificity of these
interactions. These studies provide the first evidence for G protein-coupled
receptor interactions with 14-3-3 proteins and may provide a mechanism by
which the receptors could be retained on the surface, differentially localized,
or enriched in multi-protein complexes to provide a higher order of specificity
in coupling to signal transduction pathways.

(Su/13.1/071) INTRACELLULAR LOCALIZATION AND SOLUBILIZATION

OF
THE
HOMOPHTHALAZINE
BINDING SITE G Szabt, K Tihanyi Egls Pharmaceuttcals.
Btochemtstr_v Dept , Budapest. POB 100, H-1475 Hungary

Homophthalazines (structurally they are 2,3-benzodiazepines) have diverse

effects in the CNS Some members of this family such as GYKI-52466
show an AMPA antagonist effect while others like tofizopam (Grandaxin)
and girisopam (EGIS-5810) have anxiolytic and antipsychotic properties
High affinity (Kd about 10 nM) homophthalazine binding site has been
described in the striatum [I,2]. Our aim was to characterize this binding
site of [~H]-girisopam by intracellular localization and solubilization We
have separated the subcellular fractions according to Gray and Whittaker,
and localized this binding site mainly in the microsomal fraction of the rat
striatum The binding site was solubilized and the kinetical parameters and
other properties of the solubilized binding site determined The solubilized
proteins were futher purified by column chromatography The results may
help us to understand the mechanism of action of homophthalazines in the
CNS
[1] C. Salamon, E J Horvath, M Fekete, P Aranyi, FEBS-Lett 1992 308
215-7
[2] E. J Horvath, J Hudak, M Palkovits, Z Lenkei, M I Fekete, P
Aranyi, i993 236 151-3

[1] Keefer, J. et al., J. Biol. Chem., 269, (1994) 16425.

(Su/13.1/072)

Stimulation of MAP kinases and morphogenic changes

induced by extracellular ATP in pituicytes cultured from
adult rat neurohypophysis
S. Thmon, J.D. Troadec, B. Derijardand P. Poujeol

(Su/13.1/073)

Regulation of the stimulus-secretion coupling

by extraceilular ATP in the rat neurohypophysis
J.D. Troadec, S. Thirion, D. Petturiti, P. Poujeol
UMR CNRS 6548 Lab. Physio. Cell. Molec., 06108 Nice. France

UMR CNRS 6548 Lab. Physio. ('ell. Molec., 06108 Nice, France

In the neurohypophysis, we have previously shown that ATP, co-released

with neurohormones, regulates secretion via a presynaptic P2x2 receptor [ l 1.
Furthermore, we established the presence of a P2Y receptor on pituicytes, the
major glial cells in this organ. In these cells, ATP stimulates a |Ca2*]t
increase, thereby inducing a sustained K + efflux via CaZ+-activated K
channels 12]. In the present study, we examined the effects of ATP on the
morphology of pituicytes in the context of the known plasticity of these cells
in vivo [3 I.
The predominant cell type of primary cultures of rat pituicytes was
flattened, with some cells having one or more processes. Short-term (1 h)
treatment of these cells with extraeellular ATP induced a stellate morphology
in most cells, characterized by generation and elongation of several finely
branching processes and a phase-bright rounded cell body.
In addition, we tested the effects of ATP on MAP kinases activation.
After treatment with the stimulatory agent, the cells were lysed, and a western
blot was performed with an anti-active MAP kinases (p42/p44). We found
that treatment with 100 ~uM ATP of quiescent primary cultures of pituicytes
activates mitogen-activated protein kinases (ERKs) with a maximal effect after
5 minutes. The effects of ATP on the morphogenic changes and on the
activation of MAP kinases were mimicked by PMA (100 nM).
It appears that ATP could have some morphogenic and trophic roles on
pituicytes, via the activation of the MAP kinases. This action of ATP could be
responsible for the morphological plasticity of the rat neurohypophysis
observed under intense stimulations of the gland, and shown to facilitate the
secretion. These results are in agreement with the hypothesis that the
purinergic pathway is involved in the regulation of neurohypophysial
secretion, via glia-neuron interactions.
[11 : Troadecet al., J. Physlol (Lond.),511 (1998) 89.
[2] : Troadccct al., PflugersArchav.,437 ( 19991in press'.
[31: Hatton, J.Exp. Biol., 139 (1988)67.

ATP, an intracellular molecule of fundamental importance for energy

metabolism, can also play a role as an intercellular mediator. Its actions as a
neurotransmitter are well known in the peripheral nervous system, but little
data are available in the central nervous system I 1]. The neurohypophysis is a
neuroendocrine organ formed principally by neurosecretory nerve terminals
and glial cells called pituicytes. We have studied the effects of extracellular
ATP in two complementary models: isolated neurosecretory nerve terminals,
and pituicytes in primary culture.
ATP triggers an increase in [Ca2[~ and evokes vasopressin (but not
oxytocin) release via the activation of a P2x2 purinergic receptor [2]. Using a
bioluminescence technique, we have demonstrated the co-release of ATP with
the neuropeptides during neurohypophysial secretion. In
situ,
neurohypophysial glial cells are located very close to the nerve terminals, and
so are in a prime location for receiving any secretion products from these
terminals. We have also tested the effects of ATP on pituicytes. In these cells,
ATP evoked a [Ca2+]i increase by purinergic P2Y receptor activation [3]. The
use of a radioactive K tracer, 86Rb+, showed that the ATP-evoked P2Y
pufinoceptor activation triggered a K + efflux from these cells. This study led
to the conclusion that Ca2+-acfivated K channels are present.
In the neurohypophysis, extracellular ATP could regulate autocrine
and/or paracrine secretion by acting directly on neurosecretory nerve
terminals. In pituicytes in vivo, ATP-dependent K + efflux would promote a
local increase in extracellular potassium concentration, resulting in a
modulation of the excitability of neighboring nerve terminals. These results
are in agreement with the hypothesis that purinergic pathways are involved in
the regulation of neurohypophysial secretion.
[ 1] : Zimmermann, Trends Neurosci., 17 (1994) 420.
[2] : Troadec et al., J. Physiol. (Lond.), 511 (1998) 89.
[3] : Troadec et al., Pfltigers Archiv., 437 (1999) in press.

Abstracts FEBS'99

s 111

16.1 Enzymatic mechanisms and catalysis

(Su/16.1/074)

Cells, Erythrocyte membranes and plasma lipid peroxidation :

prooxidant role of T-glutamyltransferase
H. Aberkane, M. M. Galteau, M. Wellman

(Su/16.1/075)

L~tanbu/7i:chmcal ~;mverst(v. ('hemtcal PDTgmeertngDepartment.

?';0626, ~laslal~ IstanbuL Turkey

C e n t r e d u M 6 d i c a m e n t U P R E S , F a c u l t y o f P h a r m a c y , U H P . N a n c y , France.

Gamma-glutamyltranspeptidase (GGT) is a very well know cell plasma

membrane and serum circulating enzyme involved in glutathione (GSH)
metabolism, associated in general with antioxidant properties. However,
recently it has been reported that in the presence of chelated iron, GSH
metabolism by GGT leads to reactive oxygen species (ROS) generation (1-3).
This process was explained by the autooxidation of the GGT generated
metabolite of GSH, cysteinylglycine (CysGly), which is able to form thiyl and
.
.
.
oxygen radtcais
m. reactmn
with Fe +3 and 02 (1-2-3).
The aim of this work was to investigate first the relation between human
plasma GGT activity level and the degree of lipid peroxidation. Plasma lipid
peroxidation was mesured in different EDTA collected blood samples. Plasma
GGT activity was found significatly correlated with the degree of lipid
peroxidation as expressed by TBARS. The level of lipid peroxidation was also
well correlated with the degree of GGT expression in several cultured cell
lines:A2780, KYN2, Mia-Pa-Ca2, V79 and V79GGT. We further tested
GGT/GSH/Fe 3 as oxidant system in vitro at pH 7.4 using erythrocyte
membranes as substrate. The level of TBARS was found to be dependent of
GGT activity, of Fe +3, of glycylglycine and of GSH concentrations. Tire
maximal oxidation (more than 20 times of control) was found in the following
.
+3
condmon:GGT 200 U/l, GSH 2 mM, Fe 0.15 mM and glycylglycine 20 raM.
The addition of GSH and transition metal ions to the cultured cells results in an
increase of extracetlular TBARS concentration (x9) observed only in GGT
posinve cells. These results fully confirm the hypothesis about prooxidant
properties of GGT/GSH system. The most general question, in which
physiopathological conditions does the GGT play a prooxidant role and which
other biological macromolecules are potential substrates for this reaction, still
remains open and is under current investigation. The possibility that the
GGT/GSH prooxidaut system plays an important role m the
prooxidant/antioxidant balance of cardiovascular system must first be
considered.
I- Drozdz R. et al. Free. Rad. Biol. Med. 1998,25,786
2- Paolicchi A. Free. Rad. Biol. Med., I997, 2~2,853.
3- Stark A. Mutagenesis, 1991,6,241.
(Su/16.1/076)

Glycerolysis of Palm and Palm Kernel Oil by Novozym 388

H. A. Aksoy, M. Triter, A. Akova, A. Gtintekin and F. Ozbir
lstanbul Technical University, Chemtcal Engineering Department,
80626, Maslak, lstanbul, Turkey

The objective of the present work was to evaluate the usefulness of

Novozym 388 for the solvent-free glycerolysis of palm oil and palm kernel
oil at 40 C and to investigate the fatty acid profile of each triacylglycerol
(TAG), diacylglycerol (DAG), and monoacylglycerol (MAG) fractions of the
glycerolysis products. Novozym 388 is a fungal lipase from Mucor miehei
expressed in a selected strain of Aspergillus oryzae grown in submerged
fermantation and was a gift of Nova Nordisk.
Reactions using 50g oil and 500 Units Lipase/g oil were carried out at 40 C.
The molar ratio of oil: glycerol was chosen as 1:2 using anhydrous glycerol.
The reactions were followed by a thin-layer chromatography-flame ionisation
(TLC-FID) technique. After 24h, the glycerolysis product of palm kernel oil
consisted of 19.3% TAG, 20.2% MAG and 36% DAG. Under the same
reaction conditions the glycerolysis product of palm oil contained 25.7%
TAG, 12.5% MAG, 31.8% DAG.
Separation and the recovery of the TAG, DAG and MAG fractions from the
reaction products were carried out by column chromatography on florisil .
The fatty acid compositons of the pure fractions were determined by
capillary gas chromatography in a Hewlett- Packard 5890 series 2 apparatus
fitted with a FID and a data processor. TAG, MAG and DAG fractions of the
glycerolysis product of palm oil were enriched in oleic acid. Palmitic acid
content of the MAG fraction of the same product was less than original oil.
Under the same conditions, MAG fraction of the palm kernel oil glycerolysis
product was enriched in myristic, palmitic, and stearic acids.

Characterization of Immobilized Lipase

from Nigella Sativa Seeds
A Akova, G Ustun

The application of lipase biocatalysts in industrial processes (e.g, fat

hydrolysis, triglyceride modification, and ester synthesis) is now of major
commercial importance, and this interest is expected to grow over the coming
years as a wider range oflipase catalysts becomes available To date, most of
the industrial lipase enzymes have been produced from fungal resources and
they are well characterized However, lipases from higher plants have not yet
been investigated in this respect
Nigella sativa L, also known as black cumin, is a member of Ranunculaceae
family and dormant seeds have been shown as a good source of acid lipase
In this study, immobilization by physical adsorption of acid lipase from
Nigella sativa on Celite 535 was investigated Immobilization was carried out
by direct addition of the activated celite to the crude enzyme extract, obtained
from seeds with phosphate buffer (pH 7 2) The adsorption conditions were
optimized after investigating the effects of adsorption pH, temperature and
preheating temperature of Celite on the specific activity and protein loading
capacity of the immobilized enzyme
The highest recovered hydrolytic activity, 48% of the initial activity of the
enzyme solution, was found when the lipase was immobilized on Celite
preheated to 110 C using an immobilization buffer adjusted to pH 6 at room
temperature The immobilized lipase exhibited a specific activity as high as
4 6 U / m g protein and showed K,,1 and V,,,,, values lower than those of the
flee enzyme The optimum pH (pH 6) and temperature (40 "C) of the free and
immobilized enzymes remained unaltered The thermal stability of the
Nigella sativa lipase was greatly improved by immobilization

(Su/16.1/077)

lntraprotein electron transfer between tyrosine and

tryptophan in DNA photolyase from Anacystis nidulans
Corinne Aubert', Paul Mathis', Andr6 P. M . E k e r b & Klaus BretteL'
~SecUon de alo~nerg~tlque (CNRS URA 2096), CEA Saclay. 91191 Olf-sur-Yvette
Cedex, France
q)epartmeat of Cell Biology and Genetics, 3000 DR Rotterdam. The Netherlands

DNA photolyase is a single polypeptide of 50 to 65 kDa,

which uses blue or near UV light to repair UV-induced DNA lesions.
This enzyme contains a flavin adenine dinucleotide (FAD) as the
essential catalytic cofactor, and a second chromophore (either a
reduced folate or a 8-OH-5-deazaflavin) which has the sole function
of absorbing light and transferring excitation energy to the FAD
cofactor [1]. The FAD cofactor must be in the two-electron reduced
form (FADH-) for enzymatic activity. In the purified enzyme, the
flavin adenine dinucleotide is usually present in the blue semiquinone
form FADH. This radical is stable in the dark but can be reduced to
FADH- by illumination by visible light.
In the present work, we study flavin photoreduction by flash
absorption spectroscopy in DNA photolyase from Anacystis nidulans.
We show that after selective excitation of FADH by a 7ns laser flash,
fully reduced FADFF is formed in less than 500 ns by electron
abstraction from a tryptophan residue. Subsequently, a tyrosine
residue is oxidised by a tryptophanyl residue with t~z2 = 50 Its. The
newly discovered electron transfer between tyrosine and tryptophan
occurred for about 40 % of the tryptophanyl radicals, while 60 %
decayed by charge recombination with FADH- (t~;2= 1 ms). The
tyrosyl radical can also recombine with FADH'- but at a much slower
rate (tv2 = 80 ms) than Trp . In presence of an external electron donor,
however, TyrO" is re-reduced efficiently in a bimolecular reaction
which leaves FAD in the fully reduced state FADH-. These results
show that electron transfer from tyrosine to Trp is an essential step in
the process leading to the active form of photolyase. They provide the
first direct evidence that electron transfer between tyrosine and
tryptophan occurs in a native biological reaction
[1] Sancar, A., Science, 272, (1996), 48.

s112

Abstracts FEBS'99

(Su/16.1/078) Structure changes of annexin VI upon ATP binding

J.Bandorowicz-Pikula t, A.Wrzosek', S. Pikula'& R. Buchet 2

(Su/16.1/079)

INencki htstitute, 02-093 Warsaw, Poland. 21~tb. Phys. Chim. Biol.,

Universit~ Lyon I-CNRS, 69622 Vtlleurbamle, Fratlce

Secondary structure changes induced by nucleotide-binding to porcine

liver annexin VI (AnxVl) were probed by reaction-induced difference
spectroscopy (RIDS).
Photorelease of the nucleotide from
ATP[Et(PhNO2)] in the presence of Ca 2", produced RIDS of AnxV[
characterized by small but reproducible changes in the amide-I region,
and an appearance of a new band at 1582 cm a. The magnitude of the
infrared-changeT, was comparable to RIDS of other ATP-bmdmg"
" proteins,
such as Ca -ATPase, creatine kinase (1) and arginine kinase (2). These
results suggested that the ATP binding to annexin VI was unlikely to Ix:
caused by an accidental binding site, confirming the previous
fluorescence results (3). Analysis of RIDS in the region of amide-I
indicated that 5-6 amino acid residues were directly involved during ATP
binding site. One likely interpretation of the RIDS (decrease of the 1656cm ~ band and increase of the 1621-cm 4 band) is a slight distortion of ethelix structure and/or formation of hydrogen bonds. The ATP-binding
induced secondary structure changes can be detected by circular
dichroism (CD), although the magnitude of CD changes were greater than
these observed on the RIDS. Supplementing the Ca 2 assay medium with
ATP caused a 5% decrease in o~-helix content in the protein in favor of
unordered structure. The involvement of Tyr residues in the vicinity of
ATP binding site of AnxVI was observed by intrinsic fluorescence
spectroscopy. The inrinsic Tyr fluorescence was quenched by the
addition of ATP and, to a lower extent by ADP and cAMP. Taken
together, these results provide strong evidence for ATP being a ligand for
AnxVI hz vitro, leading to secondary and tertiary structure changes of
AnxVl. The nature of these structural changes and the functional
implications of this phenomenon in vivo remain to be elucidated.
(1) C. Raimbault et al., Eur. J. Biochem. 240 (1996) 134.
(2) C. Raimbault et al., Eur. J. Biochem. 244 (1997) 343.
(3) J. Bandorowicz-Pikula et al., Eur. J. Biochem. 248 (1997) 238.

(Sud16.1/080)

n~a~

ann a ~ - a a n ~ m ~ a e ~ a ~ r ~ e n z y m e s

O. 13ellen,, IL D'eniela,R Gareilb,J. Jo~ef~vic~

aURM2, L.R.M. UMR 7540 CNRS, 93430 Villetaneuse. France
bENSCP, UMR 7575 CNRS, 75231 Parts cedex 5, France

Fucoidan, a polysaccharide extracted from brown algae, is mainly

composed of sulfated L-fucose. Fucoidan as shown to display a wide range
of
biological properties
(as antithrombotic
and
antitumoral
activities)However the structure of this polysaccharide is still debated,
particularly the nature of the glycosidic bonds and the distribution of the
sulfated groups. Because of its polydispersity and its structural
heterogeneity, the study of fucoidane requires its degradation in order to
obtain oligosaccharides easier to study. Different structural models have
been proposed based on the study of chemically degraded fucoYdans.
These degradations proceeding under conditions likely to alter the overall
polysaccharide structure, ve have implemented an enzymatic way of
degradation.
Enzymes acting on the fuco]'dan were searched in digestive gland
of the manne mollusc Pecten maximus. We have purified enzymes with
fucosidase and sulfatase activities. Their structural and functional
characteristics were studied. We have developed a method to measure the
sulfatase activity using capillary electrophoresis. This method allow to
follow the enzymatic desulfation of the fucoYdan, and to determine the
regioselectivity of this enzyme on standards of sulfated fucose. The
fucosidase activity on fucogdan was assayed by high-performance liquid
chromatography with a pulsed amperometric detection, and with a
spectrophotometric method using an enzymatic test.
The purified enzymes are considered as potential tools for the
structural study of the fuco]'dan, by operating a selective degradation of the
polysaccharide. Moreover obtaining a new type of enzymatic drgraded
fucoi'dan will lead to have polysaccharides endowed with different
biological properties from those already described.

Kinetic properties of the Malic enzyme purified from a C4

plant (sugarcane).
P. Baret I, C. Queiroz~', C. Rouch I, J.C. Meunier 2 and F. Cadet I.
1
2

Umv Rdumon. Labo de Btochtmw. 97715 St-Dents. Reumon

I N A.P G Labo de (Tzmue Btologtque. 78850 FhtvervaI-Grtgnon. France

In the Hatch and Slack photosynthetic cycle, NADP dependant

Malic enzyme catalyses the decarboxylation of L-malate into pyruvate. We
have purified this enzyme from sugarcane leaves (C4 plants) alter sucessive
chromatographic columns (ion exchange, hydroxyapatite and molecular
sieving columns). The purification procedure is almost identical to that
described by lglesias and Andrea [1] but we subsequently observed
significant differences in the kinetic properties of the enzyme.
When we compared the kinetic parameters of the malic enzyme we
purified at pH 8.0 to those of Iglesias and Andrea [2], we interestingly
observed that (i) Km towards L-malate and NADP were higher (570 p.M
against 120 gM for L-malate and 110 gM against 4,6 I.tM for NADP), (ii)
specific activity was higher (137 gmol/min/mgagainst 71 gmol/min/mg)and
(iii) Ka towards Mg 2. was greater (53 ~tM versus 1 I.tM). At pit 7.0, similar
results were obtained but we did not observed any inhibition of the enzyme
with excess substrate.
Main kinetic study results will be presented and the differences
observed between our malic enzyme and that of Iglesias and Andrea [2] will
be discussed.
References
[1] lglesias A. A. and Andrea C S., l'hmt ('ell Pt~vsiol. 30, (1989), 399.
[2] Iglesias A A. and Andrea C. S.. Plant Pig,sial, 92, (1990), 66.

(Su/16.1/081)

Active sites of angiotensin-converting enzyme are strongly

dependent
P. Binevski, E. Sizova, O. Kost
Chemistry Department. Moscow State Universio', 119899 Moscow, Rusvu~

Angiotensin-converting enzyme (ACE, EC 3.4.15.1) is a Zn 2+peptidase, which possesses a wide specificity as it hydrolyses a range of
natural and synthetic peptides. Molecular cloning of the somatic form of
ACE demonstrated that the enzyme is composed of two homologous
domains referred to as N- and C-domains, each domain containing a
catalytic site [1]. Functioning of two domains is not yet properly understood
and it is still unclear whether they act independently in the whole ACE
molecule.
We used three bovine ACE isoforms - full-length pulmonary form,
testicular form (which represents C-domain of somatic ACE) and N-domain
which was obtained by limited proteolysis by trypsin of full-length enzyme to investigate how do domains function within full-leng~h isoform. We have
shown that the hydrolysis of several synthetic tripeptide substrates by all
three isoforms was characterised by different catalytic parameters. The
kinetic parameters, kcat and Km, of the hydrolysis of Hip-His-Leu and FaPhe-Gly-Gly by all three ACE forms were found to be very similar. We
didn't fred any significant difference in Michaelis constants values in the
reaction of hydrolysis of Cbz-Phe-His-Leu, Fa-Phe-AIa-Ala and Fa-PhePhe-Arg by two-domain and single-domain enzymes but kcat value appeared
to differ markedly. The value obtained for somatic form was mean quantity
of constants determined for single-domain enzymes. The analysis of
different kinetic schemes allowed us to demonstrate that N- and C-domains
in fulMength somatic enzyme are strongly dependent, that is binding of
substrate molecule on one of the active sites of ACE prohibits simultaneous
binding of another substrate molecule on another active site.
References:
[l] F. Soubrier, et al., Proc. Natl. Acad. Sci. USA, 85, (1988) 9386.

Abstracts FEBS'99

(Su/16.1/082)

s113

Catalytic mechanism of an enantioselective and enantioconvei'eenteDlant epoxide hvdrolase

E. Bl6e~, S. Summerera,H. l~o~zniauxb. A. V,an Dorsselaer t~,
G. Hann-Archipoff, a, S~Utsumfe, F. SchubeO

alBMP-UPR 406, France; UMR 7509, S~rasbourg, France; CRes. lnst.

for food science, Kyoto University, Japan;a, UMR 7514, lllklrch, France

(Su/16.1/083)

Inhibiting protein-protein association : the example of

interface inhibitors of HIV-1 protease
N. Boggetto ~, A. Bouras b, E. De Rosny~, Z. Benatalahb, M.
Reboud-Ravaux~ & S. SicsicL

"lnstitut J. Monod, Univ. Paris 6 & 7, 7"43, 2 place Jussieu 7.5251 Paris Cx

05; t'Biocis-CNRS, 5 rue J. B.Cldment, 92296 Chgttenay.MalabryCx, France

Multiple forms of epoxide hydrolases (EHs) exist in plants, depending on

the species and the subcellular fractions examined. These hydrolases differ
by their substrate specificity and their enantioselectivity. For example,
while soluble EHs from maize, rice or wheat hydrate at the same rate both
enantiomers of 9,10-epoxystearic acid, those from potato or soybean
present a strong enantioselectivity in favor of 9R,10S-epoxystearic acid.
But, all the plant EHs examined so far share a strict and rare
stereoselectivity i.e. : they hydrolyze their substrates with a very high
enantioconvergence yielding a single chiral diol product of R,R
configuration even when starting from a racemic fatty acid epoxide. We
have proposed a model in which the oxirane ring, regardless the nature of
its substituents, is docked in a unique position within the active site that
permits the inversion of configuration to take place only on the carbon that
has the (S) configuration. An efficient yeast expression system for the
enzyme from soybean that facilitates the production of native and mutant
enzymes for mechanistic analysis is described. The catalytic triad residues
of soybean epoxide hydrolase are proposed to be Asp 126, His 320, and
Asp 285. Replacement of these residues to Leu, Leu and Tyr respectively,
resulted in a dramatic loss of activity for 9,10-epoxystearate. D 126N and
D285N mutants undergo hydrolytic autoactivation, confirming that these
Asp are in the active site. The reaction mechanism of soybean EH proceeds
v/a a covalently bound ester intermediaire, as shown by experiment with
the His 320-->Tyr mutant of EH in which the ester intermediate could be
trapped and visualized using electronspray mass spectrometry.

A novel approach to inhibitor design for oligomeric enzymes is

based on dissociation of enzyme subunits. Disruption of the dimer interface
of HIV-1 protease destroys the active site and results in loss of viral
proteolytic activity. Our strategy was to target the extended antiparallel 13sheet formed by interdigitation of N-terminal (residues 1-4) and C-terminal
(residues 96-99) 13-strands of each monomer (Figure). We designed
conformationally constrained hairpins consisting in two peptidic strands
attached to an aromatic scaffold Ar susceptible to interact with the Cterminal end of the enzyme monomer preventing the formation of the active
dimer.
+~lJ, IIIV.I prl~t~

Inactive n ~ n m r - m h d ~ l l ~
~mp~x

Innl~tor

I I:::: I

anllparalkl [~-sh~t

1o~,

HO~FNLT

The proof that these molecules act by inhibiting the dimerization

interface was obtained through kinetic analyses. The inhibition was
influenced by the nature of the spacer (2,7-naphtalenediol > 2,6pyridinediol >> resorcinol), the length of the peptidic chain (tripeptide >
tetrapeptide >> dipeptide) and their sequence. Molecules with only
tripeptidic strands which did not reproduce the N- and C-termini of HIV- I
protease are among the best values described for inhibitm-s of dimerization
(K~d = 0.56 gM).
These new molecules which are structurally simple and easy to
synthesize constitute an alternative to combat resistances observed with
antiproteases targeting the active site.

(Su/16.1/084)

Biochemical, Genetic, and Structural Studies on

Thymidylate Kinases
R. Brundiers and M. Konrad
MPIfor Biophysical Chemistry, Dept. of Molecular Genetics, 37077

(Su/16.1/085)

Mutageuesis study of residues involved in substratebinding in the chitosanase from Streptomyees sp. N174
R. Brzezinski, H. Tremblay
Biologie, U. de Sherbrooke, Sherbrooke (Quebec)J i g 2R1 Canada

GOttingen, Germany

Thymidylate kinase (TMK) catalyzes the phosphorylation of dTMP to

form dTDP, an essential step in the biosynthesis of the DNA precursor
dTTP, Thus, TMK may be an interesting target enzyme for antiviral
and
anticancer
therapy.
Phosphorylation
of
3"-azido-3"deoxythymidine monophosphate (AZT-MP) by TMK has been
implicated as the rate-limiting step in the activation pathway of the
anti-HIV prodrug AZT.
Yeast TMK1 has 44% sequence identity with the corresponding
human enzyme, the latter being capable of fully suppressing the lethal
phenotype of a TMK1 deletion strain. We have carried out structural,
kinetic, and genetic studies on the yeast enzyme which was
overproduced in E. colt [1,2]. Its tertiary structure has been
determined with either dTMP or AZT-MP bound to the active site.
Our kinetic data show very similar Km values for both substrates, but
AZT-MP has a 200-fold reduced phosphorylation rate, due to a P-loop
movement which changes the relative orientation of the phosphoryl
donor (ATP) and acceptor (AZT-MP) and increases the activation
energy for phosphoryl transfer step. All residues which are important
for binding of substrate and catalysis are conserved in human TMK. In
contrast to yeast and human TMK, E. colt TMK is much more
efficient in AZT-MP phosphorylation [3].
Conditionally lethal yeast strains were created to allow for detection
of suppressor activity of various mutant enzymes.
[1] A. Lavie et al. 1997 Nature Struct. Biol. 4:601-604
[2] A. Lavie et al. 1998 Biochemistry 37, 3677-3686
[3] A. Lavie et al. 1998 Proc. Natl. Acad. Sci. USA 95, 14045-14050

Chitosan is a polycationic carbohydrate polymer derived from chitin and can

be hydrolyzed by a variety of enzymes including chitosanase. In this study
we examined the properties of two carboxylic residues localized inside the
substrate-binding cleft of the chitosanase from Streptomycessp. N174.
Previous modelization has shown that the residue D57 could be involved in
substrate binding at subsite C, while the residue E197 would be localized at
subsite D.
Six mutated enzymes were produced by recombinant Streptomyces lividans
strains and purified: E197D; E197Q, EI97A; E197N; D57N and D57A,
Kinetic constants were measured for each mutant. We also established the
patterns of oligomeric product generation from hexaglucosamine substrate.
All these properties varied considerably among the mutants indicating that
both residues participate in substrate binding. On the other hand, thermal
unfolding properties of the mutants were very similar to wild type enzyme
showing that D57 and E197 are not involved in interactions important for
the structural stability of the chitosanase.

s 114

(Su/16.1/086)

Abstracts FEBS'99

How h u m a n topoisomerase I recognize D N A

D.V. Bugreev, E.L. Vasyutina, V.N. Buneva, G.A. Nevinsky
Novosibirsk Institute of Bioorganic Chemistry, Novosibirsk 630090,
Russia

The data of physico-chemical investigation of the mechanism action of

eukaryotic topoisomerases I (tope) have been summarized and the efficiency of
all contacts between the enzyme and specific DNA revealed by X-ray analysis
was estimated. The main factors providing the high affinity of tope to sc DNA
and the specificity of DNA cleavage have been revealed as: i) non-specific
interaction, ii) specific interaction, iii) conformational changes and iv)
topological stress. Tope was shown to interact with any non-specific ds DNA
forming weak additive electrostatic and hydrogen bonds with intemucleoside
phosphate groups and hydrophobic and/or Van-der-Waals contacts with the
bases of 10 DNA nucleotide units, which provide about 5 orders of magnitude
(Kd-10 5 M) of a total topo's affinity to sc DNA (Ka~10 -t M). When passing
from non-specific to specific (ApApGpApCpTpTpApG)-sequence, the affinity
increased due to formation of stronger contacts with phosphate groups of
pentanucleotide (GpApCpTpT) and the T-base. The enzyme contacts with
G p A and pT units of scissile strand are very important as for initial step of
recognition so as for following change of DNA structure, and for catalysis of
DNA cleavage The contribution of non-scissile strand is remarkably smaller
Specific contacts give about two more orders of magnitude (K0-10 7 M) of the
total DNA affinity. DNA-backbone conformation changes were shown to
provide the distortion bringing and ,,orbital steering~, in the active site, which are
very important for catalytic cleavage This factor gives one additional order of
magnitude of DNA affinity (Kd-10 8 M). We suggested that the curvature region
of sc DNA specific sequence can interact with linker domain of tope. This
interaction was estimated to increase the affinity by 100-fold. The unique role of
tope linker domain in sc DNA binding and recognition of DNA topological state,
and also in catalytical stage has been proposed. Finally, high affinity of tope to
scDNA is achieved by all foregoing factors: 10 5 M ~10 -2 e l 0 -I .10-~=10 -~ M.
To summarize all available data, a possible mechanism of recognition and
cleavage of sc DNA by tope has been advanced.

(Su/16.1/088)

Catalytic and structural properties of carbonic

anhydrase from Neisseria gonorrhoeae
L. Chirica, B. Elleby, B.-H. Jonsson, S. Lindskog
Dept. of Biochemtstry, Ume&Universitk; Umett. Sweden

Carbonic anhydrase (CA), which catalyzes the interconversion between

CO 2 and bicarbonate, is a ubiquitous enzyme present in animals,
plants, algae and some bacteria. Three evolutionarily unrelated
classes of the enzyme are known and have been designated c~ [3.
and ~,. The c~-carbonic anhydrases were thought to belong
exclusively to the eukaryotic world, but it was recently demonstrated
that the enzyme from Neisseria gonorrhoeae (NGCA) is of the c~ type
and, thus. homologous to carbonic anhydrases from animal sources.
The CA gene from N. gonorrhoeae encodes a 252-residue polypeptide,
but a 26-residue signal peptide is cleaved offwhen the enzyme is
expressed in E. coli, and most of the enzyme appears as a soluble, 226residue protein in the periplasmic space. The catalysis of CO z hydration
has been characterized using stopped flow spectrophotometry and
~SO exchange between CO, and water. The enzyme is a high-activity
CA with a CO_~hydration turnover number of 1.1 106 s -~ and a KM of
20 mM at pH 9 and 25C. A role for His66 in transferring protons
between the zinc-bound water and solution was confirmed by the 25-fold
lower activity of the mutant of NGCA containing the replacement
His66--+Ala. Also the crystal structure of NGCA has been solved to a
resolution of 1.78 A. The fold of the N gonorrhoeae enzyme is very similar
to that of human isozyme II (HCA II) although long deletions in the bacterial
enzyme relative to HCA II have resulted in considerable shortening of three
surface loops.

(Su/16.1/087)

Kinetic properties of the PEPcase enzyme purified from a

C4 plant (sugarcane).
F. Cadet j, P. Baret I, C. Queiroz-', C. Rouch ~ and J.C. Meunier 2.
1
2

Untv Rdumon Labo de Btochlmte, 97715 St-Dents, R~umon

t ,V ,4 P G , Labo de ('htm~e Btologtque 78850 Thlverval-Grlgllon, France.

Even if CO2 transformation inevitably proceeds through the BensonCalvin cycle, in some angiosperms that are mostly found in dry and warm
regions (C4 plants), the first step of CO2 assimilation is performed by the
phosphoenol pyruvate carboxylase (PEPcase) enzyme. Here we describe a
purification procedure tbr this enzyme in the C4 sugarcane plant. Kinetic
study results are also presented.
SugarcanePEPcase has been extracted with a citrate-phosphate 100
mM buffer pH 6.8. Protein extract was precipitated with ammonium
sulphate (15-50%) and purification was performed through successive
chromatographic columns (ion exchange, hydroxyapatite and molecular
sieving columns). The ultimate purification coefficient topped to 38.3 and
final yield was approximately 3.2%. The specific activity of the enzyme
reached 268 nkat/mg. The molecular mass of the PEPcase, 398 kDa, has been
estimated by molecular sieving chromatography and by electrophoresis.
PEPcase activity was monitored using two methods: reaction
coupled with MDH and OAA production monitoring at 270 nm. Both
methods gave similar results. The kinectlc parameters (Kin. spec. act, %
activation by G-6-P) of our PEPcase were similar to those described in the
litterature. We also showed that inhibition by L-malate was of competitive
type. Similarly. we found that sugarcane PEPcase activity was optimal at
pH 8,2 and that zt decreases at Mg- concentratzon higher than 25 mM.

(Su/16.1/089)

Crystal structure of wild-type and active-site mutants of

thioredoxin h from the green alga C. reinhardtii
C. Cat/~ v. M e r ~ C. ~
J.V.Jsalua* and A Attty Zta43~LX,~70.~
~ .
t#-LNa~yZ~2~, 54.~6V ~ y
Ca~ hnr~

Thioredoxins are ubiquitous small globular proteins involved in multiple

reactions which require reduction of disulphide bonds on selected target proteins.
The mechanism proceeds via a mixed disulfide intermediate resulting from the
nucleophilic attack of the Cys 36 thiolate (Chlamydomonas remhardtu sequence
numbering) on the target protein. The chemical basis of catulysis is still not fully
understood as well as the role of some invariant residues close to the active site
that might contribute to catalysis. Among these residues, a buried aspartate (D30)
exhibits an unusual weak acidity and could be involved in a general acidic
catalysis. The invariant Trp (D35) is held at the surface of the protein, close to
the most solvent exposed active site Cys (C36). Its side chain could be involved
in a hydrogen bond between Ne and the carboxylate of an exposed Asp residue
(D65). It was suggested that the active site could be a catalytic triad composed of
the (D65,W35) tandem, the redox disulfide and the protonated D30 [1].
In order to further investigate the role of these residues, we have undertaken the
crystallization of the wild-type (WT) and two mutants (D30A and W35A)
thioredoxin h from C. reinhardtn. Data collections were carried out at room
temperature using the laboratory X-Ray source. Two crystal forms were
obtained : the WT and D30A proteins crystallized in the trigonal space group
P3121 with two independent molecules per asymetric unit and W35A crystals
belong to the space group P312 with one molecule per asymetric trait. The
crystals of WT, D30A and W35A diffracted to 2.1, 2.2 and 1.9 A resolution,
respectively. Although a NMR smacture was already available for the WT protein
[2], attempts to solve the crystal structure by molecular replacement using the
NMR model were not successful and the structure was finally solved using
human thioredoxin as a starting model (40% sequence identity). The poster will
present the significative differences between the NMR and the X-Ray structures
and the catalytic mechanism will be discussed.
[1] I. Kfimm et al. (1998) Eur. J. Biochem. 255. 185-195.
[2] V. Mittard et al (1999) Eur. J. Biochem. 243, 374-383.

Abstracts FEBS'99

(Su/16.1/090)

Lipid-protein interaction in BDH as studied by ESR

S. De', S. Hahna, M.M Jehla, J.-O. McIntyreb, J G Wisea and
W.E Trommer'

s115

(Su/16.1/091) Enhancement of catalytic efficiency by the combination of

point mutations in a carbonic anhydrase-related protein
B. Elleby, B. Sj6blom, S. Lindskog
Dept. o f Biochemistry, Umed University, Umed, Sweden.

~JDepartment o f Chemistry, Universi& o f Kaiserslautern, Kaiserslautern

b)Department o f Molecular Btology, Vanderbilt Umversity, Nashville, TN

R-3-Hydroxybutyrate dehydrogenase (BDH) is a lipid-requiring mitochondrial

enzyme bound to the matrix face of the mitochondrial inner membrane with a
specific and absolute requirement of phosphatidylcholine (PC) for function PC
is required for tight and functional binding of the coenzyme NAD to BDH and,
w c e v e r s a , complex formation with NAD in binary and ternary complexes
enhances the interaction of the enzyme with PC. PC bound to BDH was also
found to be in rapid exchange with the bulk lipid on the ESR time scale. With
excepUon of the C-terminal region, the enzyme is highly homologous to various
hydroxysteroid dehydrogenases The C-terminal region, however, is essential
for lipid binding and enzyme activation. NAD~ derivatives spin-labeled at N6 or
C8 are enzymatically active and have been successfully employed with BDH in
these as well as in related studies. Investigations were hampered, however, by a
slow reduction of the spin label by at least one reactive cysteine SH group of
BDH Although chemical derivatization of these cysteines is accompanied by
loss of enzymatic activity, they appear not to be involved in catalysis.
The enzyme from human heart was recently expressed both in insect cells and in
E. c o h
The molecular mechanism of the lipid dependence is now being
investigated by site-specific spin labeling of various mutants via spin-spin
interaction with spin-labeled NAD Single or double mutants, in which alanine
as well as serine substitute for the reactive native SH groups Cys69 and Cys242,
were now shown to be enzymatically active. Serine substitution at position 242,
however, leads to a tenfold increase in KM for the substrate

In addition to the functional carbonic anhydrase (CA) isozymes, a

number of homologous protein sequences have been found. One of
those, CARP (Carbonic Anhydrase Related Protein) or CA VIII, is
expressed in Purkinje cells of the cerebellum. The gene for CARP
is the most conserved of all the CA-homologous sequences.
The protein does not have a detectable COz hydration activity and
its function is still unknown. However, a single point mutation,
Arg117---~His, is sufficient to convert CARP to an active enzyme.
The activity is strongly inhibited by the powerful and selective
carbonic anhydrase inhibitor, acetazolamide. Most of the putative
active-site residues in the CARP mutant are found in one or more
of the CA isozyme forms, but not all. By introducing additional
mutations in those positions and combining up to four mutations in
the same protein, the catalytic activity was incrementally increased
by up to a factor of 10 on the basis of kcat/KM at pH 9. This shows
that it is possible to engineer a protein of enhanced activity by
combining a series of rationally designed point mutations.

In addition, a clone was constructed that encodes the C-terminal amino acids in
order to express this region separately for structural studies.

(Su/16.1/092)

Non-enzymatic and enzymatic decomposition of sucrose.

S. Farine, A. Puigserver, A. Ferjancic-Biagini
Laboratolre de Biochtrate et Btolog2e de la Nutrition, CNRS-UPRESA 6033. Faculte des
Sctences et Techmques de SamtJdr6me, 13397.Viarsetlleccdex 20, France

An accurate carbohydrate analysis method, namely High Performance Anion

Exchange chromatography with Pulsed Amperometric Detection (HPAEPAD) [1], was used to monitor sucrose degradation and subsequent
production of glucose and fructose in a 65% (w/w) sucrose containing
aqueous model solution during storage at 65C, 75C and 85C, and at pH
values ranging from 4 to 9. This allowed us to estimate the extent of nonenzymatic degradation of sucrose under defined experimental conditions in
order to find out those required for optimum liquor storage in a local cane
sugar refinery. Sucrose was also enzymaticaUy processed with invertase to
produce fructooligosaccharides (FOS). FOS are known as new food
ingredients which are able to stimulate selectively the growth and/or activity
of colonic bifidobacteria (prebiotics), and to improve the bioavailability of a
number of nutrients in the host-organism The kinetics of FOS formation in
an aqueous medium (50 mM sodium acetate buffer, pH 4.5, and 180 units of
I~-D-fructofuranosidase activity per g sucrose), as well as in water-free
toluene containing 620U enzyme activity/g sucrose were performed at 55C
and showed the formation of 6 different glycosidic structures as determined
by I-IPAE-PAD. The mechanism whereby the synthesis takes place appears
not to be of the same type in aqueous and organic media. Using commercial
standards, one &the FOS products was identified as kestose. The structure
of the others five new products, which were eluated neither with nystose nor
fructofuranosylnystose, two standard FOS compounds is still under
investigation
[1] Farine et al. Int J Biol, Macromol., 21, 1997, 109

(Su/16.1/093)

Mechanism of biotin synthase :

A novel function for an iron-sulfur center
D. Florentin, B. Tse Sum Bui, F. Escalettes, O. Ploux, A. Marquet
Laboratoire de Chimie Organique Btologique - UMR CNRS 7613 Universitd Paris VI - 4, place Jussieu - 75005 PARIS - F R A N C E

Biotin synthase, the enzyme which catalyses the conversion of

dethiobiotin into biotin
O

O
HN"JJ"NH

Biotin synthase

ONe'S3 (CH214COOH

HN~j..NH

"S ~" "'(CH2)4COOH

is an [Fe-S] protein which has an absolute requirement for AdoMet. The

monoelectmnic reduction of AdoMet by NADPH through an electron
transport system generates beside methionine a deoxyadenosyl radical
equivalent, responsible for the homolytic cleavage of the C-H bonds at the
carbons to be funcfionalized [1]. We have recently brought the first
experimental evidence of the involvement of the deoxyadenosyl radical by
showing that deuterium was transferred from the labeled substrate to
deoxyadenosine [2]. We have also shown using an [Fe-34S] reconstituted
enzyme that the sulfur source was indeed the [Fe-S] center itself [3]. Thus,
the enzyme is at the same time substrate and this explains why no turn-over
has ever been observed i n v i t r o .
[i] Guianvarch D. e t al., Biochem. Biophys, Res. Comm., 236 (1997) 402.
[2] Escalettes F. e t a l . , J. Am. Chem. Soc. (1999) in press.
[3] Tse S u m Bui B. et al., FEBS Lett., 440 (1998) 226.

s116

(Su/16.1/094)

Abstracts FEBS'99

Inhibition study of the purified carrot phydroxyphenylpyrate dioxygenase by the

diketonitrile of isoxaflutole

(Su/16.1/095)

1. Garcia and M. Matringe

CNRS UMR 19321 Rh~ne-Poulene Agro 69 009 Lyon

p-Hydroxyphenylpyruvate dioxygenase (HPPD; EC 1. 13. 11.27) is

an extradiol, non-heine, tx-keto acid dependent enzyme which catalyses
the formation of homogentisate (HGA) from p-hydroxyphenylpymvate
(p-HPP) and molecular oxygen. In plant, HPPD is involved, as in
most organisms, in the tyrosine catabolism but also in the biosynthesis
of c~-tocopherols and plastoquinones. The lethality of HPPD inhibition
by new families of herbicides (triketones-isoxasoles) have proved the
crucial role of the enzymatic activity. To improve our knowledge of
this herbicide target we have isolated the carrot HPPD cDNA [1],
overproduced the corresponding protein in E. coil and purified the
enzyme to homogenity. Carrot HPPD behave as an homodimer with 48
kDa subunits and is inhibited by the diketonitrile of isoxafiutole (DKN;
5-cyclopropylsoxasol-4-yl 2-mesyl-4-wifluromethylphenyl ketone), a
new pre and early postemergence herbicide that belong to the benzoylisoxasole family. To understand the mechanism of interaction of plant
HPPD with DKN, we have realized inhibition kinetics and binding
studies. We observed that DKN act as a slow thight binding inhibitor
for carrot HPPD (kon = 3.8 x 104 M -1 s-1), competitive with respect to
HPP. Moreover, we have demonstrated that HPPD shows only an half
site-rectivity versus DKN. Modification of one active site by DKN is
sufficient to inactivate the dimeric enzyme.
[1] Garcia et al. (1997) Biochem. J. 325,761-769

Phenol hydroxylase from Acinetobacter radioresistens:

isolation of an intermediate component
E.Griva, M.Cavaletto~, S.Divari, F.Valetti, E.Pessione, C.Glunta
Dipartimento dt Biologta Animale e dell 'Uomo. 10123 Torino. Italy
~Dipart#nenlo di Sctenze e Tecnologie Avanzate. 15100 Alessandrla, Italy

A. radioresistens cells grown on phenol as the only carbon source,

synthesize a monoox-ygenase, phenol hydroxylase (PH), which is involved

in the initial metabolic reaction used to degrade the substrate phenol. PH
from A. radioresistens has a multicomponent organization [1], it consists
oE an oxygenase component (PHO) containing a binuclear iron centre
responsible for activation of oxygen before insertion into the substrate; and
a reductase component (PHR) containing FAD and a 2Fe-2S centre for
electrons transfer from NADH to the active site of PHO
PH was purified from the cytoplasmic extract of A. radioreststens ceils,
during all the purification steps PH activity was monitored by evaluation of
oxygen consumption with a Clark electrode. Samples were fractionated by
anion exchange chromatography on a DE52-cellulose column, followed by
a strong anion exchange (Source Q15) with a linear gradient from 0.05 M
to 0.5 M Na2SO4 in 0.05 M Hepes. pH 7. In this way a low molecular
weight protein, that we called intermediate component (PHI), was isolated,
and it was shown to be associated with PHR. PHI plays an important role
in catalysis, since hydroxylating activity was detected only when it was
present together with the purified PHO and PHR~ giving rise to a ternary
complex PHO-PHR-PHI. PHI was further purified by FPLC gel filtration
and sequenced. PHI has a molecular weight of 10 kDa, and shares 89% 67% residue identity with the corrisponding proteins from A. calcoaceticus
and Pseudomonas CF600 respectively; sequence similarities in conserved
positions with component B of methane monooxygenase from
M.capsulatus were also detected. On the basis of these similarities, PHi
does not seem to be directly involved in redox reaction, but it could play a
regulatory role by inducing conformational changes on PHR and/or PHO
or by affecting the substrate-enzyme interaction.
[1] Pessione, E et al., Eur. J. Biochem, submitted.

(Su/16.1/096)

NMR study of the protein-protein interactions in the

glyeine decarboxylase complex of pea n~itochondri~a.
L. Guilhaudis a, J.-P. Simorre a, M. Neuburger , R. Douce o, D.
Marion a and P. Gansa
aIBS-LRMN, bDBMS-LPCI( CEA-Grenoble. France

The mitochondrial glycine decarboxylase multienzyme system contains

four components enzymes named H (a lipoamide containing protein), P (a
pyridoxal dependent enzyme), T (a tetrahydrofolate requiring protein) and
L (a dihydrolipoamide dehydrogenase) [1]. The H protein acts as a mobile
cosubstrate which commutes between the other three proteins during the
course of the catalytic breakdown of glycine. Its lipoyl moiety undergoes a
cycle of reductive methylamination, methylamine transfer and electron
transfer. Crystallographic data of the H protein and methylamine loaded H
protein indicate that the lipoamide cofactor is bound to a lysine residue
located in the loop of an hairpin configuration at the surface of the protein
[2,3]. However once loaded with the methylamine group the lipoamide arm
rotate around the amide bond to place the methylamine group in an
hydrophobic cleft. The transfer of the one carbon
unit to the
tetrahydrofolate cofaetor implys a docking of the T protein in order to
unlocked the methyamine group and favor the channeling of the carbon unit
to the tetrahydrofolate cofactor. Complete understanding of the reaction
mechanism requires a determination of the structure of the complex. To
gain some insights into the structural and/or dynamic consequences of the
interaction between the two partners, lwe have.performed an NMR study
of the H-T complex. Variations of the H and "~N chemical shifts of the Hprotein alone and in complex with the T-protein have been analysed.
Interestingly, the interaction surface for the H-protein is not localised near
the cleft where the methylamine group is locked but in the linking region of
the lipoate arm. This led us to investigate the stability of the Hmet alone
and in presence of the T-protein. Determination of the activation energy of
the unloading reaction have been performed. For the Hmet alone, we found
an activation energy of 37 kcal/mo1-1 indicating a high stabilisation of the
methylamine group inside the protein structure. In contrast, in presence of
the T-protein, the activation energy is reduced by about 20 kcal/mo1-1.
This suggests that the role of the T-protein is not only to locate the
tetrahydropteroyl glutamate cofactor in a position favourable for a
nucleophilic attack of the methylene carbon but also to destabilise the Hprotein in order to facilitate the unlocking of the arm and initiate the
reaction.
[1] Douce, R and Neuburger, M. Ann. Rev. Plant Physiol. Plant Mol.
Biol., 40, (1989), 371.
[2] Cohen-Addad et al., Nature Structural Biology, 2, (1995), 63.
[3] Pares et al., Acta Cryst., D41, (1995), 1041.

(Su/16.1/097)

Purification and localisation of two forms of carbonic

anhydrase of leaves of Amaranthus cruentus L.
N.M. Gufiev, H.B. Babayev, Sh.M. Bayrarnov, J.A. Aliev
Institute of Botany, Patamdar Shosse 40, 370073 Baku, Azerbaijan

The methods obtaining of isolated photosynthesising tissues (mesophyll

cells and bundle sheath cells) and purification carbonic anhydrase
(carbonate hydro-lyase EC 4.2.1.1) of C4-plant Amaranthus eruentus L.
leaves were elaborated. Two forms of carbonic anhydrase were found in
leaves of this plant. One form of the enzyme is localised in soluble lkaction
of mesophyll cells. The other one is localised in chloroplasts of bundle
sheath cells. Carbonic anhydrase form localised in cytoplasm of mesophyll
cells has molecular mass approximately 250 kDa, and enzyme form
localised in chloroplasts of bundle sheath cells has molecular mass
approximately 150 kDa. The carbonic anhydrase forms are characterised by
Stokes' radius 5.3 and 3.75, diffusion coefficient 3.77 and 5.33, friction
ratio 1.07 and 0.87 correspondingly. Both forms of enzyme have
quaternary structure, contain many SH-groups and have also metal (zinc),
which plays key role in appearance of catalytic activity. Catalytic properties
of carbonic anhydrase of mesophyll ceils to certain degree differ from
carbonic anhydrase of bundle sheath cells, what can be explained by
structural peculiarities of the enzymes. But both forms of the enzyme don't
appear allosteric properties to CO2. The enzyme-substrate interaction
occurs according to Michaelis-Menten mechanism. A molecule of CO2
combines with each molecule or each active site of molecule of the enzyme
provided these sites don't interact with each other.

Abstracts FEBS'99

(Su/16.1/098)

Biosynthesis of the prosthetic group of malonate

decarboxylase from KiebsieUa pneumoniae
S. Hoenke, M. Wild and P. Dimroth

s117

(Su/16.1/099)

lnsfitut fiir Mikrobiologie der ETH Ziirich, Switzerland

Malonate decarboxylase of Klebsiella pneumoniae consists of four

different subunits and catalyzes the cleavage of malonate to acetate and
CO2 [1]. The smallest subunit MdcC serves as acyl carrier protein (ACP)
and contains 2'-(5"-phosphoribosyl)3'-dephospho-coenzyme A as
prosthetic group, which is attached by a phosphodiester linkage to a serine.
During catalysis, an acetyl residue bound to this group is exchanged in a
cyclic manner for a malonyl moiety, which is subsequently decarboxylated
to yield the initial acetyl-S-enzyme [2,3].
We investigated the biosynthesis of the prosthetic group and identified two
gene products MdcB and MdcG involved in the conversion of apo-ACP to
holo-ACP. After expression of all three proteins together, holo-ACP was
obtained, whereas expression of the ACP with only MdcG yielded ACP
with an incomplete prosthetic group, as judged by mass difference. The
synthesis intermediate was hydrolyzed from the ACP and identified as
AMP by biochemical, chromatographic and spectrometric methods. The
first step in the prosthetic group biosynthesis is the MdcG-catalyzed
adenylation of apo-ACP with ATP as substrate. This reaction has been
demonstrated in in vitro assays. MdcB catalyzes the replacement of the
adenine moiety of the protein-bound AMP by dephospho-CoA, which
completes the synthesis of the prosthetic group.
[1] Schmid et al., Eur. J. Biochem, 237 (1996), 221-228.
[2] Hoenke et al., Eur. J. Biochem. 246 (1997), 530-538.
[3] Hoenke and Dimroth, Eur. J. Biochem. 259 (1999), 181-187.

Purification and charaeterisation of extracellular

[3-glucosidase from Aspergillus carbonarius
Sz Jager and L. Kiss
Institute of Biochemlst~T, Lalos Kossuth Untversltv. P.O.B 55
tt-4010 Debrecen, Hungary

13-glucosidase was extracted from Aspergillus carbonarius grown on

glucose culture. The enzyme was purified by ammonium sulphate
precipitation, hydrophobic chromatography, chromatofocusing and affinity
chromatography Each purification step was followed by SDS-PAGE
gelelectrophoresis. The purified enzyme showed only ]3-glucosidase
activity.
The kinetic parameters were determined The Kx~value for p-nitrophenyl-13D-glucopyranoside was 1,1210 -3 M. The v,~a~/KM value was 6,92
~tmol/min/M
The pH and temperature optimum of the enzyme are pH=4 and 60C, its
isoelectric point (determined by chromatofocusing) is pH=4,2.
Substrate specificity studies were made using different p-nitrophenil-13-Dglycosides.
Inhibition kinetic experiments were carried out to explore the active site of
the enzyme using l-thio subsrate analogues and glucose.
The possible role of tyrosine side chains in the substrate binding was
investigated by specific chemical modification using N-acetylimidazole [ 1].
Specific
substrate
analogue
inactivator,
N-bromoacyl-[3-Dglucopyranosylamin was synthesised to investigate the catalytic amino acid
side chains in the active centre of the enzyme Preliminary investigations
showed that the enzyme was inactivated by this specific inactivator similarly
as previously described in the case of manioca 13-D-glucosidase [2]
[1] L Kiss et al, Biochim Biophys Acta, 662, (1981) 308
[2] Zs. Keresztessy et at., Arch Biochem Biophys., 315, (1994) 323

(Su/16.1/100)

Purification and characterisation of extraceHular

~-xylosidase from Aspergillus carbonarius
T. Kiss and L. Kiss
Institute of Biochemiswy, Lajos Kossuth ~]niversl.ly, P O.B 55,
1t-4010 Debrecen, Huny~arv

A [3-xylosidase (13-D-xylosidase xylohydrolase, EC 3.2.1.37) extracted from a

wheat bran culture of Aspergillus carbonarius was purified by ammonium
sulphate precipitation, hydrophobic chromatography, gel filtration,
chromatofocusing and affinity chromatography. Success of purification steps
was controlled by SDS-PAGE The purified enzyme showed [3-D-xylosidase
and low a-L-arabinosidase activities
The kinetic parameters showed that the enzyme bound p-nitrophenyl-13-Dxylopyranoside (PNP-Xyl) more tightly than PNP-ct-L-arabinopyranoside
(PNP-Ara) (KM = 2,1x10"4 M and 4,2x10 "3 M for PNP-Xyl and PNP-Ara,
respectively); and the enzyme was more effective toward PNP-Xyl than PNPAra (v~JKM = 49,5 ~tmol/min/M and 3,7 ~tmol/min/M for PNP-Xyl and PNPAra, respectively).
The pH and temperature optimum of the enzyme were pH=4 and 60C,
respectively. The isoelectric point determined by chromatofocusing was
pH=4,4.
Inhibition kinetic experiments with 1-thio substrate analogues and xylose
were carried out to explore the active site of the enzyme and to compare with
a previously investigated 13-D-glucosidases. The results indicated that - in
opposition of previous inhibition investigations for [3-D-glucosidases [ 1] - the
carbohydrate moiety of substrate is the major determinant in the formation of
the enzyme-inhibitor (-substrate) complex.
Specific substrate analogue inactivator, N-bromoacyl-13-D-xilopyranosylamin
was synthesised to investigate the catalytic amino acid side chains in the
active centre of the enzyme. Preliminary investigations showed that the
enzyme was inactivated by this specific inactivator similarly as previously
described in the case of [3-D-glucosidase [2].
The transglycosylase activity of the enzyme was also investigated.
[1] 1. P6csi et al., Biochem J., 256, (1988) 139
[2] Zs. Keresztessy et al., Arch. Biochem. Biophys., 315, (1994) 323

(Su/16.1/lO1)

Induction of CYP1A1 Protein, Catalytic Activity and

mRNA in the Liver Microsomes of Lisa saliens
E.Su, S.Kocablylk, E.Ann9
Dept. of Biol., Middle East Technical University, 06531 Ankara, Turkey.

Cytochrome P4501AI (CYPIA1) is known to play an important role in the

biotransformation of foreign compounds including drugs, pesticides and
pollutant chemicals and in the activation of many chemical carcinogens in
mammals and in fish. CYPIA1 and its associated enzyme activities,
ethoxyresorufin O-deethylase (EROD) and benzo(a)pyrene hydroxylase
(AHH) are induced in respose to polycyclic aromatic hydrocarbons (PAl-Is),
polychlorinated bipheuyls (PCBs) and other toxic chemicals. This study
further extends the biological impacts of aquatic PAH pollution on
enzymatic and genetic parameters associated with fish CYP 1AI system. In
the study, the test area was Izmir Bay in the Agean Sea Coasts of Turkey,
and the test species was Lisa saliens (leaping mullet). The induction of
CYP1A1 at the enzymatic level was assessed by determination of L.saliens
liver microsomal EROD activities. Fish caught from the Outer Bay, which
is accepted as a reference section in this study, had a very low EROD
activity (11 _+ 2 pmol/min/mg). However, CYP1A1 associated EROD
activity increased about 90-fold in the liver microsomes of fish samples
obtained from Pasaport, which is the most industrial and urbanized section
of the Bay. Similarily, polyclonal anti-mullet P4501A1 /gG displayed
stronger cross-reactivity with the liver microsomes of L.saliens from
Pasaport,compared to the microsomes of the samples from the reference
station ,as revealed by Western Blotting and Hybridization. This result
indicated the induction of CYP 1A 1 protein of mullet from Pasaport by PAH
and/or PCB type pollutant. To monitor the CYP1A1 induction at the mRNA
level, a synthetic 33 nueleotide DNA probe was designed based on a
completely conserved region of all the fish CYP 1A1 proteins. A major
band was produced with this probe at a position corresponding to 1.28 kb
mRNA,which should be representing L.saliens CYP1AI mRNA. Northern
Blotting and Hybridization results showed that chemically mediated
induction of CYP1A1 transcription is higher in the samples from Pasaport
than that of the samples from reference station.

s118

(Su/16.1/102)

Abstracts FEBS'99

Structural and functional investigations of

aeetylcholinesterase antiidiotypic antibody.
~A.V. Kolesnikov, aA.V. Kozvr. bE.S. Alexandrova, F.
Koralewski, ~A.V. Detain, "M.I~ qfitov, dD. Thomas, aA.G.
Gabibov, dA. Fnboulet

"Shemyakin & Ovctunnikov ht~t of Btoorganic Chemt~trv, RAS, '~Engelhardt h~st

of Molecular Biolog), RAS,"'hist. of Gene Biology, Moscow - Russfa.a UPRES-A
6022 CNRS UTC, CompiOgne-France,

Monoclonal antibody IF5 was selected by immunizing mice with AE-2,

a m o n o c l o n a l antibody directed a g a i n s t the active site of
acetylcholinesterase. In accordance with the idiotypic network theory,
monoclonal antiidiotypic antibody 1F5 displayed internal image
properties of the original immunogen, the acetylcholinesterase active site.
This antibody was cloned and its aminoacid sequence was determined.
Peptide mass mapping followed by MALDI MS has shown ~dentity of
experimentally examined antibody with the cloned protein. Modification
of 1F5 with a specific irreversible organophosphorous inhibitor of
esterases was studied. Comparative peptide mass mapping of modified
and unmodified protein has revealed an active site serine residue. The
existence of a catalytic dyad Ser-His was corroborated by computer
modelling. Interaction of idiotypic (AE-2) and antiidiotypic (1F5)
antibodies using surface plasmon resonance was investigated.

(Su/16.1/103)

(S)-PMPA, a weak bisubstrate inhibitor of E. coil PNP

EKulikowska a, A,Bzowska a, A Brysiaka, A.HoI9 b, D.Shugar ~
~Department of Btophystcs, Umversity of Warsaw, Warsaw, Poland
blnstltute of Orgamc Chemtstry and Biochemistry, Praha, Czech Republic

Several antiviral 9-(2-phosphonylalkoxy)alkyl purines have been

reported as bisubstrate analogue inhibitors (competing vs. both substrates of
phosphorolysis, nucleoside and P,) of trimeric mammalian purine nucleoside
phosphorylase (PNP, E C . 2 4.2. I) with Kiapp in the p.M range in presence of 1
mM P, [1]. We have now studied one of these, (S)-9-(2phosphonylmethoxy)propyl adenine, (S)-PMPA, as an inhibitor of hexameric
PNP from E. coil, with Ino (in presence of 1 mM Pi) and P~ (in presence of
0 5 mM lno) as variable substrates. Inhibition is much weaker (ICs0 ~ 0.5
raM) than for mammalian enzymes. Fitting the data to the classical
Michaelis-Menten equation did not establish the type of inhibition. However,
competition of (S)-PMPA with both Ino and P, was found with a model
involving two active sites, with different kinetic constants, for analysis of the
kinetic data
V ~ l co
V~-'CO
Vo(C~) = co + K~I + co + Kin.'
(eq. 1),
since both Km~ and Kin2 increase in presence of the analogue, while V,,,,x
values are unchanged. Hence (S)-PMPA may be considered a bisubstrate
analogue inhibitor of the E. cull enzyme, as for mammalian PNPs It is well
known that the classical equation does not adequately describe the kinetics
vs phosphate [2], in agreement with two dissociation constants for P, from
fluorescence titrations [3]. Hence our mode/ (eq. 1) is consistent with
previous data using P, as variable substrate. The unexpected finding with
nucleoside as variable substrate requires further analysis. Relevance to the
known differences between E. coil and mammalian PNPs will be discussed.
Acknowledgements"Supported by Pohsh Commitlee for Scientific Research (KI3N grant 6
P04A 043 12) and m part by Howard Hughes Medical Institute (grant HHMI 75195-543401)
and Grant Agency of the Czech Republic (grant 203/96/K001).
[1] E. Kuhkowska et aL, Adv Exptl. Med. Biol, 431. (1998) 747
[2] K.F. Jensen, Eur. J Biochem., 61. (1976) 377.
[3] B Kierdaszuk et al.. Biophys. Chem.. 63, (1997). 107

(Su/16.1/104)

Interactions of 5-Substituted N~-Hydroxy-2'-deoxycytidine

5'-Monophosphates with ThymidylateSynthase

T Kullko~skP. J M Dzlk b, K FelczakL A MlazgaL W Rodet'

~/nsntute o f Blochemtstrv and B~oRhvstcs P.4S ~farszawa, Poland
bNenckl lnsntute o f ExpOrsmental Bfologv PA~ I~i~rszawa,Poland

Thymidylate synthase (TS, E.C 2 1 1 45) catalyzes the reductive methylation

of dUMP in the de novo biosynthesis of dTMP and is a useful target in
chemotherapy with the use of 5-substituted dUMP analogues [1]. We
synthesized and investigated the structure and biological properties of a
range of 5-substituted Nqhydroxy-2'-deoxycytidines, untypical dUMP
analogues with CH3, CH2OH, F, C1, Br, and I substituents, exerting different
electronic, steric and hydrophobic properties.
Inhibition was studied of two TS forms differing in sensitivitiestowards FdUMP
inhibition, isolated from parental and FdUrd-resistant LI210 cell lines. N 4hydroxy-dCMP and all its 5-substituted analogues were competitive vs. dUMP
slow-binding inhibitors with K i values ranging from 10.8 M for N4-hydroxydCMP and its 5-fluoro congener, 10.6 M for the 5-iodo- and 5-hydroxymethyl
derivatives, and 10-5 M for the 5-chloro and 5-bromo analogues, to 104 - 10.3
M for the 5-methyl-substituted compound. None of the 5-halogeno analogues
became dehalogenated in the TS reaction. Trials to correlate the latter results
with the five substituents physical properties pointed for the decisive role of
hydruphobicity, as compounds with substituents of low hydrophobic constants,
such as -F, -H and -CH2OH were stronger TS inhibitors than those with highly
hydrophobic -Br, -C1, and -CH3 substituents Certain effect of electronic
properties should also be considered
Supported by grants KBN 4P05F030I lp01 and 4P05F0301 lp02
[1] J BalzarM et al, Biochem Pharmac., 31 (1982) 3673

(Su/16.1/105)

Domain mobility in flavocytochrome b 2

as studied with a monoclonal antibody.
K. H. Di~p L~ and Florence Lederer
Laboratoire d'Eazymologie et Biochimie Structurales, C.N.R.S.
91198 Gif-sur- Yvette Cedex, France

Each subunit of flavocytochrome b 2 (Flb,; L-lactate dehydrogenase) is

composed of two domains, a heine-binding domain (residues 1-99) and
a FMN-binding domain (FDH, residues 100-511). The flavin catalyses
the dehydrogenation of lactate to pymvate, and transfers electrons to
heme b 2 in the same subunit; the heine-domain is then reoxidized by
cytochrome c, the physiological acceptor. The artificial acceptor
ferricyanide can also be used to reoxidize the enzyme. In the enzyme
crystal structure, two hemoprotein domains out of four are mobile
relative to the tetrameric FDH assembly [1J.
We have previously isolated a monoclonal antibody (Mab), elicited
against the holoenzyme. It prevents electron transfer between reduced
flavin and oxidized heine and consequently inhibits cytochrome c
reduction. It only recognizes the berne domain and its epitope is
conformatioual [2]. Site-directed mutagenesis and heme replacement
with its dimethyl ester indicated that residues E63 and N69, as well as
one or both heine propionates belong to the Mab epitope [3]. We have
now made the following new mutant Fib2: P64Q, P64R, A67L, A67Q
and V70M. It appears that the residues at these three positions are also
part of the epitope, as indicated by competition ELISA tests and
cytochrome c inhibition assays. It appears in addition that the mutation
at position 67 affects the flavin to beme electron transfer rate and hence
the cytochrome c reduction rate in the absence of the antibody . In the
crystal structure, all the mutated positions lie close to or in the interface
between the domains and at the mouth of the heine crevice.
Our results confirm that the herne-binding domain is mobile in solution.
When it has rotated away from the FDH, the mouth of the heme crevice
becomes accessible to the Mab. Its binding prevents reformation of a
functional interface between the two domains and inhibits intramolecular
electron transfer in flavocytochrome b 2 .
[1] Xia, Z.X et al. (1990) J. Mol. Biol., 212, 837.
[2] Miles, C.S et al. (1998) Biochemistry, 37, 3440.
[3] L~, K.H.D et al. (1998) J. Protein Chem., 17, 530.

Abstracts FEBS'99

s119

(Su/16.1/106) Equistatin and saxiphilin, members of thyropin family

B. Lenar~i~~, E. Moczydlowski b, V. Turk a
adept, of Biochem and Mol. Biol., J. Stefan Institute, 1000 Ljubljana,
Slovenia
bDept,of Pharmacology,Yale, Schoolof Med., NewHaven,CT06510

Thyroglobulin type-1 domain was originally found in thyroglobulin and is

present in various proteins of different function and origin. The role of these
domains is not clear yet, although it is believed that they could be involved
in the control of proteolytic degradation of proteins. A characteristic feature
of equistatin, MHC class II-associated p41 Ii fragment and chum salmon
egg cysteine proteinase inhibitor (ECI) is the ability to inhibit the papainlike cysteine proteinases with Ki values from pM to nM range. They have
been classified as thyroglobulin type-1 domain inhibitors, also called
thyropins.
Equistatin is a 199-residues protein that was isolated from sea anemone
A c t i m a equina. It is composed of three-repeated thyroglobulin type-1
domains. Here we present an additional function of equistatin, being also a
potent inhibitor of aspartic proteinase, cathepsin D, with Ki value in nM
range. Enzymes, papain and eathepsin D can be bound and inhibited
simultaneously by equistatin, indicating for the physical separation of the
two binding sites.
Saxiphilin is a protein found in plasma and tissues of various animals and is
characterized by the ability of binding a neurotoxin, saxitoxin (STX). The
identification of thyroglobulin type-1 domain being the inhibitor of cysteine
or aspartic proteinases suggests that saxiphilin, having two type-I domains,
may have other biochemical functions beside its known role in binding
STX. The inhibitory properties of the recombinant saxiphilin toward papainlike cysteine proteinases (papain and cathepsins B and L) were investigated.

(Su/16.1/108) Activation of human pro-gelatinaseA by trypsin

R.I. Lindstad, J.-O. Winberg
Department of Biochemistry, IMB, University of Troms~
N-9037 Troms#, Norway

GelatinaseA (EC 3.4.24.24) belongs to a family of zinc-containing

matrix metalloproteinases (MMPs) involved in degradation and
remodelling of both normal and pathogenic tissues. In cancer,
MMPs are instrumental for processes such as tumor invasion,
metastasis and angiogenesis. MMPs are secreted into the
extracellular matrix as inactive pro-enzymes and activated
extracellularly by removal of a 10-kDa pro-peptide. It has
previously been reported that purified pro-gelatinaseA (pro-MMP2)
can not be activated by serine proteinases such as trypsin [1], but
only by membrane-bound metalloproteinases (MT-MMP) or
organomercurials. However, studies on conditioned cell media
indicate that this enzyme can be activated by trypsin [2]. The aim
of the present study was to investigate whether trypsin could
activate purified pro-gelatinaseA. The results, using [3H]-gelatin or
the fluorogenic peptide (7-methoxycoumarin-4-yl)acetyl-Pro-LeuGly-Leu-[N-3-( 2,4-dinitrophenyl )-L-2,3-diaminopropionyl]-Ala
Arg-NH2-CH3COOH as substrate, showed that the 72-kDa proenzyme was activated by bovine trypsin. Gelatin zymography and
Western blotting of activated gelatinaseA showed that the
activation is accompanied by the appearance of 42-kDa and 46-kDa
forms of the enzyme. Unlike the situation with aminophenylmercuric acetate (APMA), the trypsin-mediated activation of progelatinaseA apparently does not involve the well-known conversion
of the latent 72-kDa enzyme into the active 62-kDa form.
[1] Okada, Y. et al. Eur. J. Biochem. 194 (1990), 721.
[2] Winberg, J.-O. et al. Biochem. Genet. 30 (1992), 401.
The financial support of the Norwegian Cancer Society is gratefully
acknowledged.

(Su/16.1/107)

Structure/function relationship of the hydroxylase from the

soluble methane monooxygenase enzyme
C. Lesieu and H.Dalton a
Umversity of Warwick. Department of biological Sctences, Coventry, U.K.

Soluble methane monoxygenase (sMMO) from M e t h a l o c o c c u s

capsulatus (Bath) is known to catalyse the NADH-and -oxygen dependent
oxidation of a wide variety of substrates, sMMO consists of three
components: the binuclear iron-sites-containing hydroxylase (250 kDa), the
reductase (40 kDa) and the regulatory protein B (16 kDa). The hydroxylase
is a complex protein, it is a dimer (c#~'/)2 in which each monomer is
composed of three subunits, ~, ~ and z Each ct subunit contains two irons
atoms involved in the catalytic activity. Here, we have studied the
mechanism of unfolding of the hydroxylase and the relationship between its
structure, its prosthetic group and its catalytic function.
We have found that in contrast to other known dimeric proteins, the
hydroxylase does not unfold either in a one-step (native dimer to unfolded
monomers) or two-step (native dimer to monomers to unfolded monomers)
mechanism. The hydroxylase unfolds in a complex process of at least three
steps. We have studied unfolding by means of urea/GudCl denaturation
combined with cross-linking experiments. As the concentration of
denaturant increases, partial unfolding of individual subunits occurs before
they are fully dissociated. Indeed, high concentration of denaturant are
required to induce dissociation of the (c~[3y)2dimer and of the c~, [3 and 7
subunits of each monomer, suggesting that the complex as well as the
different subunits are tightly associated. In a first step, the (cq3y)2 dimer
dissociates into (c~l~,) monomers, followed in a second step by dissociation
of the ~,subunit from the (,[~,) monomer, and finally by dissociation of the c~
and I~ subunits. The results also indicate that dimerization strongly
stabilized the enzyme.
Effect of the dissociation and unfolding of the hydroxylase on the
activity of the enzyme is investigated. We show that cross-linking of the
hydroxylase inhibits the enzyme activity. Consequences for the enzyme
activity are discussed.

(Su/16.1/109)

The chemical mechanism of non-phosphorylating aldehyde

dehydrogenase
S. Marchal and G. Branlant.
Maturation des ARN et Enzymologie MolJculaire - UMR 7567 CNRS-UHP Faeult~ des Sciences. B.P. 239 54506 Vandoeuvre-les Nancy C~dex, France

Aldehyde dehydrogenases (ALDH) NAD(P)-dependent catalyzes the

irreversible oxidation of a wide variety of aldehydes into acidic compounds, via
a two-steps chemical mechanism. First, the acylation step involves the
formation of a thiohemicetal intermediate which precedes the hydride transfer
that leads to a thioacyl intermediate and NAD(P)H. Second, the deacylation
step implies the nucleophilic attack of a water molecule towards the thioacyl
intermediate. From a chemical point of view, the fact that most of ALDHs
described so far have high catalytic efficiencies at physiological pH implies to
fulfill several conditions. First, the essential Cys should be in a thiolate form to
attack efficiently the aldehydic function. Thus, the protein environment should
be such that the Cys pK~ppis decreased thereby increasing the nucleophilicity
of the essential Cys at neutral pH. Second, the hydride transfer from the
thiohemicetal intermediate towards the C-4 position of the nicotinamide ring of
the cofactor should be efficient. This implies a base catalyst to favor the
hydride transfer. An alternative would be the presence of an oxyanion hole,
similar to that observed in protease active site, sufficient to decrease the pKa of
the hydroxyl of the thiohemicetal intermediate and thus to permit a hydride
transfer without base-catalyst assistance. Third, the attacking water should have
its nucleophilic character strongly increased. This necessitates the involvement
of a base catalyst. Fourth, the thioacyl intermediate can be stabilized by
hydrogen bond. This interaction can not only favor an efficient positioning of
the thioacyl intermediate relative to the attacking water molecule but also
increase the electrophilic character of the carbonyl of the thioacyl intermediate.
In the present communication, all the structural and molecular factors involved
in the different chemical steps of the oxidation of glyceraldehyde-3 phosphate
by an ALDH isolated from Streptococcus mutans have been characterized by
site-directed mutagenesis. The results support a local conformational
rearrangement occuring during the catalytic process which includes at least
reorientation of side chains of essential amino acids and repositioning of the
nicotinamide ring of the cofactor that permits the chemical activation of the
essential Cys and the formation of an efficient ternary complex. This will be
discussed in relation not only with the data already published on ALDHs and
the available X-ray structures but also from an evolution point of view.

s120

Abstracts FEBS'99

(Su/16.1/ll0) ADP binding site in wild-type MM-creatine kinase

and in a W210,217,272Y active mutant.
O. Marcillat, R. Buchet & C. Vial

(Su/16.1/lll)

UFR Chinl-Biach Universitd Lyon I-CNRS. 69622 Vilh,urbamw. Fram'~

Peptide deformylase: design, synthesis and

mechanism of action of substrate analogue inhibitors
T. Meinnela, L. Patiny a, P. Mouchetb, S. Ragusa a,
C. Lazennec a, V. Dive b and S. Blanquet a
aEcole Polytechnique, Palaiseau, France bC.E. Saclay, Gif/Yvette, France

Earlier results suggested that a W residue is close to the active site of

creatine kinase (CK) (1). The sequence of rabbit muscle CK contains
fourW residues located respectively at positions 210, 217, 227 and 272.
Total inactivation (2) and lack of nucleotide binding induced infrared
changes except for a small intensity change around the 1638-cm -1 band
(3) were reported for the mutant W227Y. This nucleotide binding
induced change in intensity around 1638 cm -I suggested that another W
residue or other residue could play a distinct role in the ADP-binding site.
To precise the role of other tryptophan residues in the nucleotide binding
site of rabbit muscle creatine kinase (CK), structural changes produced
by the ADP-binding to CK and to an active W210,217,272Y mutant were
compared. The mutant activity corresponded to about 41% of the full
activity of native CK. The structural changes were probed by reactioninduced difference spectroscopy (RIDS). The reaction was initiated by
the photorelease of nucleotide from caged-nucleotide. Small infrared
changes caused by ADP binding to the enzyme are observed in the amide
I region, corresponding to structural changes affecting 3-4 amino acid
residues. Two positive bands located at 1666-1668 cm -I and 1657 cm 4,
and the four negative bands located at 1648 cm-I, 1625-1626 cm-t 1612
cm "l and 1581 cm -j resulted from the photorelease of ADP in thc
presence of either wild type or of active mutant. The RIDS of
W210,217,272Y mutant is similar to the RIDS of wild type (3) except for
the 1637-1640-cm-I positwe band. This band was strongly reduced in thc
case of W210,217,272Y mutant as compared to that of wild type and to
that of W227Y mutant. We propose that 1637-1640-cm 4 positive band
reflects a distortion of a carbonyl group of peptide backbone adjacent to a
W residue other than W227. While the W227 residue is essential for the
activity of CK, the other W residue, not essential for the activity, may
help to steer the nucleotide by forming stacking interactions between its
indole group and the purine moiety of the nucleotide.

Peptide deformylase (PDF) is an attractive target for the synthesis of new

antibacterial agents [1]. For the purpose of a rational design of such molecules,
the substrate specificity of Escherichia coli PDF was first investigated by
measuring the efficiency of the enzyme to cleave formyl-peptides of general
formula Fo-Xaa-YaaNH2. Catalytic efficiencies of deformylation were improved
by 3 orders of magnitude with substrates having bulky hydrophobic side chains
at Xaa (PI'). Next, the influence in catalysis of the second side chain (P2') was
studied after synthesis of 20 compounds of formula Fo-NIe-YaaNH2.
Positively-charged side-chains improved the rate by up to one order of
magnitude. The role of the main chain of the substrate was also assessed.
Analysis of a series of derivatives indicated the requirement for at least two
main chain carbonyl groups. On the basis of these results and given that
thermolysin and PDF display functional resemblance [1] as well as structural
homologies [2], thiorphan, a known inhibitor of thermolysin, was assayed as an
inhibitor of deformylase. The inhibition was competitive and depended on the
hydrophobicity of the first side chain as well as on the ligation of the sulphur
group to the catalytic metal in the enzyme active site. Consequently, a small
thio-pseudopeptide was derived from Fo-NIe-ArgNH2, the above-determined
best substrate of PDF. This compound (TNR) behaved as a competitive inhibitor
of PDF with K/=2.5 gM. A fingerprint of the binding of TNR with the residues
of the enzyme was obtained by NMR. In agreement with the above results, the
resulting 3-D model of the interaction revealed the occurrence on the enzyme of a
deep and hydrophobic S l' pocket as well as the absence of a true $2' pocket. A
set of likely interactions between deformylase and its substrates was deduced
and assessed with the help of site-directed mutagenesis. As a whole, this
analysis lays the basis for a rational design of powerful inhibitors of PDF, using
TNR as the lead for instance. Future prospects in this regard will be discussed.
[1] Meinnel et al., J. Mol. BioL, 267, (1997) 749.
[2] Dardel et al., J. M o L Biol., 280, (1998) 501.

(1) J. H. R. Kagi et al., Biochemistry 10 (1971) 1007.

(2) C. Perraut Ph. D thesis Lyon 1995.
(3) C. Raimbault et al., Eur. J. Biochem. 244 (1997) 343.
(Su/16.1/112)

Crystal structure of a mutant NDP kinase with a novel

imidazole kinase activity
P. Meyer a, B. Schneider b, M. V6ronb, D. Deville-Bonne b,
and J. Janin a. "Laboratoire d'En=~bmologie et Biochimle Structurales,

CNRS, 91198 Gif sur Yvette, France, Unit~ de ROgulation En:ymatlque

des ActivitOs Celhdaires, Institut Pasteur, 75724 Paris Cedex 15, France

NDP kinase catalyzes the transfer of a ,/-phosphate from a nucleoside

triphosphate to a nucleoside diphosphate. The reaction involves the
formation o f a phospho-histidine intermediate. To investigate the mechanism
of phosphoryl transfer, a mutant lacking the nucleophilic histidine (H122G)
has been designed. It has no NDP kinase activity, but it phosphorylates
imidazole and other amines or alcohols efficiently at the expense of ATP [i].
With imidazole as a substrate, the catalytic efficiency (kcat/Km ATP) of the
phosphoryl transfer is only 50-fold less than in the wild type enzyme.
We present here the structural result of co-crystallization of the H 122G
protein with ATP. The structure has been solved at 2.5 ~ resolution. It is
very similar to that of the wild-type NDP kinase in complex with the
transition state analog ADP.AIF3 which crystallizes in the same space
group [2]. The electron density at the active site reveals the presence of
ADP.Pi rather than ATP. This is presumably due to hydrolysis of ATP
during crystallization, in accordance with the observation that the mutant
has a much higher ATPase activity than wild type NDP kinase [1]. The
mutation has little structural effect beyond the deletion of histidine side
chain. In the wild type enzyme, a hydrogen bond with the carboxylate of
Glu 133 positions the imidazole group of His 122. As the Glu 133 side chain
retains its position in the mutant, a similar interaction with the noncovalently bound imidazolc substrate presumably helps maintaining the
nucleophile in its binding pocket in the correct orientation and position for
phosphate transfer from bound ATP.
[ 1] Suzanne J. Admiraal et al., BiochemisO T, in press.
[2]Yingwu Xu et al., Proc. Natl. A c a d Sci. U S A , 94, (1997) 7162.

(Su/16.1/113)

Novel inhibitors of glucosamine-6-phosphate synthase

S. Milewski, A. Janiak, R. Andruszldewicz,
R. J~drzejczak, M. Wojciechowski, E Borowski
1)epartment of Pharmaceuttcal Technology dr Blochemtst rv.
Techmcal ~niversttv of (Mahsk. 80-952 Gdahsk. Palatal

Glucosamme-6-phosphate synthase (GImS) catalyses a complex reaction

involving au amino group transfer fi'om glutamine amide to D-fi'uctose-6phosphate followed by an isomerisation of the intermediate fiuctoseiminephosphate to D-glucosamine-6-phosphate. The enzyme is known as a potential
target in antifungal chemotherapy and oligopeptides containing glutamine
analogs inhibiting GlmS demonstrated high antifimgal activity [ 1].
Several novel potential inhibitors of GImS have been designed,
synthesised and used to study the active centre and mechanism of reaction
catalysed by Candida albicans GImS. 3-amino-3-deoxy-D-glucose-6phosphate (kanosamine-6-phosphate), formed m situ in microbial cells treated
with kanosamine, behaved as a reversible enzyme inhibitor, similarly as N-acyl
derivatives of 2-amino-2-deoxy-D-glucitol-6-phosphate (ADGP). On the other
hand, N-haloacetyl derivatives of ADGP and N-oxoacyl derivatives of
L-2,3-diammopropanoic acid inactivated the enzyme in a time- and
concentration-dependent fashion. Differences in inhibitory and inactivatory
properties of the studied compounds, as well as molecular modelling and
molecular mechanics calculations, provided new data supporting a recently
suggested idea of close proximity of the glutamine-binding and D-fructose-6phosphate-binding domains ofC. albtcans GImS [2, 3].
[1] Andruszkiewicz, R. et al., J. Med. Chem., 33 (1990) 132
[2] Beame, S.L., J. Biol. Chem., 271 (1996) 3052
[3] Leriche, C. et al., Eur. J. Biochem., 245 (1997) 418

Abstracts FEBS'99

(Su/16.1/l14)

D-Glueosyltranferase: Substrate Binding and Proposed

Catalytic Mechanism
S. Mor~ra I , A. Imberty2, W. RtLger3, P. Freemont4
ILEBS, UPR 9063 CNRS. Bdt.34, 91198-Gif-sur-Yvette, France
2CRMV, CNRS BP53 F, 38041 Grenoble Cedex 9, France
3Arbeitsgruppe Molekulare Genetik, Ruhr Universaat, Bochum, Germany
4MSFL ICRF, 44 Lincoln's Inn Field, London WC2A 3PX, UK

~-Glucosyltransferase (BGT) is a DNA-modifying enzyme encoded by

bacteriophage T4 which catalyses the transfer of glucose from uridine
diphosphoglucose (UDPG) to 5-hydroxymethylcytosine (HMC) in doublestranded DNA. The glucosylation of T-4 phage DNA is part of a phage DNA
protection system aimed at host nucleases. We previously reported the first
three-dimensional structure of BGT determined from crystals grown in
ammonitun sulphate containing UDPG (Vrielink et al., EMBO J. Vail 3 pp 34133422, 1994). In this previous structure, we did not observe electron density for
the glucose moiety of UDPG nor for two large surface loop regions (residues 6874 and 108-122). We report two further BGT co-crystal structures, in the
presence of UDP (Form 1) and UDPG (Form II) respectively. Form I crystals
are grown in ammonium sulphate containing UDP and the structure has been
determined to 1.88 A resolution. Form II crystals are grown in
polyethyleneglycol 4000 with UDPG and the structure has been solved to 2.3 A
resolution. The Form I structure is isomorphous to our previous BGT structure
in UDPG. The Form I[ structure however, has allowed us to model the two
missing surface loop regions and thus provides the first complete structural
description of BGT. In this low salt crystal form, we also see no electron density
for the glucose moiety from UDPG, and thus is irrespective of the crystallisation
conditions. Biochemical data shows that BGT can cleave the glucose moiety
from UDPG in the absence of DNA acceptor, which probably accounts for the
absence of glucose in our UDPG substrate structures. The complete BGT
structure from Form II crystals, has provided us with a basis for detailed
modelling of DNA bound to BGT. Furthermore, using the structural similarity
between the catalytic core of glycogen phosphorylase and BGT, we have been
able to model the position of the missing glucose moiety from UDPG. From
these two models, we now propose a catalytic mechanism for BGT and identify
residues involved in both DNA binding and in stabilising a "flipped-out'
hvdroxvmethvl-cvtosine nucleotide.

(Su/16.11116)

Characterization of pectinlyase of commercial pectinases

for the clarification of apple juice
M Perez-Mateos, S. de Diego, M D Busto, N Ortega

s121

(Su/16.1/115)

~IBB, PAS, Pmt'mskiego 5a, 02 106 Warsaw. Poland

blB).I("CNRS, 15 rue Rene Descartes; 67084 Strasboorg cedex, France

Rn nuclease I isolated from rye germ nuclei has been characterised as a very
useful tool for secondary and tertiary structure investigation of RNAs.[ 1,2]
The main feature which makes this enzyme ideally suited to structural studies
is its ability to hydrolyse nucleic acids both in the presence and the absence of
divalent cations However using spectrophotometric assay we observed, that
the rate of RNA hydrolysis by Rn nuclease I was lowered by the presence of
divalent cations such as Mg-- Recently we have used yeast tRNA P~ and
yeast tRNA '~p to study the Mg -~ ions dependence ofRn nuclease 1 activity, it
is known from the crystallographic studies (1) that two magnesium ions are
bound to the anticodon stem and loop of the tRNA r'hc We have shown that
incrising Mg-" ions concentration inhibited Rn nuclease I cleavages observed
in the anticodon loop of tRNAVhL In contrast, the primary cleavages
introduced by Rn nuclease I to the anticodon loop of tRNA ~ were not
inhibited in the presence of magnesium ions. We conclude, that the inhibitory
effect of Mg'- ions on the RNA hydrolysis by Rn nuclease I is caused by the
change in the RNA structure rather than in the enzymatic protein molecule.
This also confirmes that Rn nuclease is a very sensitive structural probe.
[1] Przykorska A et al., Nucl. Acids Res ,20,(1992)659.
[2] Przykorska A eta[, Eur. J Biochem, 108,(1980)285.
[3 ] Westhof E, Sundaralingam M., Biochemistry 25, (1986)4868.
Supported by KBN Grant No 6 PO4A 063 10 and Polish-French
Biotechnology Centre.

(Su/16.1/117)

Department of B:otechnologv and Food Sctence, Universl~' of Burgos

Plaza 31isael Ba~uelos s n, Burgos 09001 Spare.

Clarified apple juice concentrate is one of the most consumed fruit juices in
the world. Problems in industrial clarification of apple juice are caused
mainly by the presence of pectic substances which remain as suspended
pulp particles. Commercial pectic enzymes (pectinases) are used in the
apple juice manufacturing to depectinize pressed juices in order to remove
turbidity and prevent cloud-forming [1]. Pectin lyase (PL, EC 4.2.2.10)
seems to be the only pectic enzyme capable to break down pectin with a
high degree of esterification into smaller molecules [2].
The objective of this work was to determine the activity and kinetic
properties of PL contained in four different commercial pectinases.
Rapidase C80 (Gist Brocades), Pectinex 3XL (Nova Nord:sk), Biopectinase
CCM (Biocon) and Grymdamyl 3PA (Grmdsted) In this sense, various
parameters, such as enzyme or substrate concentration and effect of pH
were studied.
All the preparations presented PL activity, exhibiting Rapidase C-80 the
highest activity levels. The kinetic constants of Michaelis (K,, and V,,~
values) were determined by measuring the enzymatic reaction rates at
different apple pectin concentrations (0.02-1%). PL activity did not follow
pure Michaelis-Menten kinetics in any of the enzyme preparation assayed.
The form of the graphs obtained was typical of enzymes exhibiting
substrate inhibition at high substrate concentrations. The determination of
apparent K,, and Vm~ constants was possible by using the linear portion of
the curve at low substrate concentrations [3], and the Km was calculated to
be 2 4; 34 3, 0.3 and 0.9%, whereas the Vm,, values were 19.0; 45 9, 3.4
and 10 9 U ml "~, respectively.
The pH-dependence of the pectinase activities was also tested. The optimun
pH was approximately 5.5 in the Rapidase C-80 assays and 6.0 in all the
other preparations.
[1] Ceci et al, Food Chem., 61, (1998) p. 237.
[2] Serra et al., Alimentacion, Equipos y Tecnologia, 8, (1992) p. 127
[3] Parr, Enzyme Microb. Technol, 5, (1983) p. 448

The influence of Mg + ions on the Rn nuclease I activity.

K. Olszak", G Keithb, A. Przykorska a.

Mechanistic studies on 8-amino-7-oxopelargonate

synthase, an unusual PLP-dependent enzyme
O. Ploux
Universit,5 Paris 6. CNRS UMR 7613, Bolte 182, 4 Place Jussieu 75252
Paris Cedex 05, France

Among pyridoxal-phosphate (PLP) dependent enzymes that catalyze

reactions at the at position of the amino acid substrate, the t-amlnoketone
synthases are unique since they catalyze a decarboxylation (the breakage of a
carbon-carbon bond) and the formation of a carbon-carbon bond by acylation.
The four enzymes that belong to this class are involved in early steps of metabolic
routes leading to important molecules (biotin, heine, sphingolipids and amino
acid).
We have been interested in the study of 8-amino-7-oxopelargonate
synthase which catalyzes the first commined step of biotin biosynthesis in bacteria
and plants as a model for this type of reaction (Scheme I) and because this unique
enzyme is a potential target for a new class of herbicides. We have thus undertaken
a thorough investigation of the enzyme isolated from Bacillus sphaericus, a biotin
producing strata, including mechanistic [I] inhibition [2] and structural studies [3]
as well as site directed mutagenes]s.
A general reaction mechanism for the ot-aminoketone synthases, involving
an intermediate, will be presented and discussed in light of the recently solved
three-dimensional structure of the E. coil 8-amino-7-oxopelargonate synthase.

CoA'S.~..F'....f~CO
0
Plmeloyl-CoA

O" +

H NH3+
H3C*~COO"

L-Alanlne

H3C

Scheme I.
[1]
[2]
[3]

+ .......

02

8-Ammo-7-oxop61argonat

Ic
~_

H ,,NH3+
H~C'~"~/~CO0"
O

Intecmedlate [

COO

-1
J

Reaction catalyzed by the PLP-dependent 8-amino-7-oxopelargonate

synthase.
O. Ploux et al (1996) Ear. J. Biochem. 236 301-308.
O. Ploux et al (1998) Eur. J. Biochem. 138 1-8.
S. Spinelli et al (1996) Acta Co'st. D 52 866-868.

S 12 2

Abstracts FEBS'99

(Su/16.1/118) Purification and partial characterization of the 13-glucosidaseof

vanilla. Implications in the preparation of vanilla
C. Robert, X . Ranjoanizafy, E. Odoux, P. Baret, F. Cadet
Laboratolrede Bloehmueet Gen~tiqueMol~:culatre,Universltede la Rrunion
97715 Saint.DenisMessa8. Cedex09

The preparation of the vanilla starts by a scalding : the pods are

plunged into water (60-65C) during 2 or 3 minutes. This step has
the objective to stop the vegetativ life and supposedly to let the
activity of the enzymes which are responsible for the appearance
of vanilla aroma. Among these enzymes, the most important one
is the 13-glucosidase which catalyses the reaction synthetizing the
vanillin, the principal aromatic chemical constituents of vanilla
aroma.
An extraction and a partial purification with different
chromatographies (DEAE-cellulose, hydroxyapatite, steric
exclusion) show that the [3-glucosidase has the optimun
temperature which is 47C and a heat stabilitie at 60-70C very
bad. The kinetic parameters (Km=5,7 mM and Vmax-438
~tmol/mirdl) and the molecular weight (250kDa) was determined.
The optimal pH is 7,0. The substrate used is the PNPG.
Considering the thermolabile nature of the 13-glucosidase, the
scalding would be a very inhibitory step of the synthesis of the
vanilla. In this case, the technique of scalding would be called
into question.

(Su/16.1/119)

Enzyme kinetics data of or-amylase extracted and purified

of marine molluscs Mytllus ~alloorovineialis Link. and
Mva Itrenaria L.
N. Rosoiu, l.Aschie, M.Aschie - Manescu,
Faculty of Medicine,"Owdim"University,,8.700 Constanta,Romania

The purified a-amylase of the hepatopancreas and whole body of Mytilus

~alloDrovincialis Link. has an optimum activity at pH 6,0 and 35C, and the
purified ct-amylase of the hepatopancreas and whole body of Mya aranaria L.
has an optimum activity at pH 6,9 - 7,0 and 32C.
Similar data obtained on unpurified protein extracts from hepatopanereas and
whole body, as well as on the purified a-amylase from hepatopanereas and
whole body, indicate the presence of single enzyme with amylolytic activity in
both bivalves. On the other hand, the more intense enzymatic activities, which
were proved the hepatopancreas, show that this is
the organ which
concentrates the amylase. Amylasie activity was discerned also in nondigestive
organs, such as mantle and branchie, and also in hemolymphin in direct
correlation to the glycogen content and closely connected with the internal
biological rhytm of the mollusca during the osmotic control process. Our data
pointed out an increased amylolytic degradation of glycogen, in hipo- and
hypersalinity stress physiological states of the mussel.
The amylasic activity in Mytilus galloprovincialis (1,102 mU/mg/minute) is
comparatively higher than the amylasic activity in Mya arenaria (587
mU/mg/minnte)
The purified a-amylase it self shows good time - stability; after four months
refrigerating (+4C), 100% of its enzymatic activity was still found, in the
absence of any ingredient. CaCI2, NaCI and MgCI2 in concentrations between 1
- 10 mM activate the a-amylase and protect it against thermal inactivation.
The Ca 2+ and CI are essential for the enzymatic activity of marine mollusca 0~amylase.
The amylasic activity in Mytilus galloorovincialis and Mya arenaria, from
Romanian Bleak Sea Coast is similar to that in the benthonophage fishes and
the higher mammals - rabbit, cow, dog - and it is several times greater than in
the crustaceans and the predatory fishes [1].
[1] Rosoiu N., Iaeovache C , Rev.roumBioehim, 17, 4 (1980), 285

(Su/16.1/120)

Adenine-N6 DNA methyltransferases also modify cytosine

residues at position N 4.
M. Roth, F. Christ, M. Fatemi & A. Jeltsch
lnstitut fi~r Biochemie, Heinrich-Buff-Ring 58, 35392 Giessen, Germany

Methylation of DNA is important in many organisms and essential in

mammals. Nucleobases can be methylated at the adenine-N 6, cytosine-N4 or
cytosine-C 5 atoms by specific DNA methyltransferases. We show here that the
M . E c o R V , M . E c o R I and E.coli dam methyltransferases as well as the N- and
C-terminal domains of the M . F o k l enzyme which formerly all were classified
as adenine-N 6 DNA methyltransferases also methylate cytosine residues at
position N 4. Kinetic analyses demonstrate that the rate of metbylation of
cytosine residues by M . E c o R V and the M.FokI enzymes is reduced by only 12 orders of magnitude in relation to methylation of adenines. This result shows
that although these enzymes methylate DNA in a sequence specific manner,
they have a low substrate specificity with respect to the target base. This
unexpected finding has implications on the mechanism of adenine-N 6 DNA
methyltransferases. Sequence comparisons suggest that adenine-N 6 and
cytosine-N 4 methyltransferases have changed their reaction specificity at least
twice during evolution, a model that becomes much more likely given the
partial functional overlap of both enz4yme types. Interestingly, methylation of
adenine residues by the cytosine-N methyltransferase M . B a m H I was not
detectable, presumably, because it is more easy to form an active site that
accepts cytosine but excludes the larger adenine than vice versa. On the basis
of our results we suggest to group adenine-N 6 and cytosine-N4
methyltransferases into one enzyme family.

(Su/16.1/121) Site-directed mutagenesis of human aromatase.

G.E. Srralini a, P. Auvray a, R. Bureau b, S. Rault b, P. Sourdaine a
"UPRES EA 2608, Umversit( de (ben, I4032 Caen. France
CERMN. UmversitO de Caen. 14032 Caen, Frame.

Human aromatase (hP450arom) is a cytoehrome responsible for

estrogen synthesis and is mainly implicated in reproduction and
estrogen-dependent tumour proliferation. In order to understand
better the structure-activity relationships for aromatase, we
mutated 18 residues on the basis of sequence differences between
hP450arom and equine aromatase (eP450arom). This equine
protein is characterized by substrates specificites and differential
inhibitions with hP450arom [1,2]. Catalytic efficiencies of
mutants were evaluated in transfected human 293 cells using
androstenedione, testosterone or nor-testosterone as substrates.
Aromatase levels were evaluated by ELISA and, when necessary,
the corresponding mRNA were checked by RT-PCR.
Our results suggest that D309 and H475 are directly implicated in
the androgen aromatisation. Moreover, E302, K130 and D476
stabilised the androgen close to H128, T310, D309 and the heine
iron. However, the aromatisation mechanism with nor-androgen
was different since E302 interacted with C(2) rather than D309.
Furthermore. we showed that M85, K119 and D232 were crucial
residues for the access channel. These results, together with
computer modelling, allow us to develop new inhibitors useful as
molecular tools for active site understanding of this crucial
cytochrome P450 and possibly in the treatment of estrogendependent cancers.
[ I] : Moslemi S. et al. Ann. N.Y. Acad. Sc., 839' 576, 1997
f2] : Auvra~ P. et al. Eur. J. Med. Chem, 33: 451. 1998

Abstracts FEBS'99

(Su/16.1/122)

s123

Stopped-flow fluorescence studies of DNA base flipping

by the HhaI methyltransferase
S. Serva I. E. Weinhold 2 and S. Klima~auskas I

(Su/16.1/123)

~hT.~tmaeo/Btotechnology, Vdmus, Llthuama

:,~lPl hit MolekularePhvstoloeie. Dortmund. Germany

Rotation of a nucleotide out of the DNA helix (base flipping) has been
first observed for the Hhal methyltransferase followed by numerous
other DNA modification and repair enzymes. MHhal catalyzes
transfer of a methyl group from cofactor S-adenosyI-L-methionine
onto the C5 position of the first cytosine in the target sequence
GCGC. In this study, stopped-flow and rapid-quench techniques
were employed in combination with fluorescence detection for kinetic
characterization of individual steps on the reaction pathway of
MHhal. Selective labeling of the DNA substrate was achieved by
synthetic incorporation of 2-aminopurine at the target or neighboring
base positions. Association, dissociation and single-turnover
experimental setups permitted direct determination of kinetic
parameters for DNA binding, flipping of the target base,
conformational transitions in the protein, methyl group transfer and
release of methylated DNA. In conjunction with structural data, our
results suggest detailed catalytic mechanisms for DNA base flipping
and catalysis by DNA cytosine-5 methyltransferases.

(Su/16.1/124)

The Functional Models for Cateehol Dioxygenases

L. Tchertanov*, a and J.-J. Gircrd b
a CNRS ICSN, UPR 2301, 91198GifSur Yvette, Cedex, France,
bURA CNRS 420, Universitd Paris-Sud, 91405 Orsay, France

The design of biomimetic catalysts is intensively studied in various areas of

chemistry. The major aim is to design artificial systems that reproduce
characteristics of enzymatic actions such as complex formation with
substrates, large rate acceleration, and high selectivity. Intradiol-cleaving
catechol dioxygenase [1] represents a family of bacterial enzymes that
catalyzes the oxidative cleavage of intradiol C-C bonds of various catechol
derivatives by molecular oxygen, yielding the corresponding muconic acid.
The proposed active site of the enzyme has a five-coordinate iron(Ill) core
structure with two histidines, two tyrosines and a water molecule or a
hydroxide ion in a trigonal-bipyramidal geometry [3]. We attempted to
synthesize highly catalytic biomimetic model systems for the study of the
reactivity of intradiol-cleaving catechol dioxygenases. The aim of our work
was to further contribute to understanding of the structure-function
relationships of this industrially important family of compounds. A series of
metal complexes was obtained by modification of both the cation (iron and
manganese) and liganding elements. Over the past years we prepared and
investigated a variety of iron catecholate complexes with the amino-phenolate
[3] and amino-pyridine ligands [4]. We also studied a series of Mn/Fe
catecholate complexes containing the tetraazamacrocyclic ligands (L=N,N 'dimethyl-2,11-diaza[3.3](2,6)pyridino- phane) as a coligand because it was
recently reported that these complexes act as efficient catalysts [5]. Binding
properties of the catechol were studied by statistical analysis of crystal
structures in the Cambridge Structural Database [6].
References :
I. Que, L., Jr., in Iron Carriers and Iron Proteins (Ed. : T. M. Loehr). VCH,
New York, 1989.
2. Ohlendorf, D. H.et al., ~ Mol. Biol., 244, (1994), p. 586.
3. Mialane, P. et al., lnorg. Chim. Acta, 263, (1997), p. 367.
4. Mialane, P. et al., Inorg. Chem. (1999). Accepted for publication.
5. Koch, W.O. and Kriiger, H.-J. Angew. Chem. Int. Ed. Engl. 34, (1995), p.
2671 ; Koch, W.O. et al Chem. Eur. J. 4, (1998). p. 1255.
6. Allen, F. H. and Kennard, O. Chem.Design Aut. News 8 (1), (1993), p. 31.

tH NMR study of the metal environment in Ce(ll)atrolysin

B from diamondback rattlesnake (Crotatus atrax) venom.
A. Sigmundsson, J.B. Bjamason, S. Jonsdottir and S. Olafsdottir.
Science Institute Universityof Iceland, Dunhaga3, 107 Reykjavil~Iceland

Atrolysin B is a hemorrhagic zinc metalloprotease in the reprolysin family of

proteases, M12, subfamily B. Crystal structures of adamalysin II and
atrolysin C, both representatives of this subfamily, show that the active site
metal is coordinated to three histidine residues and one solvent molecule that
is sandwiched between the metal and a conserved glutamic acid residue
leaving the fifth coordination site open to the solvent [1, 2].
Atrolysin B was isolated using ion exchange and gel filtration
chromatography [3]. The Zn(II) was stoichiometrically substituted by direct
exchange with the paramagnetic probe, Co(II), in order to study the structure
and function of the active site. Co(II)-atrolysin B was found to retain full
activity against azoeasein with a pH optimum at 9.3, same as that of the
native enzyme. Kinetic measurements against the fluorogenic substrate 2aminobenzoyl-AGLA-4-nitrobenzoylamide at pH 7.0 yielded a KM of 0.3 mM
for both the native and the Co(II) substituted enzyme. These data suggest
that the metal substitution causes tittle or no distortion of the active site
structure of atrolysin B. The inhibition constant (KI) of the thermolysin
inhibitor phosphoramidon on Co(II) atrolysin B was found to be lxlff s M.
~H-NMR spectrum of the Co(II) substituted enzyme showed at least 15 well
resolved paramagneticaUy shifted resonances in the regions from 10 to 80
ppm and -2 to - 12 ppm corresponding to hydrogen nuclei in the vicinity of
the para_rnagnetic metal. Variation of pH in the range from 6.0 to 10.0
resulted in a further shift of some of these resonances.
[1] Gomis-Rtlth, et al., E M B O , 12, (1993) 4151.
[2] Zhang, et al., Proc. Natl. Acad. Sci. USA, 91, (1994) 8447.
[3] Bjarnason and Tu, Biochemist2/, 17, (1978) 3395.

(Su/16.1/125)

Structure of a covalent intermediate trapped to CGTase

J.C.M. Uitdehaag a, R. Mosi b, K.H. Kalk a, B.A. van der VeenL
L. DijkhuizenL S.G. Withers b, B.W. Dijkstra a
aLab of Bwphystcal Chem , Umv. of Groningen, Nqenborgh 4, 9747 AG
the Netherlands bDept, of Chemistry, Umv of British Columbta, Vancouver, Canada CMtcroblology, Umv of Groningen, the Netherlands

Cyclodextrin glycosyltransferase (CGTase) is an enzyme of the

a-amylase family, which uses a double displacement mechanism to process
ct(1->4)-linked glucose polymers. In this mechanism the sugar in the catalytic
site (labelled-l) is processed in the following order: substrate -> [transition
state] -> intermediate-> [transition state] -> product. In the past we have
studied this catalytic process by X-ray structures of CGTase in complex
with a substrate (maltononaose) bound to CGTase [1]. Here we report the
1.8 A structure of a covalent intermediate bound to CGTase [1 ].
To trap the intermediate, we used the compound 4-deoxymaltotriosyl
ct-fluoride (4DG3ctF), from which the fluoride group leaves easily in the first
step of the double displacement reaction. However, the compound prevents
the second reaction step through the absence of an acceptor 4-OH group,
instead the very poor acceptor water has to be used by CGTase. Activation
of water by Glu 257 is prevented by use of the E257Q mutant CGTase.
Mass spectroscopy experiments indicated that this experimental approach is
indeed succesful in trapping a covalent intermediate [2], and we proceeded
with a crystallographic experiment. Crystals of the Bacillus circulans strain
251 E257Q CGTase grow in the presence of 5% maltose which is a very good
acceptor. Therefore we replaced the maltose by repeatedly washing the
crystals with 1% 4-deoxymaltose over 3 days. Finally the crystal was soaked
for 16 minutes in a freshly prepared solution of 60% MPD, 100 mM MES
buffer, pH 6.1 and 125 mM 4DG3ctF, after which it was frozen to 100 K for
datacollection to 1.8 A.
The electron density maps show an unequivocal covalent 13(1->4)
glycosidic bond between the glucose in subsite -1 and the nucleophilic aminoacid Asp 229. The conformation of the intermediate is not distorted, in
contrast to the conformation of substrate (as seen in another CGTase
complex). Comparison of both structures for the first time shows in atomic
detail how catalysis in the ct-amylase family proceeds [l].
1.
Uitdehaag,J C.M, et al Catalysis in the s-amylase family. X-ray structuresalong
the reaction coordinateof cyclodextringlycosyltransferase NatureSlrucl Btol repress
(1999),
2.
Mosi, R, He, S., Uitdehaag, J, Dljkstra, B.W. & Wtthers, S.G Trapping and
characterization of the reacnon intermediatein cyclodextrmglycosyltransferaseby use of
activated substratesand a mutant enz3'me Btochemistry36, 9927-9934 (1997)

s 124

Abstracts FEBS'99

(Su/16.1/126)

Stabilization of Lipase from Nigella Sativa Seeds

By Polyethylene Glycols

Probing RNA structure with IgG and synthetic RNases

G. Ustun, S Unsal, A. Akova, S Turkay

A. Vlassov', M. Helm b, D. Konevets', V. Sil'nikov',

M. Zenkova ~, G. Nevinsky', C. Florentz b, R. Gieg&

lstanbul Techmcal Umveesitv, Chemtcal Engineering Department,

80626,/vlaMak, L~tanbul. Turkey

"NIBC, 8. Lavrentiev ave., 630090 Novosibirsk, Russia

blBMC/CNRS, 15, rue Descartes, 67084 Strasbourg, France

Lipases are one of the most important class of industrial enzymes, which are
used intensively in the biotransformation of fats and oils, in household
detergents, and especiallly in the synthesis of very important pharmaceuticals
and agrochemicals. Most of the industrial lipase enzymes have been
produced and stabilized from microbial resources. Although cereal grains and
oilseeds are cheap and alternative sources of lipases, they have not yet been
studied in this regard and they could be exploited for industrial or technical
purposes
Nigella sativa L. is a member of Ranunculaceae family and contains an active
lipase even in dormant seeds. In this study, the stabilization of Nigella sativa
lipase in the presence of polyethylene glycol (PEG) compounds was
investigated.
To see the effect of PEG compounds ( PEG 1000, PEG 6000 and PEG 8000)
on the thermostability of Nigella saliva lipase, PEG compounds at various
concentrations from 0.1 to 5 0 % (w/v) were added to the crude enzyme
solutions which are extracted from seeds by phosphate buffer (pH 7 2). After
heat treatment of enzyme solutions with or without PEG compounds at 30-60
C for 30 min, optimal concentrations of PEGs that provide the highest
thermostabilization of enzyme, were found to be 2 5, 1 3. and 1 0 % for PEG
1000, PEG 6000 and PEG 8000, respectively. The greatest enhancement of
thermostability was observed with PEG 6000, as a nearly 5-fold increase
above 50 ~JC The addition of PEGs did not cause any change in the optimal
temperature and pH value of free enzyme, but the parameters V,,~, K,~, the
activation energy were decreased in some extent because of the
uncompetitive inhibition by PEG compounds

(Su/16.1/128)

(Su/16.1/127)

Site-directed Mutagenesis of Porcine Pyridoxal Kinase

H.Y. ~'ong. S.C.L. ko. F. Kx~ok. J.E. Churchich. Y.C. I eang
The Hong K~m,4Po(i techm~ ( nn'crsi 0 H(nl~ h'r,l~,. (7tt,a

lgG purified from sera of patients with autoimmune and viral diseases are
shown to present RNA hydrolyzing activities that are different from the weak
RNase A-type activities found in the sera of healthy donors. Further
investigation brings evidence for two intrinsic activities, one observed in low
salts conditions and another specifically stimulated by Mg 2 ions and
distinguishable from human sera RNases. Cleavage of RNA substrates by the
latter activity is not sequence-specific but sensitive to subtle conformational
and folding changes, as evidenced by comparative analysis of couples of
structurally well-studied RNA substrates. These include yeast tRNA Aspand its
in vitro transcript and human mitochondrial tRNALys-derived in vitro
transcripts. The discovery of catalytic antibodies with RNase activities is a
first step towards creation of a new generation of tools for the investigation of
RNA structure.
RNA cleaving molecules mimicking catalytic center of ribonuclease A were
synthesized. The compounds have imidazole residues, conjugated to cationic
structures with different number of charges. In experiments with human
mitochondrial tRNALyS-derived in vitro transcripts and yeast tRNA Phe it was
found that some of the artificial nucleases cleave RNA in physiological
conditions (37C, pH 7). displaying similar cleavage specificity with some
variations. Sensitivity of phosphodiester bonds to the compounds decreases in
the order: CA>UA>CG>CU, CC. Synthetic ribonucleases display strong
preference to the single-stranded RNA regions. The compounds provide new
probes for investigation of RNA structure in solution and potential reactive
groups for antisense oligonucleotide derivatives.

(Su/16.1/129)

USING THE PHOTOREACTIVE ANALOGS OF dNTPs

FOR INVESTIGATION OF THE DNA POLYMERASES
A. L. Zakharenko, S. N. Khodyreva, N. I. Rechkunova, I. V.
Safronov, D. M. Kolpashchikov, O. I. Lavrik
Institute of Bioorganic Chemistry, Novosibirsk, Russia

P xridoxal 5-phosphate (PLP) is tile metabolically acti~ e

lbrm of ~itanfin B,, and acts as an essential coenz.x me in the
sxnthesls, catabolism, and interconversion of amino acids in
cellular metabolism. Pyrido'~al kinase (PK) catalyzes the
formation of PLP from pyridoxal (PL). ATP and a divalent
cation (Zn 2.) [l]. flowever, the amino acid residues in PK that
are responsible for this important biochemical reaction ha,,e
not been determined. Recently. the porcine PK gene has been
cloned in our laboratory [2]. With the gene in hand. it is now
possible to use the powerful site-directed mutagenesis method
to change the potentially important amino acids in this enzyme
so that their structural and functional relationships can be
studied in a more logical manner.
Site-directed mutagenesis was performed and three
potentially important mutants (G240A. G242A and Y135F)
have been constructed and confirmed by sequencing the DNA.
The mutants and wild-type PK have been successfully
expressed in E. coli BL21 (DE~) expression system, in which
the PK gene was put under the control of a T7 phage promoter.
Activity of the expressed enzymes in the cell extract was
detected by using phenylhydrazine assay and fluorometric
assay. The G240A and G242A mutants totally aborted the PK
activity while the Y 135F mutant lost about 80% of the activity.
To conclude, our data suggest that both Gly240 and
Gly242 are crucial for the proper binding between PK and
substrates. Moreover. Tyr135 might have a role in maintaining
the optimal folding of the PK protein.
[I] McCormick. DB. et al.. Proc Natl Acad. Sci. USA. 45. (1959) 1371
12] Gao. Z G. et al. Int. J. Brochem.Cell B., 30. (1998) 1379.

The interaction of DNA polymerase 13and DNA polymerase from

Thermus thermophilus B-35 (Tte DNA pol) with dNTPs was investigated
by the method of photoaffinity modification using two ways. The first
one suggests modification of the enzyme by primer synthesized in situ
via incorporation of photoreactive dNTP into the 3'-end of primer, The
second variant consists in irradiation of the enzyme with photoreactive
dNTPs followed by incorporation of covalently bound dNTPs into
primer. It was shown that the photoreactive group of the dNTP analogs
under UV-irradiation can yield some long-living reactive products which
still are capable of modifying proteins and give certain contribution in the
case of second way of labeling. All analogs of dNTPs used were shown
to be substrates of DNA polymerases. DNA polymerase 13 was modified
by NABdUTP, sTTP and IdUTP. It was shown that the efficiency of
modification depends on length of linker of analog used [1].
Photoaffinity modification of Tte DNA pol was carried out by
FABdCTP, AIFABdCTP, NABdCTP [2], FAP-7-dUTP and FAP-8dUTP [3]. The complementary addressed modification of the DNA
template was carried out by use of primer synthesized in situ by Tte DNA
pol with FABdCTP and A[FABdCTP as substrates.
The comparative analysis of substrate properties of dCTP~ TTP
and their photoreactive analogs in DNA synthesis catalyzed by IMV-1
reverse transcriptase and two its mutant forms (Y115W and Y115A) was
carried out. The wild type and Y115W forms of reverse transcriptase can
use both pyrimidinic dNTP and their analogs as d T T P and dCTP. Mutant
form Y115A seems to be less elToneous than those having in position
115 an aromatic amino acid are. When the dNTP analogs replace their
natural counterparts all the enzyme forms tend to pause DNA synthesis.
[I] 0. 1. Lavrik et al., Molekulyarnaya Biologiya, 32, (1998) 621, in
Russian
[2] A. L. Zakharenko et al., Biochemistry (Moscow), 63, (1998) 929,
translated from Biokhimiya, 63, (1998) 1090
[3] D. M. Kolpashchikov et al., Bioorganicheskaya Khimiya, (1999)
(accepted), in Russian

gEI s

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sX~p ao~o oql aoj punoj sllnso~I "uo!lt.qFtm" olgalsqns ol OA!~!SU~SU!lSOtUI~S~/~
lOamOO u! lu~soad atuAzuo atll al!qta uo!l!q!qu! ~ n s q n s o~ 1!A!I!SUOStl~.tq
po~aoqs 91 eP le ,uasaad otuXzuo aqz "s!soqmdq s ! ~ pauu~uoa 01) lumsuoa
La!OOla^ uo!mt~oa oql j uou~m.uu~mt2 'uo!~lnmt3sounurm! aapun mososd
'IAItu t," I -+t,l Jo anleA aamoI e ql!~ auo aaqlotre ptm 'suo!l!puoo letmou aapun
mosoad 'IAItUg"I-+tr~ jo anl~A ttr~ aoq~.rqql!A~ouo :s~!d tzou!n~jo A~oa.mtuouoq
aql m. ostq'm,eo aql aoj sotuXzuoos! oA.u~tseoI ~ttzoq plnoqs oaoql l~ql pa~,so$~ns
slInsoa asoq.L '(KIOtqmadsaa 'IAlm L'0-+g'81ptre lAitu L'0-+ffg~) Lm!potmom!
o.la~ son[VA UI~ '0E pu~ El sXep .tod "po.lOAO:)O.l S~AX I~Im g [ -+t~E
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'(~P 1V "puno3 s~A~lArtu ;'i-+~E3o Onlt~AU' 8 pu~ ~, s,(up soJ s~ lla~ se lOnUOO
oq* zo3 ~mq1potaoqs sonluA ttr-A jo uo.t,~u!tmom(i "0E up lu anita I~l.1!u! oql
qo~o.t ,qqSno.t ol past~aaap Ll!^!lo'e oq~ '.to~J~a.toqz "91 gep :l.'e~u!h~eodos~.tou!
a~!ssoa~oad ~ q poa,,OIlO3 ~ ~p ltz oseosoop ~ potaoqs uo.qoo[u] lsa~ aql aotJ~
s,(ep snO.LIeA :V~ptre lOXlUOOaq~ u! poanseatu ~!A~O~OSelm~D -uop,oa.fu! lsag
oql ao~je sep 0 ol dn pou!tm*lap oao~ otu/,zuo aq~,jo so!laodord o!lOUpl OtlZ
tau 0I'E le EOCH jo ootrea~oddes!p oql ~u!~ollo3 q XII~O!aotuoloqdo~loads
pau!tuaolop s ~ 1!A!lO~ aS-elm~D 'qsxeosoa s!to u! papnlou.t mu aaoA~ 0~
'aSelm~a u!muoo ,,uop si!qdo~ q oam.S 'sl!qdosmou aoj 0Z ptm 91 sep ~u!pnlau!
~ o d t~ ptm Slt.qdom.sooaoJ 0Z t~p ,e )l~ad e ~u.tonpoad 'postzoaout. UOtll pue 1ss~3
le poseoaaop sl!qdoamou ptm sl!qdom.soo jo oSmuaasacl oql lwql pot~oqs saeotus
~oJaem ouoq jo uo!l~m.umx~I "suo!~u!tuaalop ot.l~tu~o aoj saidttms oat~doad
o~ ptm uo.tl~u!ttmxo o!doosoao!m aoj s.moms osdoad o~ posn S~A~S~OI pm.q
ptm luoaj mo.Ij pOAOLUOaA~o.t,mulouoq '.pa0g.uo~s ~ao~ SlVm!tre 'uo!lao[ut. lsa~
oql aolJ~ sg~p 0t~ o~ ~ moaj ~u!~tma sam D sno.u~A ~V 'st.solXoOlntres~aoj pam.ud
s~!dt~au!n8 'sXep ~[ soj poolq daaqs aloq~a [m auo jo suop,aa[u! snoatmmoqns
gl!eP ,(l~I "suo!mtzaa auntum! jo smnpoad ap!s oxe qot.qA~ sol~!pattualut.
uo~,~xo aA!la~aa Su!i]uaA~as m. a^!laoj.3a s! 'tuo~s,is os~p!xoaod auo!qlmni~
aql ptre as~mtustp ap!xoaodns ~u!pniau! 'sltmp!xot.lu~ sa~o o)[1.1 'as~IeltzD
UOal 'uvatta 'uva~tal fo eOt.saaa!ul"l
'go!~,frldolffpwo ~als!utatlOO!gaof almltsu I
!ueqa'>I "~ '.ttreAtI-.t~loolqxe::tq~1 " '!a.re2 "V'IAI
uo.tl~lnm!lsounmm! ,~q paanpu!
~oJaem auoq ~!d ~amn.Bm. aSelmma io som~zos I
(O~[/I'9I/ns)

66,S ~tK_q s l a e a s q v

s126

Abstracts FEBS'99

2.1 Control of DNA replication

(Su/2.1/131)

Fidelity of replication of leading and lagging DNA strands

in the Escherichia coli dnaX mutator.
D. Gaweq ~, P. Jonczyk ~, I. J. Fijal'kowska ~, R. M Schaaperb

(Su/2.1/132)

]nstt~te of Biochemistry and Biophysics, Warsaw,Poland

bNational Institute of Environmental Health Sciences, USA

The mechanisms that control the fidelity of DNA replication are being
investigated by a number of approaches. Important tools in these studies are
mutant versions o f D N A polymerases that affect the fidelity of DNA
replication.
Replication ofEscherichia coli chromosome is performed by the DNA
polymerase 1II holoenzyme (Pol III HE). Pol III HE is asymmetric complex
containing a total of 18 subunits (10 distinct). It has been suggested that proper
interactions within the polymerase III holoenzyme could be essential for
maintaining the optimal fidelity of DNA replication. We have been particularly
interested in elucidating the physiological role o f t subunit (product ofdnaX
gene). The z subunit dimerizes DNA polymerase III via interaction with the ot
subunit, allowing DNA Pol lII HE to synthesize both leading and lagging
strand simultaneously at the DNA replication fork.
To gain insights into the functional and structural importance of proper
x - ot interaction in maintaining the high fidelity of replication we examined'
a/the ability of the ot protein to interact with two x mutant proteins (DnaX36,
DnaX2016) using yeast two-hybrid system,
b/the fidelity of synthesis of the leading and lagging DNA strands in E. coli
strains carrying mutant dnaX alleles.
Our results indicate that dnaX36 and dnaX2016 mutations result in an
altered strength of the ct subunit-x subunit interaction. E. coli strains carrying
either dnaX36 or dnaX2016 allele exhibit moderate mutator phenotype.
Our previous data [ 1] suggested that in the wild-type strain of the E.
coli the lagging-strand replication is more accurate than leading-strand
replication. The presence ofdnaX36 and dnaX2016 mutator alleles changes the
mutational strand bias between leading and lagging DNA strand. A possible
role of'c subunit in controlling fidelity of DNA replication will be discussed.
References:
[1] I. J. Fijal'kowska et al Proc. Natl. Acad Sci. USA Vol. 95, 1998, pp. 9718

(Su/2.1/133)

DNA sequence analysis of XsusOs revertants UV-induced

in a host permissive or nonpermissive for DNA replication
J Krwawlcz, 1 Pietrzykowska
Institute of Biochemistry aml Biophysics. PAS,

Most known archaeal DNA polymerases belong to the type B family, which
also includes the DNA replication polymerases of eukaryotes, but maintain
high fidelity at extreme conditions. We describe here the first crystal
structure of an archaeal DNA polymerase, at 2.5 A resolution, from the
hypertherophilic Archaea Thermococcus gorgonarius and identify structural
features of the fold and the active site that are likely responsible for its
thermostable function. Comparison with the mesophilic B type DNA
polymerase gp43 from the bacteriophage RB69 highlights thermophilic
adaptations, which include the presence of two disulfide bonds and an
enhanced electrostatic complementarity at the DNA-protein interface. In
contrast to gp43, several loops in the exonuclease and thumb domains are
more closely packed. This apparently blocks primer binding to the
exonuclease active site. A physiological role for this 'closed' conformation
is unknown but may represent a polymerase mode, in contrast to an editing
mode with an open exonuclease site. This archaeal B DNA polymerase
structure provides a starting point for structure-based design of polymerases
or ligands with applications in biotechnology and the development of
antiviral or anticancer agents.
References:
[1] Hopfner KP, et al, Proc. NatL Acad Sci. USA (1999), in press.

(Su/2.1/134) A Novel Conformation of the Herpes Simplex Virus Origin

of DNA Replication Recognized by the Origin Binding Protein
Alireza Aslani, Bertil Macao, Stina Simonsson and Per Elias.
Department of Medical Biochemistry, Medicinaregatan 9, Box 440, 405 30
Goteborg, Sweden

Pawinsklego 5A Sir., lSarsaw, Poland

Studies on the mechanism of UV-mduced mutagenesis independent of DNA

replication have been continued We have previously shown that this
mutagenic pathway daffers in several aspects from that occurmg under
conditmns permissive for phage DNA rephcation in E. coil C600 su" host
The main d~fference is related to the requirment for UmuD and UmuC
proteins and the refractory to the mismatch repair system. Mismatch repair
deficiency in E. coh 594 mutL host, nonpermissive for kin.sOs phage, does
not affect spontaneous and UV-anduced L~usOs mutagenesis. By contrast,
in E. coli C600 .m-mutL a high increase m spontaneous, and a moderate in
UV-mduced mutagenesis was observed.
Sequence analysis of ks'u.~Os revertants have reveled that most (63%) of
UV-induced mutations in the host nonpermisstve for phage DNA replication
(594su-) consists oftransversions. On the other hand, m the host permissive
for DNA replication (C600su +) the oposite is observed, 60% are
transitions. Deletion of umuDC operon m the nonpermlssive host
completly inhibited UV-mduced mutagenesis, but not in the permissive one.
In the last, 70% of UV-mduced mutants are transitions.

Crystal structure of a thermostable type B DNA polymerase

Hopfner, K.-P), Eichinger, E.t, Engh, R.A. 12, Laue, F.2,
Ankenbauer, W.2, Huber, R), Angerer, B.2
i Max-Planck-lnstitutfur Biochcmie,I)-82152 Martinsried, Germany
'- RocheDiagnostics,D-82372 Penzberg, Germany

Initiation of replication of herpes simplex virus type I is dependent on

cis-acting elements, oriS or oriL, and trans-acting factor the origin binding
protein or UL9 protein. The ability of OBP to bind cooperatively to oriS
and the replication efficiency of plasmid DNA containing oriS varies
periodically with the distance between the recognition elements. A model
has been proposed suggesting that ATP-dependent cooperative binding of
OBP to oriS induces a conformational change followed by an OBP-guided
deposition of the single-strand DNA binding protein ICP-8.
OBP is a dimeric sequence specific DNA binding protein and an ATPdependent DNA helicase.
Recently we have seen that the dimeric full length protein binds a novel
conformation of oriS referred to as oriS*. This DNA consists of a hairpin
structure with the AT-rich sequence presented in the loop whereas the
stem contains recognition sequence. The hairpin appears to be flanked by
5'- and 3" single-stranded tails. It appears that OBP interacts not only with
its recognition sequence but also with a novel structural element in oriS*.
The OBP-oriS* complex is exceedingly stable with a half-life of 25
minutes at 37C. Mutations in oriS that inhibit replication also prevent the
formation of a OBP-oriS* complex.
Based on these observations we suggest a model for the activation of
or~S in which OBP facilitates the extrusion of hairpin or cruciform
structures.
Using individually expressed domains of OBP we are currently
attempting to identify contacts between OBP and oriS*.
References
Boehmer, P. Aet al. (1997) Annu. Rev. Biochem. 66, 347-384.
Elias, P et al (1986) Proc. Natl. Acad. Sci. USA 83, 6322-6325.
Makhov, A.M et al. (1996) EMBO J. 15, 1742-1750.

Abstracts FEBS'99

(Su/2.1/135)

Unequal fidelity of leading strand and lagging strand DNA

replication in SOS-induced Escherichia coil cells.
M. Maliszewska-Tkaczyka, p. Jonczyk~, M. Bialosk6rska~,
I. J. Fijalkowska~, R.M. Schaaper b

s127

(Su/2.1/136)

Functional aspects of yeast telomerase activity

Petro\ A V' Dokudoxskaxa S $ ' Sakolox K A '.
Fax re A :. Dontsox a O'A '. Bogdanox A A '
~ DeparUnellt of Chenusn'.. Mosco~x State UlllVCfsln. N|oscov~. Russia
_ lasl'lla Jacclues Mollod. Pans. France

~Insntute of Biochemistry andBiophysics, Poland, bN I E H S, USA

To improve our understanding of the role of DNA replication in

mutagenesis we investigated the mutagenic potentials of leading and lagging
strands during DNA replication in the model organism Escherichia coil One
major pathway of mutagenesis in E. coli depends on the SOS response. The
current model of SOS mutagenesis invokes the interaction of RecA* and the
Umu(D')2C {"mutasome"} with DNA polymerase III. Despite progress in
understanding the components of SOS mutagenesis, the mechanism of the
error-prone replication is still unknown.
In this work we studied mutagenesis on the E. call chromosome in the
recA 730 strain. Strains carrying recA 730 allele exhibit constitutive expression
of the SOS system and spontaneous mutator effect without activation by
external D NA damage.
We measured the differences in the fidelity of synthesis of the two
strands during SOS induced chromosomal replication in viva. The possibility
of differential replication fidelity arises from the distinct modes of replication
of the two strands. The different mechanisms o f replication of two DNA
strands may create different ability to form mutasome. For this purpose we
constructed pairs of strains differing in the orientation of a mutational target,
the lac operon, with respect to the origin of chromosomal DNA replication. In
one strain of each pair, a transcribed sequence is replicated as a leading strand,
while in the parallel strain, the same strand is replicated as a lagging strand [ 1].
To measure the specificity and frequency of mutagenesis we used several lacZ
alleles constructed by Cupples and Miller [2] These alleles represent different
missense mutations at the same coding position. Each missense mutation can
be reverted by a single, specific base substitution. Our results obtained with the
recA 730 mutator clearly indicate that there is a significant difference in the
fidelity of replication between the two replicating strands. Based on our data
we propose a model describing the mechanism of SOS mutagenesis.
References:
[1] I J. Fijatkowska et al Proc. Natl. Acad Sci. USA Vol. 95, 1998, pp. 9718
[2] J. H. Miller et al Proc. Natl. Acid. Sci. USA Vol. 86, 1989, pp. 5345
(Su/2.1/137)

Replication of Chromosomes
B. Stillman
Cold Spring Harbor Laboratory
Cold Spring Harbor, NY USA

During the cell division cycle in eukaryotic cells, chromosomes replicate

and separate once during the S phase of the cell cycle. There are two
principal steps in the control of the cell division cycle. These occur at
transitions from G1 to S phase and from G2 to M phase. However, the
assembly and disassembly of protein complexes that affect these processes
occurs throughout the entire cell division cycle. For the initiation of DNA
replication a key protein is the Origin Recognition Complex (aRC). a R C
recognizes origins of DNA replication and acts as a landing pad for a series
of protein-protein interactions that result in the assembly of a number of
higher order complexes that control initiation. The loading of proteins such
as the Cdc6p and Mini-Chromosome Maintenance (MCM) proteins is
required for formation of a pre-Replication Complex (pre-RC) and later,
other proteins are loaded onto the pre-RC to form a pre-initiation complex.
These latter proteins include the Cdc45p, a protein that interacts with the
MCM proteins. In addition to these and many other proteins required for
the initiation of DNA replication, two protein kinase systems, including the
S phase-cyclin CDKs and the Cdc7/Dbf4 protein kinases are both required
to affect initiation of DNA replication. We have characterized the steps
involved in kinase activation of the initiation of D N A replication, including
the cell cycle regulation of these protein kinases. Initiation of DNA
replication is regulated at every, single origin of DNA replication and the
frequency of initiation is controlled by the levels of the Cdc6 protein. We
have begun to understand the cell cycle dynamics of these events in human
cells, including the identification of the Origin Recognition Complex, Cdc6
and other proteins required for the initiation of DNA replication. These
and other studies will be discussed.

Telomcr.ase Is a specific ribollUcloproteul coulplex cSSeluiaI for Ielolncr~ mannainillg

ill eu,karyollC cell. h eoulauls RNA providing the template for telomere s)nthesls aud
se','eral protein subunits. R N A component o f l n a n y lelolnerases ts '.~rellcharacterized.

Recently telolnerase reverse transcriplase catalytic subunit as well as pS0

Tetroh)olella homologues were ldeutilied ul Ulall3 species. As for yeast telolnerase
only one reverse transcnplase subunat (EST2) ~as shown to be a crucial part
of tile colnple'~
We have developed a two-step procedure for telomerase punfieatlon. The crude
extract of Saccharom) ces cerel,zstae cells was subjected to ullracelUrifugatiou m
20-40 ,.,glycerol gradient. The fractious coutauung active telolnerase were collected
and used for next step of the purification ion-exchange chromatograph 3 . On
ever3 step of purification telolnerase actlx'lt5 ~ as measured by direct teloulerase
assa?, and Northern biol. After this purlficanon step Wehave found two pools of
telomerase aetlvn3, which differ in their proecesivit3.
We have found that some nou-telomeric oligonucleotldes wah GT, TG, or GTG
motives could be effecnvel3 elongated b3 3east telolnerase. We also have observed
the nucleasc actl','it} of lelolnerase complex, Thus, we conskder that the telolnerase
complex can digest the DNA-pnmer and select the proper sequence to start all
elongation reaetlOU.
To determine the protein components of xcasl Ielomerasc we have used phatoaffimt 3
cross-hnking approach. This approach Is based on the abthtx of telolnerasc to
incorporate photoreactlx'e reagent 4-thlo-dTTP in the growutg nucleic acids ehaiu.
Alter UV-lrradlatlon such modified elongated pnlner can farm crossdmks with the

eompoucnts of telomerase complex. We have observed that spectra of cross-liltkcd

proteins Ill proce~sl\ e aud uon-processi\'c telomerase fractions differ stgntficanll 3 .
Thus wc have isolated two ~; cerel,is~ae tclolncraqc comple'~c~ that arc dlffcrellt Ill
their fullCllOUa[ properIics and also S[10~ ed that 3cast telolnerasc ColUams
several protein subunlts,

s 128

Abstracts FEB S'99

7.1 The translational machinery

(Su?7J/D$)

Ribosomal protein L3 apotential target for suramin.

N. Avliyaku'lov'~, J. Luke~"~,A. V. Kajava', B. Liedberg ~,
I. Lundstrom ', S. P. S. Svensson'

(Su/7.1/139)

Dept of Pharmacology/Applied Physics2, University of Linkoping, SE-581

85 Linkoping, Sweden, Institute of Parasitologfl, Ceske Budejovice, Czech
Republic, Centerfor Molecular Modeling~CIT, NIH, Bethesda, USA

A genomic phage library from a primitive kinetoplastid flagellate

Trypanoplasma borreli was screened with the ATPase domain of
topoisomerase II. A 1.4 kb DNA fragment was identified which
contained a 416 a.a. ORF corresponding to the ribosomal protein L3
(rpl3). The cloned rpl3 showed high sequence identity to previously
cloned rpl3 but also low sequence similarity to the ATP-binding domain
of human Hsp70, suggesting that T. borreli rpl3 might bind ATP. The
cloned gene was overexpressed and purified as a His-tag fusion protein
in E. coli. Radioligand binding experiments, using 35S-'~ATP, showed
that the recombinant rpl3 binds nucleotide triphosphate (NTP) with high
affinity, however its affinity for NDP and NMP was 1000 fold lower.
These results indicate that rpl3 has affinity to the gamma-phosphate
group of NTPs. Molecular genetic and biochemical analyses allowed us
to localize the NTP binding domain of rpl3 to the N-terminal fragment
(1-296 aa). The C-terminal region (297-416 aa) failed to bind 35S~/ATP. Interestingly, the anti-trypanosomal drug suramin potently
inhibited the binding of NTP.
Binding of suramin was also investigated by surface plasmon
technology (BIAcore 2000). Rpl3 was immobilized to a carboxylated
dextran matrix on gold chip (CM5) through amine coupling chemistry.
Increasing concentrations of suramin were then injected over the sensor
surface. Our results show that immobilized rpl3 binds suramin with high
and saturable affinity. Suramin is one of the first widely prescribed
antiparasitic drugs and it has been used against trypanosomes for over
70 years. How suramin exert its trypanocidal effect is still far from clear
and a variety of proteins have been suggested as potential targets. Our
results therefore suggest that rpl3 might be a one of the target of this
drug.

(811/7.11140) A general procedure for high-level expression in E. coli and

purification of eukaryofic ribosomal proteins
G. Dieci, L. Bottarelli, A. Ballabeni, S. Ottoneno
Istituto di Sctenze Biochimiche, Universitd di Parma, 43100 Parma, ltal)

Many eukaryotic ribosomal proteins are expressed at very low levels in E.

coli. We find that this is mainly due to their extremely high content of
A G A / A G G arginine codons that are poorly utilized by the bacterial
translational machinery. In fact, we could overcome the above limitation
through the combined use of a T7 RNA polymerase expression system [1]
and a multicopy plasmid carrying the E. coli gene, argU, encoding the
minor tRNA ~ species that reads the above codoas [2]. Under these cotransformation conditions, five highly basic, arginine-rich cytoplasmic
ribosomal proteins from three different eukaryotic organisms (S. cerevisiae
$8, L13 and LI4; A. thaliana L13; H. sapiens L7) have been overexpressed
in E. coli in a histidine-tagged form and purified to homogeneity in
milligram amounts. Recombinant polypeptides accumulated to up to 40%
of total bacterial protein. Taking advantage of this high expression level, we
worked out a two-step procedure for ribosomal protein purification only
requiring metal-affinity and heparin-agarose chromatography. Due to its
versatility, this procedure can be applied to any arginine-rich cytoplasmic
ribosomal protein, thus facilitating structural and functional studies of the
eukaryotic ribosome.
[1] Studier F.W. et al., Methods Enzymol. 185, (1990), 60
[2] Brinkmann, U. et al., Gene 85, (1989), 109

Determinationof the 1~ for the [eEFIA*GDP*tRNA] complex

formation by the method offluorescence polarization
T. Budkevich, B. Ne~-utskii, V. Shalak, Z. Petmshenko, A. El'skaya
l~iltute of Molecular Btologv~mdGenelws..VationalAcadenWqfSctences
of (J,7"ame,tOev, LT,7"ame

The idea of tRNA/aminoacyl-tRNA channeling is attractive to explain

a high efficiency of protein biosynthesis in the mammalian cell.
Recently a formation of an unusual [eEF1A*GDP*tRNA] complex has
been demonstrated by several independent methods [ 1].
In the present work the binding of deacylated tRNA to eEFIA*GDP
has been studied quantitatively by a technique of steady state fluorescence
polarization. Covalent fluorescent derivative of rabbit liver elongation factor
(GDP-form) was prepared by the modification of the protein with fluorescein5-isothiocyanate (F1TC). The fluorescein-labeled eEF 1A (FITC-eEFI A*GDP)
contained about one dye molecule per the protein molecule The activity of
FITC-eEF1A*GDP was not impaired in the assays of GDP/[3H]GDP
exchange and translation ofpoly(U) in a cell flee system
The association constant (K~) for the [eEFIA*GDP*tRNA] complex
formation in Tris-HCl pH 7.5 buffer containing 50 mM NH4CI and 10 mM
MgCI2 has been determined as 5xl0~M~ It was shown that K, of mammalian
deacylated tRNA binding to the E site of homologous ribosomes at 13 mM
Mg2" was 1.7xl06M -~ [2]. In that case the affinity of uncharged tRNA to the
E site of 80S ribosome seems to be about 30 times lower than to
eEFIA*GDP These data support earlier suggestion about a possible role of
eEF1A*GDP in accepting tKNA directly from the ribosomal E site, thus
completing tRNA/aminoacyl-tRNA channeling circle during the elongation

[3]

[1] Petrushenko Z.M. et al, FEBS Lett, 407, (1997) 13

[2] Crraifer D.M. et al., BBA, 1171, (1992) 56
[3] Negrutskii B.S et al, Prog Nuel. Acid Res. Mol Biol, 60, (1998) 47

(Su/7.1/141) The jasmonate-induced 60 kDa protein of barley exhibits Nglycosidase activity in vivo
Marina Dunaeva, Cornelia Goebel, Eckart Goerschen
lnsntut fur Pflanzenbwchemle, Weinberg 3, D-06120 Halle/Saale, Germany

Upon methyl jasmonate treatment barley leaf segments express the

putative ribosome-inactivating protein (JIP60). RIPs are N-glycosidases
that hydrolyse an adenine residue from a conserved loop of the large
rRNA subunit. For the first time we successfully established two
biological systems that allowed us to investigate RIP activity in planta.
The influence of this protein on translation has been analysed by using
barley plants and tobacco plants transformed with a barley cDNA
encoding JIP60. In both plant systems JIP60 exhibited N-glycosidase
activity. The depurination of the 25 rRNA of tobacco and barley
ribosomes led to accumulation of translational inactive polysomes.
Treatment of ribosomes from transgenic tobacco plants with c~-sarcin
resulted in the appearance of a rRNA specific fragment. The alteration of
the conformation of ribosomes in transgenic tobacco due to the
expression of JIP60 was assumed

Abstracts FEBS'99

(Su/7.1/142)

Study on elongation cycle of eukaryotic protein synthesis

A.El'skaya, B.Negrutskii, Z.Petrushenko, S.Ladokhin,
V Shalak, T.Budkevich

s129

(Su/7.1/143)

1MBzG, NAS oJ L'krame, 150 Zabolotnogo St. 252143 h)ev, Ukraine

A possible role of aminoacyl-tRNA synthetases (ARS) and eEF-IA in

tRNA/aminoacyl-tRNA channeling at the elongation cycle of protein
synthesis in higher eukaryotes was postulated [1, 2] The data presented
here are in favor of this suggestion and evidence the interactions between
ARS, eEF-1A and tRNA that have never been considered in the traditional
schemes of the eukaryotic elongation cycle. Rabbit liver eEF-IA stimulates
the activity of several homologous ARS, in particular phenylalanyl-tRNA
synthetase (PheRS), in the reaction of aminoacylation. The stimulation is
already observed at the level of aminoacyladenylate formation and is caused
by both GDP-and GTP-bound forms of eEF-IA A formation of [EFIot*GDP*tRNA] complex has been proved by several independent
procedures membrane filtration, fluorescence spectroscopy, protection from
RNase digestion and tRNA chemical modification The tRNA sites involved
in the interaction with eEF-IA are positioned in the acceptor stem and
TWC-arm. The recognition sites for PheRS and EF-Ia are situated on the
opposite sides of the tRNAeh~ tertiary structure, imposing no steric
restriction on the interaction between [EF-l~*GDP*deacylated tRNA]
complex and PheRS. Such unusual quaternary complex has been detected
indeed by the band shill assay in agarose gel. Since GDP-tbrm of EF-tct is
suggested to be involved in tRNA release from the ribosomal E site
[2],
the tRNA/aa-tRNA
channeling cycle seems to include the
following steps: ribosomal
E site-*EF-lct*GDP-~aminoacyl-tRNA
synthetase---~EF-lct*GTP---~ribosomalA site.
[1] M P Deutscher, B.S Negrutskii, ProcNatl.Acad Sci.USA, 88 (1991)
499 I
[2] BS Negrutskii, AVEl'skaya, Progress in Nucleic Acid Research and
Molecular Biology, eds K Moldave et al., Academic Press, San Diego/New
York/" Boston/London/Sydney/Tokyo/Toronto, 60 (1998) 47

Selenophosphate as a substrate for mammalian

selenocysteine synthase, its stability and toxicity
Kazuo Kanaya, Chiharu Goto and Takaharu Mizutani
Nagoya City Universtty, Nagoya, 467-8603 Japan

Selenium is an essential trace element for humans, its deficiency causing

Keshan disease, a condition endemic in China. The syndrome was relieved by
administration of selenious acid. Most selenium in the body was found as
selenocysteine (SeCys) in the active sites of glutathione peroxidase, 5'D! and
thioredoxin reductase. The SeCys moiety is incorporated into selenoproteins
by a co-translational mechanism at the positional in-frame UGA codon. The
mechanism of selenocysteine synthesis on tRNAs= in mammals was
previously studied by means of HSe- as a Se donor to synthesize
selenocysteine. It has been recently established that HSe'in E. coli is activated
by ATP to become selenophosphate (SeP). In this study, we provide evidence
that [:SSe]selenocysteine is produced by bovine selenocysteine synthase from
Ser-tRNAs= and

[75Se]SeP, synthesized

from

elemental 7SSe and

Tris(tfiir~thylsilyl)phosphite. We also studied the stability of SeP by the

measurement of NMR. SeP was stable during storage under nitrogen at -80C
for 3 months in 0.2M Hepes buffer at pH 6.8. However, SeP decomposed at
0C in air (half life 32 hrs) or at 22C under nitrogen (half life 30 hrs) at pH
6.8. The half lives of SeP at - 19C in air and at 0*C under nitrogen at pH 6.8
were 740 and 840 hrs, respectively. At pH 4 under nitrogen at 22*C, the half
life was 240 hrs. The half life was only 9.2 hrs at pH 9 under nitrogen at 0*C.
Thus, SeP was proved to be stable at low temperature, under acidic and
anaerobic conditions, but labile under neutral and alkaline conditions.The LDs0
administered i.p. to mice as 37.5 mg/kg body weight.
BioFactors, IN PRESS

(Su/7.1/144)

The role of GTP

in the initiation step of Translation
S. LaalamP, M. Grunberg-Manago b and Y. Cenatiempo~
"IBM1G, 40 av.recteur Pineau, 86022 Poitiers Cedex, France
nlBPC, 13 rue P. et M. Curie, 75005 Paris, France

The first step of prokaryotic mRNA translation requires, in addition to

initiator tRNA and the three proteins known as initiation factor (IF1, IF2
and IF3), a GTP molecule. Of the three proteins, only IF2 is a GTP-binding
protein. In order to define the IF2 GTP binding domain, we have mutated
very conserved residues suspected to be involved in GTP binding or
hydrolysis. The effect of these mutant proteins was studied in vivo using a
strain carrying a null mutation in the chromosomal copy of infB. The
different mutations globally confirmed a theoretical 3-dimensional model of
the IF2 G-domain and demonstrated the crucial importance of GTP
hydrolysis in translation initiation. Eight out of eleven mutations in our
study were lethal.
Of the eleven lethal mutants, two exhibited a strong dominant inhibitory
effect towards the wild-type protein.These are V400->G and H448->E
mutations. The two mutants were studied in vitro. The behaviour of these
mutants (Vat400 -> Gly and His448 -> Glu) towards GTP allowed to better
understand the role of GTP and its hydrolysis during translation initiation.

(Su/7.1/145)

Structure of the Decoding Center of the Ribosome

I N Laxnk'. P V Scrglcx' S S Dokudoxskaxa ~ 0 ,X. Dontsoxa
.
A A Bogdanox ~. J Rmke-Appe[:. R Bnmacombc:

Depcl/'Imelll (~f ('hetlll~D'v ~lo~colr ,~'lale [ntl'er~ll~ t fO~COII

L\[a.v-Planck Institute 6o'.\[oteculare Genetlk, Berlin. German~

Decoding center of the ribosome provides the mRNA translation and

the fidelity of the codon-anticodon interactions in the course of protein
biosynthesis The detailed spatial structure of the ribosome decoding
center is still unknown To study the decoding center of E.coh ribosome
of E.coh the site-directed cross-linking technique has been applied A
number of mRNA ligands as well as tRNA analogues carrying photochemical probes at various positions have been constructed As photochemical probes different photo-affinity analogues ofnucleotides were
used namely 4-thiouridine, 6-thioguanine, diazirine derivative of 5methylenaminouridine. Using these analogues the set of the cross-links to
the ribosomal rRNA and ribosomal proteins has been obtained. The
cross-links to rlLNA have been localized by two-step technique based on
the RNase H digestion in combination with the reverse transcriptase
analysis On the basis of cross-linking results the major elements of
rRNA and ribosomal proteins participating at the formation of the decoding center were found The rRNA helixes 34, 44, 28, 18 and the ribosomal proteins $2, $3, $5. $7, SI 1, S18, $21 appeared to be these components The model of their mutual arrangement in the ribosome will be
discussed

s 130

(Su/7.1/146)

Abstracts FEBS'99

Detection of interactions between translation

elongation factors and other proteins.
F. Mansilla, C. R. Knudsen, B. F. C. Clark
IMSB, Aarhus University Gustav Wieds vej l0 C 8000 Arhus C, Denmark

Bacterial elongation factor 1A (EFIA) and its eukaryotic partner eEF1A

(previously known as EF-Tu and EF lc0 play an important role in protein
biosynthesis, where they transport aminoacylated tRNA (aa-tRNA) to the
A-site of the mRNA-programmed ribosome in a ternary complex,
EF-Tu:GTP:aa-tRNA. The newly delivered aa-tRNA remains on the
ribosome, where the amino acid of the aa-tRNA is incorporated in the
growing polypeptide chain. The translation factor is released in complex
with GDP and reactivation of EF1A or eEF1A, in order to carry out a new
round of elongation, involves other elongation factors, EF1B (previously
EF-Ts) or eEFIB (eEF-I[~8) which catalyses the exchange of GDP for
GTP. Hence EF1A and eEF1A appear to be multifunctional proteins
involved in many interactions of different types during the elongation cycle.
Besides its role in the elongation phase of translation, eEF 1A has also been
proposed to be involved in a variety of other functions in the cell. These
include proteolysis of N-acetylated proteins, binding and bundling of
cytoskeletal components (actin and microtubuli), mitosis, linkage to
transformation of cells, signal transduction and ageing. We are using the two
hybrid system as a novel technique to investigate any known or novel
protein-protein interactions linked to these functions in vivo.
We have used human eEF1A1 to screen a human brain library and the
proteins interacting with the elongation factors emerging from these studies
have been sequenced and different biochemical studies are being performed
to reveal the specifity and structure-function implications of theses
interactions.
In the case of EFIA we use the two hybrid system to extend the knowledge
of the interaction with EF1B.

(Su/7.1/148)

The last sense codon of a model protein is a significant

site of peptidyl-tRNA drop-off
J Menez~. V. Heurgue-Hamard b, M Ehrenbergh, R.H Buckingham"
aUPR9073 du CNRS, Paris, France ~'Umv ofUppsala, Uppsala Sueden

During protein synthesis, pept~dyl-tRNA dissociates from the ribosome

before normal peptide release at an average frequency of 3x10 "4 per codon in
Escherichla c o b [1]. Pept]dyl tRNA hydrolase, an enzyme essential for cell
viability, hydrolyses the ester linkage in these "drop-off' products, allowing
the tRNA molecules to be reused High level expression of a non-functional
protein (AEF-Tu), derived from elongation factor EF-Tu by the deletion of an
essentml domaine [2], greatly inhibits the growth of Eschertchia coh partly
deficient in peptidyl-tRNA hydrolase. High level expression in wild type
cells, or in cells where the mutation affecting peptidyl-tRNA hydrolase is
complemented by a plasmid carrymg the wild type gene, has little effect on
growth The inhibitory effect ~s presumably due to sequestration of essential
tRNA species in the form of pe~tidyl-tRNA, and can be partially suppressed
by the overproduction of tRNA ~-', a tRNA isoacceptor known to accumulate
rapidly as peptidyl-tRNA Ly~ m peptidyl-tRNA hydrolase-deficient cells [3]
The growth inhibitory effect can be considerably modulated by changing the
last sense codon in the gene for AEF-Tu (or a variant lacking 63 amino acids
near the C-terminus). In particular, Lys or His codons in this position
increase growth inhibition The effects of eleven changes studied correlate
perfectly with the rates of accumulation previously observed of the
corresponding tRNA species as peptidyl-tRNA. These results suggest that
peptidyl-tRNA dissociation from a ribosome paused at the stop signal can
account for a sigmficant part of such dissociation events in the cell At certain
stop s~gnals, drop-off may be a significant form of translation terminanon
producing polypetides of normal length.
[1] Menninger, J R. .L Biol. ("hem. 253, (1978)6808
[2] Dong. H et al. J. BactertoL 177, (1995) 1497
[3] Heurgue-Hamard, V e t al /A4BOJ. 15, (1996) 2826

(Su/7.1/147)

Yeast mitochondrial methionyl-tRNA

transformylase
Y. Mechulam, P. Gomez,-L. Vial, E. Schrnitt,
M. Panvert, S. Blanquet
Lab. de Biochimie, Ecole Polytechnique,91128 Palaiseaucedex, France

One possible repercussion of the study of methionyl-tRNAf Me~

transformylase is the design of specific inhibitors for antibiotherapy.
Nevertheless, inhibitors of a bacterial methionyl-tRNAfMet transformylase
can be toxic for human cells also. Indeed, initiation of translation in
mitochondria shares similarity with that occuring in procaryotes. In
particular, it includes the step of formylation of Met-tRNA~ Met. However,
since mitochondria do not possess a protein deformylase, many questions
remain open concerning the role of protein formylation in these organelles.
The Yeast system was used as model of mitochondrial translation and
the gene coding for the yeast mitochondrial methionyl-tRNAf Met
transformylase (MTF) was isolated. Inactivation of this gene in the Yeast
chromosome was achieved without effect on the respiratory growth of an
haploid strain. Overexpression of the mitochondrial MTF in an fret - E. coil
context allowed to complement for the deficiency in bacterial formylase
activity. Finally, yeast MTF purified from the resulting overproducing E. coil
strain was used to carry out substrate specificity studies.

(Su/7.1/149) Solution structure of B. st. IF-2 tRNA binding domain

S. Meunier ~, R. Spuriob, M. Czisch a, M. Guenneugues~,
C.O. Guaierzi b, R. Boelens a
a Bijvoet Center for Biomolecular Research, Utrecht, The Netherlands
b Dipartimento di Biologica MCA, Camermo, Italy

The C terminal domain of IF2 is involved in the recognition of

the formylMethionine-tRNA fMet. Further investigations indicated
that the C terminal domain consists of two sub-domains and that
only the last 85 residues are necessary for the specific binding of
N-blocked amino-acylated tRNA. 1F2-C2 (E632-A741) was
expressed and overproduced either N labelled or 10% t3C/100
% ISN labelled The three-dimensional structure of IF2-C2 has
been determined by heteronuclear NMR spectroscopy and
consists of six strands, forming an open [?,-barrel. This structure
was found to be homologous and is compared to the domains 2
(tRNA binding domains) of the elongation factor G [1,21 and of
the elongation factor Tu [3]. The high degree of homology
between these structures indicates an identical pattern of
interactions for IF2 upon the binding of the initiator tRNA on
the C2 domain However, the necessary large specificity of EFTa for all the peptidyl-tRNA implies that the residues involve in
the interaction may change. Thus a strict pattern could not be
determined for IF2-C2, nor the specificity for the N-blocked
amino-acylated tRNA. However the analysis of the three
dimensional structures indicated that the localisation of M272
(at the surface of EF-Tu,) and R700 (at the surface of IF2-C2)
could explain the affinity for free amide group amino acylated
tRNAs only.

15

[1 ] Czworkowski, j., et aL EMBO J. 13 (1994) 3661.

[2] ,'Evarsson, A., et al. EMBO J. 13 (I994) 3669.
[3] Berchtold, H., et al. Nature 365 (1993) 126.

Abstracts FEB S' 99

(Su/7.1/150)

s 131

Characterization of the guanine-nucleotid exchange

complex EF-1 in sea urchin Sphaerechinus granularis.
A. Monnier, C. Delalande, P.Cormier, .l. Morales, R.
Bell6 and O. Mulner-Lorillon
Station Biologique de Roscoff CNRS-UPR 9042, BP74,29680 Roscoff

In sea urchin, fertilization (Gl/S transition of cell cycle) induces an

important augmentation of protein synthesis (10 to 30 fold) partly due to an
increase of elongation rate. The Elongation Factor of protein synthesis EF-1 is
composed of two elements: a G protein, the c~ subunit and a macromolecular
complex EF-1 [3y~5. The G protein, is responsible of the fixation of the
aminoacyl-tRNA to the ribosome. The high molecular weight complex, EF1[$T~is the exchanger of GTP/GDP. We have analysed sea urchin EF- 1[~'/fi,
A cDNA coding for the ~ subunit was isolated and a polyclonal
antibody raised against the recombinant protein. EF-lg mRNA translated in a
reticulocytes lysate produces a 35kDa protein wich was recognized by EF-18
antibody. Furthermore the 35kDa protein was immunoprecipitated by this
antibody.
Three proteins of 47, 35 and 33kDa were immunoprecipated by
antibody from a sea urchin cytosol. The 35 and 33 kDa proteins were
recognized by the 5 antibody upon immunoblotting. Therefore the 35kDa
protein corresponds to EF-15, the 33 kDa protein is an EF-15 isoform.
Furthermore 8 subunit integrates into a complex containing the 47kDa protein.
EF-lg antibody was used as a screen for the purification of EF-16
containing complex. Cytosol was prepared from sea urchin ovocytes,
homogenized in a buffer containing antiphosphatases and clarified at high
speed (100000g during 30 rain). Proteins were precipitated with 50%
ammonium sulfate and separated on an anion exchange column (DEAE). The
fractions containing EF-15 were resolved on an high affinity anion exchange
column (Poros). After this purification step, EF-15 and the 33kDa isoform
were associated with five major proteins (110, 63, 47, 30 and 25kDa). These
proteins are under microsequencing to determine their identity and their
relationships with the exchange complex.
A purified fraction of EF-I complex will allow the characterization of
the different subunits and their regulation during early development of sea
urchin.

(Su/7.1/152)

Mammalian mitochondrial methionyl-tRNA

transformvlase
N. Takeuchi a,b, E. Schmittb, ]Vl. panvert b, T. Uedaa,
K. Watanabe a, Y. Mechulam b, S. Blanquet b.
b a aUniversityof Tokyo.Hongo,Bunkyo-ku,Tokyo 113,Japan
L b. de Biochimie,EcolePolytechmque,91128 Palalseaucedex,France

Translation in animal mitochondria is quite unique in that it appears to

use a single tRNA Met species for both initiation and elongation.
Mitochondrial initiation involves an IF2rnt-dependentbinding of formyl-MettRNAMet to the ribosome. The unformylated form of the same Met-tRNA is
used through EF-Tumt in the elongation pathway. Thus, it is likely that
mitochondrial methionyl-tRNA Met transformylase (MTFmt) plays a key
regulatory role in maintaining a correct balance between the two fates of this
single tRNA Met molecule species towards either initiation or elongation.
As a first step towards the study of the biological role of formylation
in animal mitochondria, the enzymatic properties of bovine mitocbondrial
formylase were investigated. MTFmt was purified 2200-fold from bovine
liver mitochondria. The purified protein was used to clone the corresponding
cDNA. MTFmt expressed in E. coli as a histidine-tagged protein was used
together with various mutants of E. coli tRNAsMet in order to investigate
substrate specificity and to make comparison with the MTF enzyme from E.
coli. Wathever their origin, the two MTFs perceive the same regions of
tRNA as identity elements (the acceptor stem, base pair 11-24 and the
esterified methionine group). However, the relative contributions of each
element to the enzymatic reaction efficiency widely distinguish the bacterial
and mitochondrial systems. These features will be discussed in relation with
the unique properties of the translational machinery in animal mitochondria.

(Su/7,1/153)

Does Translation Initiation factor 2 play

a role in the Myxococeus xanthus developmental process?
E. Tiennault, Y. Cermtiempo, S. Laalami
IBMIG, 40, av. du Recteur Pineau, 86022 Poitiers, France

Myxobacteria are a class of Gram-negative organisms which show a

characteristic social behaviour with formation of multicellular aggregates in
case of environmental changes. They express proteins essential for this
developmental process. In Myxococcus xanthus, the C-terminus of initiation
Factor, IF3, was shown to be necessary for a normal myxobacterial life
cycle[l]. In Stigmatella aurantiaca, the N-terminal region of IF2 presents a
remarkable composition in amino acids similar to the I1:3 C-terminal portion
which has been implicated in the developmental process [2].We have cloned
M. xanthus in/B gene, together with the 5' region of ORF3 and the 3' regina of
nusA gene, located respectively dowstream and upstream of infB. M.xanthus
injB gane encodes a 115 kda protein. This protein is able to complemerlt an
E. coli strain carrying a null mutation on the chromosomal copy of inJ~, To
study the putative role of the N-terminal region of M. xanthus IF2 il~ the
developmental process, different mutations will be generated on the
chromosomal copy of the gene (deletions and point mutations) in order to
define the portion of the protein and the residues necessary for the possible
function(s) of IF2 in myxobacterial development.
[1] Kalman et a/., J. Bacteriol., 176, (1994), 1434.
[2]Bremaud eta/., J. bacterinl.,179, (1997), 2348.

s 132

Abstracts FEBS'99

10.2 Structure and biosynthesis of glyco-conjugates

(Su/10.2/154)

Structural Investigation on the sulfated

polysaceharide fucoidan
IL D'~x~P,L ChevoloP-b,J. Abi~, N. ~ ,
J. Jozefonvicza
aURM2, UMR 7540 CNRS, 93430 Villetaneuse, France. blFREMER,
44311 Nantes cedex 3, France cCS1C, 08034 Barcelone, Espagne.
dUMR 7613 CNRS, 75252 Paris, France

Fucoidan is a sulfated fucose -based polysaccharide derived from

brown algae, which is endowed with very important biological
activities for human therapeutics like antithrombotic activity,
inhibition of complement and modulation of cell adhesion. However,
molecular mechanisms need to be elucidated, and structure-activity
relationships remain to be established. To date few structural data are
available due to the heterogeneity and the polydispersity of this high
molecular weight sulfated polysaccharide (500 000 g/mole).
To get new insight into the structure of fucoidan, we have
undertaken its chemical and enzymatic depolymerization into
oligosaccharides easier to characterise. Chemical depolymerization was
performed by a radical process. For the purpose of enzymatic
degradation we found new fucoidan depolymerizing enzyme activities
in the digestive glands of a marine mollusc.
We have performed structural analysis of the resulting sulfated
oligosacharides by electrospray ionization mass spectrometry.
Although strongly anionic with a large number of sulfate groups, these
oligosaccharides were successfully detected in negative mode with a
high sensitivity. Oligosaccharides ranging from monosulfated difucose
(M= 349) to pentasulfated pentafucose (M= 1145) were analyzed by
fragmentation upon MS/MS experiments. The sulfation ratio of these
oligosaccharides varied from 0.5 to 1,5 sulfate/sugar unit. Two
dimensional ~H-NMR experiments were also carried out and the
obtained data indicated the presence of 2-sulfated-c~-L-fucose residues
1-)3 and 1-)4 linked.

(Su/10.2/156)

Investigation of UDP-glucuronosyltransferases involved in the

biosynthesis of glycosaminoglycans using oligosaccharidic
derivatives as primers
J Gouze, S Gulberti, J Grillasca, J Magdalou,S Foumel-Gigleu~

(Su/10.2/155)

........ ~,,.

l~li~n)tlh~i~.g~l immunolocalisation of Lewis a-containing

t: /~ ~ ' N-glycans in the plant cell
A-C. Fitchette a, M. Cabanes-Macheteau a, B. Martin b, V.
Goruorda, C. Hawes b, P. Lerouge a, L. Faye a

........
-

I__~,.F_Z E~.SA=C_NRStO37~IFRMP 23, Umversit~ de)~ouen, Facultd ds

Sz"tenvesT,7"6821 Mt St Aignan cedex, France
" -"
t'l.b ;, 3[ Type W171i~a;Ol[~bltlof'Bi~f~'a~'ltC~dtl/Idldcl~t~Sciences,
Oxford Brookes
Umversay. Gipsy Lane, Headington, Oxford OX3 0BP, UK

Lewis a (Le a, Gal 13(1-3)[Fuca(1-4)]GlcNAc) is a glycan epitope which is

responsible in humans for histo-blood groups and is involved in cell-cell
recognition processes. Le a epitope has recently been identified in
oligosaccharides N-linked to secretory plant glycoproteins [1, 2]. Using
antibodies specific for the Le a epitope, named anti-plant Lewis antibodies, we
found that Lea-containing plant N-glycans are immunodetected on
glycoproteins of most higher and lower plant tested, except members of the
Crucifera family, such as Arabidopsis thaliana. By light and electron
microscopy, Le a epitope was found to be synthesized at the trans- most part of
the Golgi apparatus and are N-linked to the extracellular glycoproteins, but not
to vacuolar ones. Since we demonstrated by double immunolabelling that
vacuolar and extracellular glycoproteins cross the same Golgi compartment,
where the Le" antennae are built, before being tranported to their respective
target compartments, the different N-glycosylation patterns of these two groups
of glycoproteins do not result from two different Golgi pathways, but more
probably from rapid degradations of N-glycans in the vacuole leading to
truncated structures.
[1]: Fitcbette-Laind et al., Plant J., 12, (1997) 1411.
[2]: Melo et al., FEBS Lett., 415, (1997) 186.

(Su/10.2/157)

Proteoglycans affect testosterone production in

immature and mature rat Leydlg cells
N. Grudet, P.J. Bonnamy, D. Le Goff, S. Carreau

[RBA, Universitd de Caen, Esplanade d e la Paix, 14032 Caen.

UMR CNRS 7561- UHP Nan(v L 54500 Vandoeuvre les Nancy

In order to investigate the glucuronidation of carbohydrate containing

drugs, the in vitro formation of glucuronides on the thioxyloside ring of the
antithrombotic drug, LF 4.0212 was followed in rat and human liver
microsomes and with recombinant UDP-glucuronosyltransferases (UGTs).
Human liver microsomes glucuronidated the coumpound mainly on the 2hydroxyl position of the thioxyloside ring, whereas rat were able to form
glucuronide on either the 2-, 3- or 4- hydroxyl group of the molecule, although
to a lower extent The biosynthesis of the 2-O-glucuronide isomer was
catalysed by the human UGTIA9 and 2B4, but not by UGT1A6 and 2Bl 1.
By contrast, the rat liver recombinant UGTIA6 and 2B1 failed to form the 2O-glucuronide isomers. Interestingly, glucuronidation on the 4-position was
found in all the metabolic competent V79 cell lines considered, inc/uding the
nontransfected cells, suggesting the presence of endogenous UGTs in
fibroblasts able to actively glucuronidate the drug. This activity, which was
nonsensitive to the inhibitory effect of 7,7,7-triphenylheptanoic acid, a
potent UGT inhibitor, could reflect the existence of different enzyme
possibly involved in the biosynthesis ofglycosaminoglycans In this way, we
further investigated the use of LF 4 0212 derivatives, whose structure mimic
the trisaccharidic moiety (xylose, galactose, galactose) as model substrates for
the UDP-glucuronosyltransferase(s) involved in the biosynthesis of
glycosaminoglycans. Glucuronidation activities showed optima/ pH around
pH 5 0 and were stimulated by Mn 2- but not Mg 2~ Interestingly,
glucuronidation in these conditions was nonsensitive to the inhibitory action
of 7, 7, 7-triphenylheptanoic acid. Altogether, the data suggest that LF
4 0212 and structurally related substrates can be used as potential primers of
glycosaminoglycan
synthesis
The
regulation
of
the
glucuronosyltransferase(s) involved is currently
under investigation in
cellular models for joint cartilage pathologies
This work was supported, in part, by Laboratoire Fournier, Daix (France)

We have demonstrated that immature and mature rat Leydig cells, cultured
during 24 h, synthetize cell layer associated proteoglycans (PG) and in
particular heparan sulphate PG (HSPG) [1]. In immature animals, the PG
are more abundant than in mature rats (+20%) and among them, HSPG are
3 fold more concentrated in immature Leydig cells [1].
In order to characterize the involvement of proteoglycans in the regulation
of Leydig cell function, we have examined the effects of para-mtrophenyl~D-xyloside (PNPX), a specific inhibitor of PG synthesis and of paranitrophenyl-!8-D-galactoside (PNPG), an inefficient structural analogue, on
testosterone production by purified Leydig cells from immature and mature
rats, in presence or not of various concentrations of hCG during 24 h.
Whatever the age, addition of PNPX induces a decrease of [35S]- and [3HIlabelled PG associated with the cell layer. The latter are in lower amount (50 and -25%, in immature and mature, respectively) and are also less
sulphated (-40%). In immature Leydig cells, the PG inhibition decreases
basal and weakly stimulated-hCG or -oLH or -(Bu)2cAMP testosterone
syntheses. In mature Leydig cells, the PG inhibition has no effect on basal
(and weakly stimulated) testosterone production but increases it in presence
of subsaturating amounts of either hCG (or oLH) or (Bu)2cAMP.
Whatever the age, the PG inhibition has no effect in presence of saturating
amounts of hCG or (Bu)2cAMP. These effects are maintained in presence
of either an inhibitor of phosphodiestemse or an activator of protein kinase
C, but are not observed in presence of 22R-hydroxycholesterol. These
findings suggest that in rat Leydig cells, the PG inhibition affects the LH
stimulated testosterone output at a step distal to cyclic AMP and more
precisely, the cholesterol supply by acting on cellular cholesterol
distribution (free and esterified cholesterol) [2].
[1] Grudet N. et al., C.R,Acad. So. Paris, 319, (1996), 1101.
[2] Grudet N. et al, J. Steroid Biochem. Mol. Biol., 68, (1999), 3.

Abstracts FEBS'99

(Su/10.2/158)

lmmunochemieal study of water-soluble polysaccharide

isolated from Bifidobacterium adolescentis 94-BIM.
J. Kiibler-Kielbb,G.Novik~,B. Adamikb, N. Astapovich',A. Gamianb
Institute of.tIicrobioL :Vat.Academy of Science, Minsk, Belarus
~lnstitute of lmmunoL Exp. Therapy, Wroclml., Poland

Bifidobacteria are known to be important for human health and their

probiotic effects have been demonstrated. Polysaccharides isolated from
bifidobaeteria have been shown to present an antitumor and
immunopotentiating activities [1]. Cells ofBifidobacterium adolescentis 94BIM when analysed with electron microscope revealed to have extracellular
fibrillar structures bridging the cells [2]. The polysaccharide fractions have
been isolated from cells of B. adolescentis 94-BIM strain by sonication,
ethanol precipitation and DEAE-Sephadex A-25 chromatography. A major
polysaccharide fraction contained glucose and results of methylation analysis
indicated the presence of a terminal glucose, 4-substituted and 4,6disubstituted glucose residues in molar ratio 0.9:4.0:1.2, respectively. A
MALDI mass spectrum of this polysaccharide revealed lack of repeating unit
structure, and indicated that during biosynthesis single hexose residues are
added to the growing chain. Immune rabbit serum against whole bacterial
cells reacted with all polysaccharide fractions. The quantitative
microprecipitation and double immunodiffussion assays indicated the
presence of two populations of antibodies, from which one population could
be absorbed with yeast mannan, whereas the second was directed to the cell
surface derived glucan. Cross-reaction, however, could be observed between
anti-glucan antibody and supernatant derived mannan. Results indicate that
immunogenic polysaccharide of glucan type is present on the surface of
Bifidobacterium adolescentis 94-BIM cells.
[1]. Sekine K. et al., Cancer Res., 45 (1985) 1300
[2]. Novik G.I. et al., Microbiologiya, 63 (1994) 515

(Su/10.21160) Sialoglycoconjugates of human milk: changes along lactation

S.Martin-Sosa, M.J.M.Mart[n , P.Hueso.
Depto de Bioqu/mica ) Btologla Molecular, Universidad de
Salamanca, 37007 Salamanca, Spain

Human milk contains different sialylated oligosaccharides and

glycoconjugates (glycoproteins and gangliosides). However, little is
known about the physiological and biological relevance of these
compounds in milk. The ability of some of them to be growth factors for
bifidus flora and to inhibit bacterial adhesion to epitelial surfases suggest
a protective effect against infection. Their abundance during the firs
phases of lactation shows the great importance of these compounds in the
beginning of postnatal life.
Sialoglycoconjugates from human milk of different stages of lactation
(colostrum, transitional and mature milk) were determined. Milk was
deffated, total proteins or casein were precipitated by TCA or acetic acid
pH 4.6 respectively and the sialic acid content of the oligosaccharide,
glycoprotein, casein and ganglioside fractions determined.
Human milk has a high content of all sialoglycoconjugate fractions in the
early lactation (colostrum) then decreasing during the period studied. The
oligosaccharide-bound sialic acid content in bovine milk is lower than
that of human milk in all stages of lactation. We found more sialic acids
bound to oligosaccharides (77-87 %) than in bovine milk (20-40%). By
contrast, glycoprotein-bound sialic acids is higher in bovine (60-80%)
than in human milk (12-15%). Moreover, the sialic acid amount
complexed with casein is also lower than in bovine milk.

s133

(Su/10.2/159) Milk gangliosides: Laetationai changes in fatty acids

M.J.M.Martfn a, J-P,Zanetta b, S.Martfn-Sosaa, P.Huesoa
a Depto de Bioqufmica y Blolog/a Molecular, Universldad de
Salamanca, 37007 Salamanca, Spain
b U M R I I I du CNRS, USTL 1, 59655 Villeneuve d'Ascq C~dex, France

Gangliosides, sialic acid-containing glycoconjugates, are

important constituents of milk fat globules membranes. They are
supposed to play a role in the defense of newborn against infection. It is
also well known that milk fat composition is strongly influenced by
physiological, seasonal and nutritional factors. In this sense, the
ganglioside content of milk changes markedly during lactation; it is high
at the beginning and decrease thereafter. Medium-chain fatty acids
(MCFA: C12-C15) would be synthesized in the mammary gland from
plasmatic acetate or I~-hydroxybutyrate, and long-chain fatty acids
(LCFA: C17-C26) would be taken directly from circulating lipoproteins.
C16 fatty acids could have both origins. Here we report the changes in
the ganglioside ceramide moiety during lactation.
Gangliosides from bovine milk of different stages of lactation
(colostmm, transitional, mature, and late lactation milk) have been
obtained by acetone and chloroform/methanol extractions, Folch's
partition and dialysis. Individual gangliosides were isolated by
preparative thin-layer chromatography.
Five to seven individual gangliosides were isolated from the
different stages of lactation. Gangliosides from colostrum have a high
content of saturated fatty acids (97%). This content decreased in
transitional (68%) and mature milk (78%) and tended to increase toward
the end of lactation (83%).
MCFA and LCFA maintain their respective percentages about 13
and 49% in the three first stages, but their amount decreases slightly at
the end of lactation. On the other hand, C16 increases from 35-40% until
48% in the late lactation.
As gangliosides contribute to the structure of milk fat globule
membrane, we could expect that biosynthetic enzymes (e.g., ceramide
synthase) use preferently LCFA in order to maintain the stability of the
globules. Because of that, the increase in MCFA content reported for
milk triacylglycerols is less pronounced in milk gangliosides.

(Su/lO.2/161) t-~rohl'eratlon of D('ro~.i",nnl~.~', 133 oleic acid !n(int'lilnl

afft, ct'~ dnlichul ~}nthc'd,, m ,~.cerel'isiae
\. ~,zkol>msl-.a. ",t. Skon~-~zaa\. It.. :2,~\1~:.c~st,a
]'l~]lll/l," ,~r ~]], I~r,/?I,','~ ,!t,' [1 : ,'i~'lr , J~'," ()2- lilt, II ,1 ;,],

Yeast are single cell organisms but complexity of their structure and
t\mctions does not differ from higher eukariotes The ease of nutritional
and genetic manipulations makes S. ccrevl.~Ycw a very suitable system to
investigate gene and gene products required for lipid metabohsm In the
present paper we showed that disruption in t'ASl(peroxisome assembly)
gene lowers by 20% the total synthesis of dolichol Moreover. we proved
that oleic acid induced proliferation of yeast peroxisomes results in the
appearance #1 vivo and m vltlw of the additional family of longer (C,)~ - C
in0) than typical yeast dolichols Furthermore, we show that tile total
HMG-CoA reductase and FPP synthase activities are significantly
diminished m yeast cells deprived of functional peroxisomes We also
show that treatment of yeast cells with oleic acid increases the cellular
level of PC which is the main constituent of peroxisomal phospholipids
and may inf/uence the synthesis of longer dotichols On the basis of the
above results we suggest that yeast peroxisomes besides endoplasmic
reticulum participate in the synthesis ofdolichols

66, S ~12.q s l o g a s q v

89-6~ '(866I ) '08 'a!m!tl~O!fl 'laaqaaA 9xPnV ~' anAIo.UelAI-atmv

'n~Iqeq allapo '~pg.tUlX lam~(i 'laAll(~ Omx.pues 'tt~3eD 9 ~ I [~]
"LL~-g9g '(L661) '6 '~I!qoAlO
ptte saoua!osoo~lO m. sptmJ. "~aq~aA 9 J p w ~? ueoe3 9Ua~l [~]
'I8 'HaD 'sn.malOH .t-W ~ ~ottrIlOO:l o11#90 'laoqaH "N 'lame(/ [I]
: saolloJoja~l
ose!qol.ttlo e pu~ oseONd
jo ao!loe [e!luanbos e gq ~o asep.m~..m~soonlfi'opua tit jo uo!loe
aq~ ,~q .Iaql9 aptnxt uaaq seq uo!lqAsooAIg'ap aql ~etI1~'m.le.,,suomap p~a
flmonpaa ~.aql le anp.~saa ~VlqOlD aao ssassod sop!sotmemo~.qo asoq,L
[E '~7] 'IosolKo oq1 m. pasealoa oae sap!sommmo~'qo oql leql ptm p o l q
uo,, poAeap aq ueo sm.alo~do9,(l'$ paz~.s,oql,As ~iA~aujo so!lo!om ~e3,~l~
aql 'poapm 'l~ql polealst~omop am 'm.o,~mos.me ql.t~ s!soqluXs m.a~oad
at0 ~.m,lOOlq ~q ptn~ SOml.ilaO OHD jo m~lnm uop,eILSOO,q~ ~msn ~fl
'polso~ns uaaq
s~q so!~o!ota m~,qfl aq~ ~ta.S~OlOaas~Dgd ~.qoso~o ~jo uo!lo~ oql pu~
p a ~ s q o ~ a q s~q o]~!pam~olm, pol~I~so~,q~op ~ 'ssaoo~d rto!l~p~op
s.n0 jo ol.ts!nba~ald ~ s~ ']~ql pa~so~flns maq s~q ~I "uo!i~oss~
~.nmqns po~.~d~. ,~q Jo aouanbos p!o~ o.mm~ oql m. loojap e ,(q pomy~qo
S~A~ ms.n~qoom lOattoo ,~1.q~nb oql ~q po~npm, uo!lspe~op oql '~oti
I~.l dn po.tpnls uoaq OAglt qo.rqA~slapom oql ul 'uotl~pea~op m.oloJd oql
ol speoI soseo amos m. pile tlomnI ~I~ tl~no~ at0 aA~O| ol sm.oloJdoo~l~
paplojs.lm sluoAaad ms.m~qoom s.rqL "[1] mnlno.~loa 3!mseldopua
tl~nOa at0 m. ms.mt~ltoamioaluoo &l.q~nb ~ ol pal .gmqns aJe sm.olo~doo,q~
pu~ sin.aloud paza.saqltKs 1~aou l~t0 tt~otls OAeq so!pros lua0o~I

~uead "xoPgDtr~sV,POAnamlITAgg96g 'sl~OlOUtt:~I1,~s~OmOS

sap 9~tS.I~'A~.II-I_'11 ou "8/NTI'S~,N3 "onbfSolo!gaau~D ap aJ!o~Joqeq
ueoeD "d pue J~AI"IAIy 'leq~ael~ I 'lea~3 ,I '~aAnO 'S 'lJoqaaA V
sm.aloaflo~g# p~..saq~u,Cs gl~aU jo lO.~Uo~ ~l![nnb
aql Jo sJa~l.IEm se sopt.souucmo~!lO pa)lU!l-Nl pug aa.l~l
(~9[f~'oI/ns)

~EI s

Abstracts FEBS' 99

s ] 35

11.1 Lipids in membrane function

(Su/ll,l/163)

Cholesterol regulates annexin 11 membrane binding

and aggregation properties
J. Ayala, P. Gouache, ]P. Henry, LA. Pradel
CNRS UPR 1929-Biologie de le Sdcrdtion /IBPC
13 rue P. et M. Curie. 75005 Paris, France

Annexin II a member of the annexin family has been implicated in

calcittm-regulated exocytosis in chromaffin cells. It has been shown that
upon nicotine stimulation, a fraction of the protein becomes Triton X-100
insoluble [ll, translocates from the cytosol to the cell cortex [21 and is
phosphorylated by protein kinase C [31. This calcium-dependent
association ofannexin II with the membrane occurs via linkages to anionic
phospholipids.
We give evidence for the presence of annexin II on the chromaffin granule
membranes depleted of calcium by EGTA. The calcium-independent bound
annexin 1I is released by methylff3-cyclodextrin, a reagent which selectively
depletes the granule membrane from cholesterol. If the cholesterol content
of the depleted membranes is restored, annexin II binding is also restored.
The binding of monomeric and heterotetrarneric annexin II was also tested
on liposomes of different compositions. Surprisingly, in the absence of
calcium, annexin II binds only to liposomes contaiaing phosphatidylserine,
and the presence of cholesterol in these liposomes increases binding at very
low concentrations of calcium or without it. The aggregation properties of
the monomeric and tetrameric forms of armexin II were also studied. The
results obtained are in complete agreement with those described for binding
and demonstrate an interaction of annexin II with cholesterol.
In conclusion, it is suggested that cholesterol may regulate directly
the binding of the two forms of annexin 2. If cholesterol is concentrated in
some regions of the membranes, annexin 2 may form clusters at very lo~v
concentrations of intracellular calcium as in resting cells.
11] Sagot et al., FEBS leas., 410, (1997) 229
121Chasserot-Golaz et al., J. Cell. Biol., 133, (1996) 1217
[31 Delouche et al., J. Neurochem., 68, (1997) 1720
(Su/11.1/165)

MI~ANEPHf~PHOLIPIDASYM~ETRYINE-BETATHALASSEMIA
J. Basu and P.T. Srinivasan, Bose Institute,
Calcutta, India

The underlying
cause of the accelerated
destruction
of
erythrocytes
in the bone marrow and in the peripheral
circulation, accompanying the beta-thalassemic syndromes is
still not clearly understood. The transbilayer distribution of
phospholipids
in human beta-thalassemic
erythrocytes
also
remains to be explored in detail. We demonstrate that increased
phagocytosis of erythrocytes in E-beta-thalassemia is inhibited
by the presence of phosphatidylserine (PS) vesicles, suggesting
a PS-"BS receptor" type of interaction in premature recognition
of these erythrocyte s by macrophages. Increased exposure of
both aminophospholipids phosphatidylethanolamine
(PE) and PS
was demonstrated by fluorescamine labeling and annexin binding,
respectively. The slower rate of translocation of PS across the
bilayer suggested that this contributed towards the increased
exposure of PS in E-beta-thalassemic erythrocytes.
We also document differences in transmembrane distribution of
phosphatidylcholine (PC) between normal and E-beta-thalassemic
erythrocytes. Passive diffusion movements probably contribute
to alterations
in transmembrane distribution of PC. The
activity of the Ca-dependent phospholipid scramblase did not
differ between normal and thalassemic erythrocytes. The slower
rate of PS translocation and the enhanced passive movement of
PC into the inner leaflet of the lipid bilayer both contribute
towards a change in the transverse lipid architecture of
E-beta-thalassemic erythrocytes.

(Su/11.1/164)

Lipid rafts in ovary cancer-derived cell membranes:

effects of caveolin-1 transfection
M. Bagnoli, S Canevari, A Tomassetti, and S. Miotti.
Dept. of Exp Oncolog.v, Istituto Nazionale Tumori, via l"eneztan 1~
20133 Milan.

Lipid rafts, enriched in sphingolipids, cholesterol and GPI-anchored proteins

represent specialized membrane regions which move within the fluid bilayer
Lipid rafts are involved in the recruitment and assembly of cell signaling
molecules and caveolin l(cav-1) represents the key molecule which binds
signaling proteins in their inactive form and negatively regulate their activity.
However, microdomains devoid of cav-I also exist, and in tumor cells, a loss
of cav-1 expression was found associated to cell transformation.
Recently we demonstrated that the GPI-anchored Folate Receptor fiR),
overexpressed in human ovarian cancer, is localized in detergent-insoluble,
low-density membrane microdomains in association with the src-family
member p53-56 lyn and the C-~-3 subunit of heterotrimeric G-proteins Using
the IGROVI cell line as a model, complexes ofFR, lyn and G~,_~ were coimmunoprecipitated using either anti-FR or anti-lyn antibodies, and an in vttro
kinase assay on the microdomains immunoprecipitated by the anti-FR Mab
revealed specific phosphorylation &the p-53 form oflyn and its associated Gprotein subunit. These findings suggest that FR present in lipid rat~s can
generate intracellular signals.
Since IGROV1 detergent-insoluble domains contain low or no car-1 at all, we
transfected IGROV1 cells with cav-1 cDNA to determine whether ricoexpressed cav-1 can affect the activation of FR downstream molecules
Following transfection, firstly we observed a reduction of FR mRNA and
protein expression, associated to a reduced cell proliferation. The effects on
FR membrane distribution and functional association with its related signaling
molecules are now under investigation.
Partially supported by AIRC/FIRC grants.

s 136

Abstracts FEBS' 99

(Su/11.11167) Omega-3 fatty acids increase liver uptake of HDL

cholesteryl ester in mice.

(Su/ll.l/168)

BERARD A, DUMON M-F, PEUCHANT E, PALO~PINTO A, COSTET P, CLERC

19Land DARMON YM

* I)ep. A4tc'roblo/o10; Ak'hool qfBzohq, O,. Moscow Slate (/itlver~'tO;

I~IOXCf)U. 119899: ** (Drdtolo&'~' Research ('entel; Movcow. 121552

Laboratotre de Biochtrate, Hdpital Sud, CHR et Universit~ Bordeaux-2

In humans, diets high in fish oil (containing omega-3 fatty acids) decrease
the incidence of coronary artery diseases. This is thought to be due to a
decrease in plasma triacylglycerol concentrations and possibly to an increase
in plasma high density lipoprotein (HDL) levels.
T o better understand the effects of omega-3 fatty acids on HDL
metabolism, we fed C57B1/6 female mice with an omega-3 fatty acidenriched diet and found that, although showing a decrease in plasma HDLcholesterol concentrations, they exhibited an increase in HDL-eholesteryl
ester removal from plasma via the liver.

Moreover, as an attempt to mimic the situation in humans, we also used

transgenic mice expressing human apolipoprotein A-I and human lecithin
cholesterol acyltransferase genes. These mice actually responded to omega-3
fatty acids by increasing plasma HDL-cholesterol, but, more importantly,
they still exhibited an increase in HDL-cholesterol ester liver uptake.
Consistent with these results, we also found that the in vivo fractional
catabolic rate of HDL-cholesteryl ester was significantly higher m mice fed
an omega-3 fatty acid-enriched diet. Taken together, these results suggest
that an omega-3 fatty acid-enriched diet increases reverse cholesterol
transport in mice.

(Su/11.1/169)

Characterization of lipids associated to alkaline

phosphatase in TRMC from bovine kidney.
F. Besson, S. Bonnin. K. El Kirat, B. Roux
Lxlb. Ph~ ~lco-Chtlltle Brologlque. CNRS UPRESA 5013
Untrergit( C. Bentard-L3on L 69622 Villeurbanlw, France.

Membranes that contain glycosylphosphatidylinositol (GPI)-anchored

proteins had ever been isolated from a variety of eukaryotic cells and from
cultures of epithelial or Madin-Darby Canine Kidney cells, on the bas~s of
their surprising insolubility in non-ionic detergents such as Triton X-100.
Models for the organization of the proteins and hpids present in the detergent
resistant domains had been proposed [1-2]. The purpose of this work was to
characterize the Triton Resistant Molecular Complexes (TRMC) isolated
from bovine kidney. TRMC were purified by successive ultracentrifugations
on discontinuous sucrose gradient and their location on the gradient was
monitored by determination of the activity of alkaline phosphatase, a GPIanchored protein, and by lipid titration along the gradient. In parallel, the
lipids of whole cell membranes and TRMC were extracted to determine their
composition. We demonstrated that (i) the specific activity of the alkaline
phosphatase was sixty-fold increased during the different steps of the
purification of TRMC, (ii) the alkaline phosphatase activity was mainly
associated to the lipids of TRMC and (iii) the lipid composition of TRMC
was different of that of whole cell membranes. Furthermore, it had been
proposed that the GPI-anchored proteins, which often have saturated fatty
acyl chains, should prefer the less-fluid regions of the membranes. Such lessfluid regions would contain more saturated hpids [2-31. To verify this
hypothesis, the fatty acids of the various lipids of TRMC and whole cells
were analyzed.
[1] Simons and Ikonen Nature 387 (1997) 569.
[2] Brown and London, Biochem. Biophys. Res. Commun. 240
(1997) 1.
[31 Schroeder et al. Proc. J. Biol. Chem. 273 (1998) 1150.
Kevwords. Triton Resistant Molecular Complexes (TRMC); lipids; alkaline

phosphatase.

The NAD(P)H-cytochrome b5 redox chain protec~ biological

membranes from oxidation: role of lipid radical cycles.
LF. Dmitriev* and M V. lvanova**

The studies reported in this work represent the first &rect evidence of
protein-lipid redox interactmn: cytochrome b5 acts as an electron donor for a
redox system, m which lipid radical is an acceptor. The goal of this work was
to study lipid peroxldatlon ILPO) m liver mmrosomes (native and enriched
with tocopherol) The process of mlcrosomes ~solatmn was ran&fled to obtain
mierosomes with a low catalase activity. or microsomes depleted of cytochrome bs. Two various conditions of LPO activation in the membranes were
used: LPO was activated by either enzy.matlc (NADPH-dependent) or nonenzymatic (cumene-dependent) methods.
The result gwes rise to the suggestions: (a) LPO m the microsomal
membranes does not proceed by the chain reaction mecbamsm (the reaction
chain length is close to I ), (b) the inhibition of lipid peroxide production (the
antioxidant activ W of tocopheml) is implemented in membranes only under
the combined actmn oftocopherol and one of the member of the redox chain.
An external donor of electrons medmtes the reductmn of pernxyl
radicals and the production of anion peroxides (kO:" ---> LOT ); the latter
interacts with tocopherol. This may cause the recovery" of the lipid molecule
to the initial state as a result of one-electron oxidation of tocopheml and substitution o f Oz molecule (release of superoxide) for an H- atom (LH --> U---~
LO~ --* LO_;" ---> IM). Then. the NADPH- or NADH-dependent redox chain
~mplements specml types of interactions between tocopherol and LO/anion
m biological membranes. These interactions need the cytochmme b5 and they
are not accompanied by the tbrmatmn of LOOH as a terminal product. The
hpid hydropemxldes are not produced owing to lipid-radical cycles and hence
the lipid peroxidation in the functionally active biological membranes ~s
really blocked The lipid-radical cycles or. more precise, lipid pulsations are
probabl,, able to achvate the end member of monooxygenase system m the
microsomal membranes, cytocbrome P-450 and moreover to activate the
synthesis of proteins by membrane-associated ribosomes.

(Su/ll.1/170)

Phospholipid excess in the outer leaflet of plasma membrane

induces actin polymerisafionin platelets.
N. Bettache, J. Davet, S. Baghdiguian, A. Bienven/Je
CYRS-UMR 553~ UnwersltdMontpelher 2, 34095 ~4ontpelher France

The distribution of phospholipids between the two leaflets of the plasma

membrane of many eukaryotic cells is asymmetric. This asymmetry is
maintained in platelets as in erythrocytes by continuous transport of
aminophospholipids (phosphatidylserine and phosphatidylethanolamine)
from the outer to the inner leaflet by ATP-dependent aminophospholipid
translocase, while the choline head phospholipids remain preferentially
located in the outer leaflet. During Ca-~'-induced activation, we observed
aminophospholipids exposure and fllopod formation in platelets pre-treated
with calpeptin (a permeant inhibitor of calpain). This filopod formation
results from phospholipid excess in the outer leaflet of plasma membrane and
from cytoskeleton reorganisation induced by a polymerisation of aetin. This
study was devoted to the effects of an excess of phospholipids in the outer
leaflet of plasma membrane of resting platelcts in comparison with those
obtained in Ca>-ionophore activated platelets pre-treated with calpeptin.
Addition of short chain analogues of phospholipids in the outer leaflet of
plasma membrane of resting platelets induced a shape change resulting in
filopod formation and actin polymerisation. This process is reversible when
platelets were incubated with an analogue of phosphatidylserine. In this case.
during the first two minutes, we observed formation of filopods and actin
polymerisation and. for longer time. the platelets recovered their shape and
their initial filamentous actin content, while tins analogue was transported to
the inner leaflet by the anainopbospholipld translocase. Pre-incubation of
platelets with cytochalasin D inhibits both filopod formation and actin
polymerisation when treated with a phosphoIipid analogue.

Abstracts FEBS'99

(Su/ll.1/171)

s137

Alteration of mitochondrial functional parameters

induced by dietary fatty acids in muscovy ducklings
F. Chainier, D. Roussel, R. Meister, C. Duchamp. H Barr6

(Su/ll.1/172)

UMR CNRS-UCBLyonl. 43Bd du l lnoverabre 1918,

69622 l'dleurbam~e cede):. F)'auce

Alterat,_on of mitochondrial efficiency is one of the mechamsms involved

in avian skeletal muscle nonshwenng thermogenesis. The baochemical
background of such mechanism is still incompletely understood but the
lipad composition of matochondrial membranes is likely to play a role It
was hypothesised that dietary-induced alteration of mitochondrmt
membranes may gave us some clues to the influence of structural hpids
on mttochondnal function m birds.
During four weeks, three groups of ducklings were fed with diets
enriched with 6.5% of either hydrogenated coconut oil (HCO, rich in
saturated fatty acids), grape-seed oil (GO, rich in n-6 fatty acads) or fish
oil (FO, rich m n-3 fatty acads). Isolated mitochondria were then obtained
from skeletal muscles rich in esther fast twitch fibres Ipectoralis and
external gastrocnemius) or slow fibres (internal gastrocnemaus).
The fat~ acid composition of mitochondrial phosphohpids globally
reflected the diet composition but &fferences between groups were lower
than those in the diet composition Unsaturation index and average chain
length gradually increased from the HCO, to the GO and FO diets
Cardiohpin level of mltochondrlal membranes was lower in the three
skeletal muscles wath the HCO diet. Respiratory characteristics of
mltochondria were altered by the HCO diet. The specific (per g
mitochondrial protein) activtty of two mltochondrial enzymes
(cytochrome oxydase and ATPsynthase) was also altered by the HCO
d~et. At the t~ssue level, these differences were however compensated for
by differences in the total amount of muscle mitochondria
In conclusion, there were relatively few differences between the GO and
FO diets. ]'he HCO daet appeared as the most deleterious for
mitochondrml functional actw~ty Interestingly, mitochondrial membrane
composition appeared relatwely buffered by compensatory mechanisms
counter-acting the hpid composition of the diet. These data suggest that.
m birds as in mammals, the hpld compositmn of mltochondrial
membranes modulates their functmnal charactertstacs

(Su/11.1/173)

On the influence of linoleie acid on the phospholipid

species in bovine lens epithelial cells (bLEC)
D. Glanz~, J.-U. Lechler~, K. Lennarz~, K. Raithb
M. Trirnborn~, D. GlliBera
"Institut far Physiologisehe Chemic, Medizinische Fakultdt, blnstitut
far Pharmazeutische Technologie und Biopharmazie, Fachbereich
Pharmazie, Martin-Luther-Universitdt Halle-Wittenberg, D-06114
Halle, Germany

Unsaturated fatty acids are known to cause damaging effects on living cells.
Generally these effects are assumed to be triggered by the action of fatty acid
peroxidation products. However, results obtained with lens epithelial cells,
strongly support the notion that eis-linoleic acid (LA) itself injures the cells
without being oxidized [1]. On the other hand, free fatty acids are normally
taken up by organ cultured lenses and incorporated into triacyl-glycerides and
phospholipides of the lens cells [2].
The aim of this work was to find out to what extent LA influences the lipid
composition of cultured bLEC. We found that 75 % of the t~C-LA was
incorporated into phosphatidyl choline (PC), 8 to 10 % were found in
phosphatidyl ethanolamine (PE) and neutral lipids, respectively. After
converting PC into diaeyl-glyeerides by phosphollpase C treatment and
preparation of the corresponding dinitrobenzoyl derivatives [3] the reaction
products were analysed by HPLC. In cells which were cultivated in presence
of 10 I~mol/l LA the amount of dllinoleoyl-PC were doubled, while dioleoylPC remained equal.
Dllinoleoyl-PC is known to inhibit the acyl-CoA:eholesterol-acyltransferase
(ACAT, EC 2.3.1.26) resulting in increasing amounts of free cholesterol in
the cells [4]. Enhanced cellular concentrations of cholesterol are cytotoxic
and are also found during eataractogenesis and Niernann-Pick disease [5]. We
therefore suggest that the increase of cellular dllinoleoyl-PC might be one
reason for the especially high toxicity of cis-linoleic acid to lens epithelial
cells.
[1] D. Gl~er et al., Eur. J. Cell Biol., 71, (1996) 286
[2] B. Alhers-Jackson et aL, Curr. Eye Res. 2, (1982-83) 233
[3] M. Kito et al., J. Biochem., 98, (1985) 327
[4] S. N. Mathur et al., Biochim. Biophys. Aeta., 751, (1983) 401
[5] E. Cotlier et al., Biochim. Biophys. Acta, 530, (1978) 267

Lipid and ganglioside changes occur during retinal

ischemia and reperfusion. V. Fontaine, B. Gu6rold, C.
Fuchs, J. Sahel, H. Dreyfus. Laboratoire de P~(vsiopathologte
cellulalre et moleculaire de la retme. Strasbourg, France

Purpose. To determine the impact of retinal ischemia on

different classes of lipids implicated in neuronal structure,
survival and plasticity Methods. Retinal ischemia was induced
unilaterally in Long Evans rats by increasing intraocular pressure
to 160 mm Hg for 60 minutes Retinas were harvested after
different times of reperfusion (0 to 24 hours), lipids were
extracted and each lipid group (gangliosides, phospholipids and
neutral lipids) was isolated by solvent elution usin~ silicic acid
column chromatography, and characterized by HPTLC Results.
During the 24 hours of reperfusion following retinal ischemia the
GM1 ganglioside content increased 100% compared to 0 hour of
reperfusion or no ischemia Among the phospholipids analysed,
the highest changes were found for phosphatidylinositol
increasing 32%, and phosphatidylethanolamine andphosphatidic
acid decreasing 13% and 23% respectively among the same
period of reperfusion. Amount of cholesterol was not modified
by ischemia but that of diacylglycerol, another neutral lipid,
decreased 25% during the 24 hours of reperfusion. Furthermore
the ratio <t phospholipids/cholesterol )), which reflects membrane
fluidity, decreased 23% during the same period. Conclusions.
Our data showed dramatic lipid composition modifications
occuring during the 24 hours of reperfusion following retinal
ischemia. Such modifications may reflect the importance of lipids
implicated directly in neuronal survival and plasticity, or in the
intracellular signalisation pathways leading to apoptosis
(survival)
Acknowledgements. V F. thanks <<Fondation Berthe Fouassier ))
for personal support.

(Su/11.1/174)

Increase of caveolin-1 protein levels and parallel decrease

of G-protein (13) by 25-hydroxycholesterol in NIH3T3 cells.
C. G6mez~, M.J. Toro~, y A. Montes b
~Dept. Bioquimica y Biologia Molecular, bDept. Ftsiologia, Facultad de
Medicma, Umverstdad de AIcal6. Madrtd, Spain.

Caveolae are plasma membrane domains rich in cholesterol,

glycosphingolipids and lipid-anchored membrane proteins present in most ceils
and playing multiple functions [1]. Caveolin-l, a 22-24 kDa cholesterolbinding protein associated with caveolae, is a member of a gene family broadly
implicated in transmembrane signalling and transport [1]. Recent evidence
using human fibroblasts have shown that caveolin mRNA levels are upregulated by free cholesterol, which is correlated to the rate of free cholesterol
effiux and to the free cholesterol content in caveolae [2], supporting the
hypothesis that caveolae and caveolin are involved in cellular free cholesterol
homeostasis [2]. In that system, the effects of cholesterol were mediated by
Sterol Responsiveness Element Binding Protein 1 (SRBPI). On the other
hand, it has been shown that caveolin-I is able to interact with heterotrimeric
G-proteins, regulating its activity. Previous results in our laboratory have
shown that 25-hydroxycholesterol (25-hc) affect the levels and subcellular
distribution of G-protein ct- and fl-subunits, and the GTPase activity of Got
In this work we studied the effect of this oxysterol on the protein
levels of caveolin-1 and G~ in confluent mouse fibroblasts monolayers,
observing opposite effects on both proteins. Compared to the control, in total
homogenate 25-hc (2,5 laM) significantly increases protein concentration of
caveolinl by 2- to 3-fold as was meausured by western blotting. These
changes are dosis-dependem, and are seen at 24 and 48 h after the treatment
in a lipoprotein-deficient medium. Also, it seems the effect is mediated at a
transcriptional level because there was a similar change in the mRNA level at
48 h of treatment, as meausured by northern blotting. On the other hand, GI3
protein was significantly reduced under the same conditions. These data are
interesting because it seems to indicate: 1) 25-hc has the same effects as free
cholesterol on the caveolin-1 expression; 2) the changes in caveolin-1 levels
could influence changes in G~3 protein, which is not sorprising in view that
both proteins are present in caveolae and this domain is severely affected by
agents depleting the cellular cholesterol, as 25-hc does.
[1] Anderson, R Annu. Rev. Biochem. 67 (1998), 199.
[2] Fielding, C., et at. Proc, Natl. Acad. Sci. USA. 94 (1997), 3753.

s138

(Su/11.1/175)

Abstracts FEBS'99

Transport of newly synthesized cholesterol in BHK cells

S. Heino a, S. Lusaa, P. Somerharju b, V. Olkkonen ~, E. Ikonen~

(Su/11.1/175)

aDept. of Biochem, National Public Health lnstttute, Helsinki, Finland

bInst. of Biomedicine, Dept. of Medical Chem., Univ. of Helstnkl, Finland

Cholesterol is synthesized in the endoplasmic reticulum in a complex

biosynthetic process. From its site of synthesis, cholesterol is transported to
various cellular membranes. The movement of endogenously synthesized
cholesterol to the plasma membrane is thought to be a fast (half time of ~
10-20 min), energy requiring process which is not affected by drugs that
affect the cytoskeletal network, protein synthesis or the Golgi apparatus.
We have studied the synthesis and transport of nascent cholesterol in baby
hamster kidney (BHK) cells by pulse-labeling the cells with 3H-acetate,
chasing for increasing times and measuring the arrival of radioactive
cholesterol on the plasma membrane. Surface arrival of cholesterol was
analysed by capturing it to the extracellular acceptor methyl-13-cyclodextrin,
or by subcellular fractionation. The newly synthesized lipids were separated
by thin layer chromatography (TLC) followed by high performance liquid
chromatography (HPLC). Our results show that cholesterol synthesis in
fibroblastoid cells is relatively slow (half time of - 1 h), even when the
synthesis is upregulated by prior cholesterol depletion. The kinetics of
cholesterol transport to the cell surface was found not to differ significantly
from that observed for the viral marker protein influenza hemagglutinin
(half time of - 30 - 45 min). Moreover, cholesterol transport was partially
inhibited by disassembly of the Golgi apparatus using Brefeldin A or by
depolymerization of microtubules using nocodazole. Our data show for the
first time that part of the newly synthesized cholesterol is transported from
the ER to the plasma membrane along the same pathway as newly
synthesized proteins.

Docosahexaenoic acid supplementation of peroxisome

deficient mice does not improve postnatal survival
A. Janssen, P.E. Declercq, M. Baes and P.P. Van Veldhoven
Laborato~ of Clinical Chemtstry and Laboratory of Pharmacology,
Katholieke Universitett Leuven, 3000 Leuven, Belgium

Patients with Zellweger syndrome and other disorders of peroxisome biogenesis have several abnormalities in lipid metabolism resulting in accumulation of very long chain fatty acids (VLCFA) and phytanic acid and depletion
in plasmalogens. Docosahexaenoic acid (DHA), an important polyunsaturated-fatty-acid of brain membrane phospholipids and photoreceptor
cells, was shown to be decreased in several tissues of Zellweger patients.
Martinez et al. [ 1] supplemented these patients with DHA which resulted in
reduced hypotonicity and improved visual functions
Using our mouse model for the Zellweger syndrome, recently generated by
inactivation of the PEX5 gene [2], the following questions were asked: 1) do
newborn PEX5 ' pups have a low DHA content in their tissues; 2) can these
pups be supplemented with DHA by feeding it to the mother; 3) does this
result in clinical improvement of the newborn Zellweger mice?
Total lipid extracts of mouse tissues were separated on amino-columns. The
phospholipid fraction was transmethylated and the fatty acid methyl esters
were analysed by GC on a BPX70 column.
The DHA level in brain phospholipids of newborn Zellweger mice was 50%
reduced as compared to normal littermates. Ten mg DHA-ethylester was
administered daily to pregnant heterozygous mothers CPEX5+~') by tube
feeding from embryonic day 10 (El0) till day E~s5 or birth. The DHA level in
Zellweger mice brains normalized to the level of control animals (born from
non supplemented mothers) and was elevated in normal newborn mice. However, in PEX5 -~- pups supplemented with DHA, no clinical improvement
could be observed. Their brains will be analysed to examine whether the neuronal migration defect, that is typical for Zellweger patients and also for the
PEX5 -/- mice, is affected by the DHA supplementation.
These data suggest that DHA therapy of pregnant mothers carrying Zellweger mice, does not correct clinical abnormalities in newborn PEX5 ~ mice.
[1] Martinez M. et al., Neurology, 43, (1993), 1389.
[2] Baes M. et al, Nature Genetics, 17, (1997), 49.
Supported Biomed BMH4 - CT98 - 3569

(Su/11.1/176)

Docosahexaenoie acid and body temperature in vertebrate

brain
K. Kitajka, T. Farkas
Biological Research Centre, Hungaman A cademy of Sctences, Hungary

Docosahexanoic acid (DHA) plays an important but not completely

understood role in maintaining structural and functional integrity of brain
membranes. Similar to mammals the level of DHA cannot be influenced by
dietary means in fresh water fish brains revealed by comparison of fatty acid
composition of fish different feeding habits. Although the content of DHA in
total phospholipids does not seem to depend on environmental/body
temperature, its level in synaptic plasma membranes decreases with increasing
body temperature from fish to birds. Despite similarities in fatty acid
composition phospholipids from birds form more ordered vesicles (assessed
by DPH fluorescence polarization) than mammals or warm and cold adapted
fish in a way that anisotropy values measured at the body temperature are
almost identical. Comparison of molecular species compositions of
phosphatidylcholines (PC) and phosphatidylethanolamines (PE) showed an
accumulation of molecular species containing a monounsaturated fatty acid
(18:1) in position sn-1 and a polyunsaturated fatty acid (mostly 22:6 but also
20:4) in position sn-2 of these phospholipids. Level of 18:1/22:6 PE in
synaptic membranes and also in total phospholipids from subtropic fish to
birds was inversely related to body temperature in a mariner that the sum of
molecular species containing 18:0+I 8:1 in position sn-1 and 22:6 in position
sn-2 is almost identical in all vertebrates investigated so far.
Exposing warm adapted (22~C) fish to 5C for three days resulted in a
measurable increase of 18:1/22:6 PE and also fluidity of phospholipid
vesicles. Since surface areas of these phospholipids is 20-30% larger than that
of their saturated/polyunsaturated analogues [1] we propose that they play part
in controlling fluidity of membranes throughout the evolutionary chain.
Morever, because these species exhibit a low bilayer to non-bilayer transition
temparature [2] and therefore are highly fusogenic, they may facilitate fusion
of synaptic vesicles with postsynaptic neuronal membranes [3] and thus
contribute to normal signal transduction at the relevant body temperature.
[1]: Zabelinskii et al., Comp. Biochem. Phsiol., 111B, (1995) 127.
[2]: Giorgione et al., PNAS, 92, (1995) 9767.
[3]: Lohner et al., Chem. Phys. Lipids, 81, (1996) 167.

(Su/ll.l/177)

Threshold effect for penetration of daunorubieine into

membrane model: effect of lipid composition
M.F.Leeompte ~, J P.Jaffrrzou b, G.Laurentb
Fac.Mdd.Rangueil, 133 rte de Narbonne 31062 Toulouse Cedex, France
b INSERME9910, L CLR.. 20 rue pont St Pierre, 31052 Toulouse Cedex

The neoplastic cell has long been considered a passive target for an
antitumor drug. A simple example is the axiom (which disregards the
complexity of an eukaryotic cell plasma membrane) that molecules enter a
cell through Fickian diffusion.
Using the chemotherapeutic drug daunorubicin, we studied the
interaction between drug and a model monolayer membrane. We used
alternative current polarography, which has the specificity to allow for the
detection of the interracial process of water soluble molecule penetration
within a membrane [1]. The validity of this approach was previously
demonstrated on <<prothrombinase >~, membrane complex in coagulation
cascade. Indeed, consistent results were obtained, by complementary
methods, between monolayers, vesicles and platetets, either on structural [2]
or on functional point of view.
In a pharmacologically relevant concentration range, dannorubicin
presents different binding states, and more specifically distinct adsorption and
penetration steps. The threshold value separating these two events depends
on the lipid composition and most notably it decreases when sphingomyelin
content increases.

[ 1] M.F.Lecompte, GBouix & K.G.Mann (1994) JBiol.Chem. 269, 19051910

[2] M.F Lecompte & J.Elion (1998) Bioelectrochem & Bioenerg 47, 57-66

Abstracts FEBS'99

(Su/11.1/179)

s139

Analysis of antioxidant enzymes and lipid peroxidations

in Aizheimer patients
M.C. Martin Mateo, S. Munoz, I. Ramos, R. Fernandez

(Su/ll.l/180)

Universidad de galladolid, Prado de la Magdalena s.n.,

47005 Valladolid, Spain

Lipid peroxidations in erythrocytes of patients with Alzheimer disease has

been followed by malondialdehyde, The concentrations of the following
antioxidant enzymes were also determined; superoxide dismutase (SOD),
catalase (CAT), glutathione peroxidase (Gpx), glntathione reductase (GR),
total glutathione (GST) and reduced glutathione (GSH).
The results showed a high level of oxidative stress in Alzheimer patients,
evidenced by lowered total antioxidant state and CAT, SOD, Gpx, and GR
activity, plus a considerable increase in MDA level. This increase is a clear
indicator of greater lipid peroxidation.

PARAMETERS
MDA (_n~..gHB)
G-px (Uml RBC)
CAT (K/s)
GR (U/mlRBC)
SOD (U/ml Blood)

(Su/ll.l/181)

DONORS
~4.17 5:9.05
4.24 :t0.62
3.006 5:0.0018
3.657 5:0.07
134.33+_10.35

PATIENTS
114.52:t:20.26
2.88 + 0.34
0.0045"5:0.00072
0.63 5:0.045
113.55+_9.94

Modulation by cholesterol of the P-glycoprotein

ATPase activity
S. Orlowski, A. Garrigues, M. Garrigos
Section de Biophysique des Prot3ines et des Membranes, DBCM / CEA
Saclay, 91191 Gif/Yvene Cedex, France

The plasma membrane multidrug transporter P-glycoprotein (P-gp) is

responsible for the ATP hydrolysis-dependent efflux of various amphiphilic
molecules out of the cells. P-gp function seems to be particularly sensitive to
its membrane environment since (i) the presence and even the nature of the
lipid components are key parameters for P-gp activity, (ii) transport sites are
likely located within the P-gp transmembrane segments, and (iii) some
amphiphilic molecules indirectly modulate P-gp functions through membrane
perturbations, Cholesterol, an important physiological component of the lipid
phase, has been reported to influence the specific azidopine binding on P-gp
contained in liposomes [1]. We thus investigated wether cholesterol can
modulate the P-gp ATPase activity. This enzymatic activity was measured on
membrane vesicles containing high amounts of P-gp.
It is well-known that saponin can chelate cholesterol, resulting in
membrane permeabilization. Indeed, the saponin concentration necessary to
permeabilize our vesicles is increased after cholesterol addition to the
suspension: this indicates cholesterol insertion into the membranes.
Moreover, in agreement with its amphiphilicity, saponin modulates the P-gp
ATPase activity in the presence of some transport substrates : it increases the
stimulating effect of verapamil (with a decreased apparent affinity), whereas
the progesterone-stimulated ATPase activity remains unchanged.
Cholesterol below 200 I~M alters neither the P-gp ATPase activity
without any drug, nor the verapamil-stimulated ATPase activity (although it
induces a decrease of the apparent affmity for verapamil), In contrast,
increasing cholesterol concentrations progressively inhibit the progesteroneinduced ATPase activity stimulation, until complete collapse at 220 laM
cholesterol This observation could be explained by the related chemical
structures of the polycyclic moieties of cholesterol and progesterone.
As a whole, the modulating effects of saponin and cholesterol on the
P-gp ATPase activity cannot be reduced to a simple chelation between these
two molecules, since these effects are not opposed each other. Rather, they
seem to independently alter the P-gp function. These data suggest that
cholesterol might act as an inhibitor of the drug-stimulated P-gp activity,
which could mean a physiological relevance.
H1 Saeki et al., Biochim Biophys Acta, 1107, (1992) 105.

Continuous measurement of rapid transbilayer

movement of lipid analogues in microsomal membranes
U. Marx, G. LaBmann~, D.Wiistner, P. Miiller, A. Herrmann
Humboldt-Universitiit Berlin, In.~t.f. Biologic, D-10115 Berlin, Germany
OTechntsche Univ. Berlin, Mat-Vo[mer-Inst., D-10623 Berhn, Germany

Lipid analogues containing either a paramagnetic doxyl moiety (spin-labelled

analogues) or a fluorescent NBD moiety (NBD-labelled analogues) have
proven to be useful for the examination of transverse lipid dynamics in model
and biological membranes. The most widely used method for monitoring
analogue redistribution across a bilayer is the extraction of analogues to bovine
serum albumin (BSA), the so-called 'back-exchange' method. Usually,
centrifugation steps limit the time resolution of that method. Here, we have
developed a novel EPR stopped-flow 'back-exchange' procedure [1] which is
suited for the measurement of rapid redistribution of spin-labelled analogues
across model and biological membranes. This approach allows to follow
continuously the kinetics of transverse redistribution of glycerophospholipid
analogues in rat liver microsomal membranes. The time resolution of this
approach is only limited by the half time of analogue extraction from the
membrane leaflet exposed to BSA which is in the order of about 2 sec at room
temperature. For microsomal membranes, the transbilayer movement was very
fast and did not depend on the head group (e.g., the half-time of transverse
redistribution of spin-labelled PC and PE analogues was 15 - 17 s at room
temperature). Moreover, we used a recently developed fluorescence resonance
energy transfer approach to follow the transverse redistribution of NBDlabelled glycerophospholipid analogues in rat liver microsomal membranes,
NBD-labelled glycerophospholipid analogues redistributed much slower across
the microsomal membrane than spin-labelled glycerophospholipid analogues.
[I] Marx et al., Biophys. J., 73, (1997) 1645.
[2] Zang and Nichols, Am. J. Physiol., 267, (1994) G80.

(Su/11.1/182)

Sphingolipid: sterol brayers. Susceptibility towards detergent solubilisation.

S.K. Patra, A. Alonso, J.L.R. Arrondo, F.M. Gofii
Unidad de Biof[sica (CSIC-UPV/EHU), and Departamento de Bioqulmica,
Universidad del Pa(s Vasco, Aptdo. 644, 48080 Bilbao, Spa/n.

In an attempt to understand the phenomenon of resistance to

detergent solubilisation exhibited by putative plasma membrane
microdomains or 'mils', we have examined the solubilisation by
Triton X-100 of large unilamellar vesicles consisting of pure
egg sphingomyelin (T= = 39~C) or sphingomyelin:sterol or
sphingomyelIn:phosphatidylcholine:sterol mixtures at various
temperatures. For pure sphingomyelin, solubilisation occurs
most readily at temperatures just below Tin. In general, egg
sphingomyelin is solubilised by Triton X-100 more easily than
egg phosphatidylcholine. Aqueous dispersions of
sphingomyelin:eholesterol mixtures are notoriously resistant to
solubilisation by Triton X-100. Infrared spectroscopy has been
applied to explore the interactions of cholesterol and
sphingomyelin at the level of the lipid-water interface.
Moreover, various cholesterol analogues (cholestane,
cholestartone, androstenol) have been used in parallel
solubilisation experiments and IR observations. The results
show that cholesterol modifies the conformation (or H-bonding
properties, or both) of the sphingomyelin polar headgroup, both
above and below T o. Both the hydroxyl group at C3 and the
hydrocarbon chain at C17 of the steroid nucleus appear to be
required for insolubility to be detected. Increasing proportions
of phosphatidylcholine modify accordingly the detergent
resistance of sphingomyelin:cholesterol bilayers.

s 140

Abstracts F E B S ' 9 9

(Su/ll.l/183) Study of fatty aldehydes release from rat brain

plasmalogens subjected to an oxidative stress.
S. Stadelmann, A. Wahl, F. Chauvelon, C. Tallineau.

(Su/ll.l/184)

Centre d'dtudes et de recherche sur les x~nobiotiques, Facultd

de M~decine et de Pharmacie, BP199, 86005 Poitiers. ?'~nee

Plasmalogens (1-alkenyl-2 acyl glycerophospholipids ) are a particular

group of phospholipids characterized by the presence of a vinyl-ether
bond at the sn-1 position instead of an ester bond. The hydrolysis of
this bond leads to the release of fatty aldehydes. This reaction occurs
when plasmalogens are submitted to a photosensitized oxidation since
the ether-vinyl linkage is particularly reactive towards singlet oxygen.
Nevertheless, their biological function is still poorly known. We
focused our studies on the effects of various toxic compounds and of
free radical generating processes on the ether-vinyl linkage. In the aim
of studying the reactivity of plasmalogens in specific rat brain areas,
we developed a method by gas-chromatography / mass spectrometry
to quantify fatty aldehydes after derivatization by
pentafluorobenzylhydroxylamine (PFBHA, HCI). Cerebral cortex
homogenates were incubated for various period of times (0-6h) under
different conditions i- with antioxidants (BHT, Desferal), ii- without
antioxidants, and iii- with the oxidant system Fe2+ / ascorbate. Lipids
were extracted, then derivatized with or without priorly submitting
them to acidic hydrolysis &the ether-vinyl linkage which allowed us
to quantify the totality of aldehydes (free and bound) and aldehydes
released during the incubation period respectively.
Results : Rat cortex contains 10 3 ~tmoles 0.9 [.tmoles/g tissue of
fatty aldehydes, i e plasmalogens. Free aldehydes represent less than
1% of the totality of aldehydes. No significant modification occurs in
the presence of antioxidants. Without antioxidants, the incubation of
brain homogenates leads to an increase of TBARS but the amounts of
plasmalogens and free aldehydes are not modified. The results
obtained after the homogenates were submitted to an oxidative stress
will be discussed and compared to those obtained when homogenates
were submitted to a photosensitized oxidation.

(Su/11.1/185) Biophysical and biochemical characteristics of nuclear

membrane isolated from rat hepatoma cell line Reuber H35
M.L Tomassoni ~, D. Amori~, C Leray~, M. Micheti~, M. Viola
Magrl!- a n lnstatuteof GeneralPathology. Schoolof Medicine. 06100 Perugla.
Italy. bINSER2vlU31l, 10, rue Spielraann,67065 Strasbourg,France
.

The nuclear phospholipids (PL) are mainly localised in the nuclear

membrane and their content has been found to be inversely related to
that of the cholesterol (CH) during the cell maturation [1]. Previous
studies have shown that the rat liver nuclear membrane fluidity,
measured by fluorescent probes, changes during liver regeneration [2],
in relation to the lipid composition [ 1,2] and to the behavior of nuclear
membrane neutral sphingomyelinase (N-SMase) activity [2]. The aim
of the present research is to study how the nuclear membrane fluidity
changes in relation to the composition in PL, fatty acids and CH and to
the N-SMase activity in hepatoma Reuber H35 cell line. The results
show that sphingomyelin/phosphatidylcholiue (SM/PC) and CtUPL
molar ratios of bepatoma nuclear membrane are higher than in normal
rat liver nuclear membrane and furthermore a decreased double-bound
index in phospholipid fatty acids is also found. The changes in SM
content may be explained by a decreased N-SMase activity which is
about half that found in normal rat liver nuclear membrane. The
fluorescence
anisotropy
of
diphenylhexatriene
and
trimethylammonium-diphenylhexatriene probes, inversely related to the
membrane fluidity, is higher in H35 bepatoma than in normal nuclear
membrane in a temperature range comprised between 15 and 45C The
alteration of the lipid pattern which occurs in hepatoma nuclear
membrane could have a pivotal role in the regulation of some nuclear
functions such as the RNA nucleocytoplasmic transport that has been
recently seen to be related to the nuclear membrane fluidity [3]
Ill A/OLE et al. Cell Biochem.Funct 15, (1997) 181.
[21 Tomassoni.ML. et al. Biochcm.Mol. Biol. ha. 47 (1999). in press
[3] TomassoniML. et al, llal Biochem Soc. Trans. l I. (1998)307

Polarized Caco-2 cell culture as a model for

intestinal absorption of drugs in man: further Investigation
C.Thomas', J. Pachotb
"SBPM/DBClggCEA Saclay, 91191 Gif/Yvene Cedex, France
bMgtabolisme et Passage Intestinal, H.M.R, 93230 Romainville, France

Caco-2 cell cultures have been proposed as a model for intestinal

absorption of drugs in man [1]. Caco-2 cells are derived from a human colon
carcinoma. When cultured on porous filters, they spontaneously differentiate
into polarized enterocytes, forming a monolayer which defines a basolateral
and an apical compartments. In the present work, we have extended previous
drug absorption studies on the Caco-2 model to test for its predictive ability.
Moreover, this Caco-2 model allows to distinguish between passive and
active transport mechanisms thanks to the polarized nature of the cellular
monolayer and to the accessibility of both compartments in the Transwell
insert design. We have determined apparent permeabilities (P~p) for a series
of 28 compounds belonging to various chemical families for which the
corresponding absorbed fractions in man after oral administration have been
published. Transport studies were conducted with cultured epithelia 21 to 28
days after seeding on the Transwell inserts. As a control, transepithelial
electrical resistance and [l*C]mannitol transport were routinely measured to
test for monolayer integrity. In a typical experiment, concentration of each
compound was determined as a function of time in each compartment defined
by the Caco-2 monolayer, and subsequently used to calculate corresponding
P~p. Our results show that the diffusion across the Caco-2 monolayer of 21
compounds among the 28 tested could be interpreted by a passive transport
process, with a good correlation between their respective calculated P,pv and
the human absorbed fraction values. Indeed, completely absorbed compounds
(fraction absorbed after oral administration in human > 90%) have a P,pp >
6.0xl0 "6 cm/s, whereas poorly absorbed compounds (fraction absorbed after
oral administration in human < 20%) have a P~,p < 0.4x106 cm/s. For the
seven remaining compounds, the data suggest an active transport mechanism
which agrees with their physiological transport pathways in enterocytes. In
conclusion, an important propertie of the Caco-2 model is its ability to point
out active transport for certain drugs, particularly in the context of intestinal
absorption where the presence of various transporters, like the multi-specific
P-glycoprotein, has been reported.
[1] Artursson, Crit Rev Ther Drug Carrier Syst, 8, (1991) 305.

(Su/11.1/186)

Identification and characterisation of a mouse gent

family implicated in tissue recruitment
P. Tvrdik 1, R. Westerberg 1, A. Asadi 1, B.Carmonl,
M.Capecchi 2, G.Loison 3 ,A.Jacobsson 1
1The Wenner-Gren lnstttute, The Arrhenius Laboratories F3, Stockholm
University, S-106 91 Stockholm, Sweden, 2Howard Hughes Medical
Institute, University of Utah, Salt Lake City, UT 84112-5331, USA,
3Department of Mwrobiology, Sanofi Recherche, Lab~ge lnnopole
BP137, F-31676 Labdge Cddex. France

Cig30 was previously isolated in our laboratory as a gene specifically

expressed during brown fat recruitment, and in liver and skin. By
searching for Cig30-homologous mouse cDNAs, we have identified two
novel genes, termed SscI and Ssc2, and cloned their corresponding fulllength cDNAs. Analysis of expression in mice revealed that the Sscl
mRNA was ubiquitously expressed in all tissues tested, with highest levels
in stomach, lung, kidney and skin. In contrast, the Ssc2 mRNA was rather
tissue specific, being predominantly expressed in testis and liver. The
deduced SSC1 and SSC2 polypeptides are approximately 30% identical
with CIG30. We have performed experiments to rescue the phenotypes of
yeast null mutants in the "corresponding" genes which have been
implicated in the biosynthesis of very long chain fatty acids and
consequently the formation of sphingolipids.
In order to understand the physiological significance underlying
Cig30 gene expression, we have isolated and sequenced the entire Cig30
gene and, by homologous recombination, obtained Cig30 deficient mice.
The obvious phenotype of these mice is markedly diminished
subcutaneous fat and abnormal hair growth.
Moreover, we found that the Sscl m R N A levels were markedly
reduced in the brains of the myelin-deficient mutant mice which have been
reported to have decreased cerebral fatty acid chain elongation activity.
In conclusion, we have characterized three paralogous genes in mice
which appear to be the first mammalian genes directly involved in fatty
acid elongation to be identified.

Abstracts FEBS'99

(Su/11.1/187)

Cloning and Recombinant Expression of a Novel Mouse

Group IIA Secreted Phospholipase A2
E. Valentin, R. S. Koduri, J.-C. Scimeca, G. Carle, M. H. Gelb,
M. Lazdunski, and G. Lambeau

s141

(Su/11.1/188) Nonbilayer/bilayer lipid balance in membranes: Regulatory

glucosyl transferase responds to various cellular signals
S.Vikstr6m, L.Li, O.Karlsson, A.Wieslander
Dept. of Biochemistry,Ume~Unlvcrsi~,Um~, Sweden

IPMC, CNRS, 660 route des Lucioles, 06560 Sophla Anttpolis,, France

Secreted phospholipases A2 (sPLA2s) form a class of structurallyrelated enzymes that are involved in a variety of physiological and
pathological effects including inflammation and associated diseases, cell
proliferation, cell adhesion, and cancer, and are now known to bind to
specific membrane receptors. Here, we report the cloning and expression of
a novel sPLA2 isolated from mouse thymus. Based on its structural
properties, this sPLA2 is most similar to group I1A sPLA2 and has been
called mouse group llA-2 sPLA2 (mGIIA-2). As for the previously cloned
mouse group IIA sPLA2 (mGIIA-1), mGIIA-2 sPLA2 is made up of 125
amino acids with 14 cysteines, is basic (pI = 8.71) and its gene has been
mapped to mouse chromosome 4. However, mGIIA-2 sPLA2 has only 48%
sequence identity with mGIIA-I, indicating that the two enzymes are not
close isoforms. In further contrast with rnGIIA-1 which is found mainly in
intestine, transcripts coding for rnGIIA-2 sPLA2 are found in several tissues
including pancreas, spleen, thymus, skin, lung and ovary, suggesting distinct
functions for these enzymes. Recombinant expression of mGIIA-2 sPLA2 in
E. coli indicates that the cloned sPLA2 is an active enzyme that has much
lower specific activity than mGIIA-1 and displays a distinct specificity for
binding to various phospholipid vesicles. Finally, recombinant mGIIA-2
sPLA2 did not bind to the mouse M-type sPLA2 receptor, while mGIIA-1
was previously found to bind to this receptor with high affinity.

In the single membrane of Acholeplasma laidlawii a specific glucosyl

transferase (DGIcDAG synthase) synthesizes the major, bilayer-forming
lipid diglucosyldiacylglycerol (DGIcDAG) from the preceeding major,
nonbilayer-prone monoglucosyldiacylglycerol (MGIcDAG). This is crucial
for the maintenance of phase equilibria close to a potential bilayernonbilayer transition and a nearly constant spontaneous curvature in vivo.
The glucolipid pathway is also balanced against the phosphatidylglycerol
(PG) pathway to keep a certain lipid surface charge density.
In mixed-micelles a cooperative dependence for the purified DGlcDAG
synthase on anionic lipid activators was confirmed, with PG as the best. The
kinetics for the lipid substrate MGIcDAG exhibited cooperativity according
to Hill. At MGIcDAG above 3 tool% the rate of synthesis was constant, and
the DGIcDAG synthase Vmaxand Hill coeff, were more affected by lipid
activator (PG) amounts than by the lipid substrate at physiological
concentrations of the lipids. The enzyme was also shown to be sensitive to
curvature "packing stress" changes, i.e. was stimulated by various
nonbilayer-prone lipids but inhibited by certain others. With the two
purified MGIcDAG and DGIcDAG synthases reconstituted together in the
presence of a potent aonbilayer lipid, the MGIcDAG/DGIcDAG amounts
synthesized mimicked the compensatory responses in vivo.
Different metabolic phosphates could supplement PG, depending on type,
amount and valency. Actwation involved a conformational change of the
enzyme. Especially efficient were glycolytic key intermediates fmctose-l,6bisphosphate and ATP, active at cellular concentrations on the DGIcDAG,
but not on the MGlcDAG synthase. Potencies of different
phosphatidylinositol (foreign lipid) derivates correlated wlth numbers and
positions of phosphate moieties. Selective stimulation of the DGIcDAG, but
not the MGlcDAG synthase, by minor amounts of dsDNA corroborated
these findings. The latter were also in support of two types of activator sites
on the enzyme: (i) lipid-phosphate ones close to the membrane interphase,
and (ii) soluble-(or particulate) phosphate ones further out. Thereby,
regulation of the nonbilayer (MGIcDAG) to bilayer (DGlcDAG) lipid
balance can be integrated with the metabolic status of the cell, and
potentially also to membrane and cell division.

s142

Abstracts FEBS'99

13.2 Receptors in the immune system

(Su/13.2/189)

Monocional antibodies against tumor necrosis factor alpha

(TNFa) and its receptors p55 (CD120a) and p75 (CD120b)
in clinical diagnostics and therapy

Role of Ser 332 and Ser 334 in C5aR phosphorylation and

involvement of Ser 338 in C5aR desensitization

V. Bojani~, K. Hartman Pretnar, R. Rupreht and V. (~urin (~erbec

T. Christophe. M.D. Milcent, M.L Rabiet, M. Tardif and F.

Boulay

Blood Transfusion Centre ofSlovema, SI- 1105 L)ubljana, Slovenia

BBSI/DBMS.'CEA Grenoble, GRENOBLE, FRANCE

Introduction: Receptors of human TNFct (p55 and p75) should play an

important role in cell recognition in different normal and malignant processes.
In one of our previous studies we showed that the serum concentration of
soluble receptor p55 in melanoma patients is a good prognostic factor for
determination of the disease progression.
Materials and methods: Mouse monoclonal antibodies (MAbs) against
TNFu and p55 were prepared and ELISA tests, which determine a total TNFct
(free or in a complex with its receptors) and a total p55, were developed.
Mouse MAbs against p75 were also obtained. Serum samples of patients with
pulmonal infection, septic shock, virus infections, mammalian carcinoma and
ovarian carcinoma were measured with the already mentioned ELISAs to
determine the concentration of TNFct and its soluble receptor p55. MAbs
against both TNFa receptors (p55 and p75) were used in FC (flow cytometry)
analysis in order to measure both receptors bound to the cell. In vitro tests on
murine fibroblastoid cell line L-929 and in vivo tests on brown mice were
used to determine whether some of MAbs against human TNFct are able to
neutralise its cytotoxic effect.
Results and conclusions: It was shown in the present work that there is no
significant correlation between the serum concentration of TNFct and its
soluble receptor p55 in pulmonal infection, septic shock, virus infection and
mamalian nor ovarian carcinoma. On the other hand, the serum concentration
of TNFct and p55, measured by our ELISA is elevated in metastatic
melanoma patients. The values of TNFct in the serum of these patients
showed fluctuations during the treatment, whereas the concentration of p55
remained almost the same all the time.
FC analysis with MAbs against p55 and p75 on different types of cells
provides us with an additional information about receptors p55 and p75,
bound to the membrane.
In vivo and in vitro tests showed that one of our anti-human TNFct MAbs can
neutralise the cytotoxic effect of TNFa in a molar ratio 1:1 and can be
humanised in order to be used as a therapeutic agent in some diseases where
the action of TNFc~ is harmful.

(Su/13.21191)

(Su/13.2/190)

Activation of Lymphocytes and Macrnphages by

Carbohydrates Containing Fungal Extracts
H. Fan a, J. S. Zhang ab, Y. X. Li ac, R. Reutter"
" lnstitutfur Molekularbtologie undBtochemte, Freie Universttat
Berhn, Arnimallee 22, 14195-Berhn-Dahlem. Germany
Shanghai Academy of Agricultural Sciences, China
Nanjing Agricultural University, China

It was known that carbohydrate complex can influence the immune

activity [ 1]. Traditional Chinese Medicine has long experiences to use
fungal extracts, which contain high amount of carbohydrates, to improve
the immunoactivity of tumor patients [2, 3]. In order to determinate the
effective compounds and to understand the mechanisms of such fungal
extracts three fiangals: Coriolus versicolor, Ganoderma lucidum and
Lentinus edodes were extracted and fractionated using different
chromatographic methods. The activation of lymphocytes and
macrophages was investigated after treatment with such extracts and
their fractions in vivo and in vitro. Our results show that the
proliferation of lymphocytes from both mouse spleen and human
peripheral blood were significantly stimulated by all three extracts and
some of their fractions. It is remarkable that the lymphocytes from
Sarcorma180-bearing mice are more sensitive to the stimulation of fungal
extracts than that from normal mice. The activity of peritoneal
macrophages was also increased after stimulation by special fractions.
Flow cytometric analysis of lymphocyte subsets shows that
percentages of CD7I and CD19 positive cells increased from 10% to
50% and from 11% to 40% respectively, after stimulation with one of
the fractions. The percentage of CD8 positive cells increased only
slightly, whereas of CD4 positive cells reduced. These results suggest
that the three fungal extracts used in Traditional Chinese Medicine
stimulate the proliferation of lymphocytes and activity of macrophages.
One of the fractions shows a capacity to activate B cells.
Reference:
[1] Pilatte, Y. et al., Glycobiology, 3, (1993) 201
[2] Ng, T. B. Gen Pharmacol. 30, (1998) 1
[3] Matsuoka, H. et ah, Anticancer Res. 17, (1997) 2751

The C5a receptor (C5aR) is a 7 transmembrane domain receptor coupled to

a G, protein which mediates chemotaxis and activation of myeloid cells
during inflammatory responses. Persistent stimulation leads to attenuation
of cell responses, a process termed desensitization. Upon stimulation, C5aR
is rapidly phosphorylated on six serines in the intracellu[ar C-terminal tail
[ 1], internalized, dephosphorylated and recycled to the cell surface [2].
To determine the influence of receptor phosphory[ation on desensitization,
a series of Ser to Ala mutant receptors have been analysed after transient
expression in COS-7 cells. The mutant receptors SA332-334. SA334-338
and SA332-334-338 have lost their ability to be phosphorylated after
stimulation by C5a. Mutations of these serines in aspartic acids restore the
ability of the C5aR to be phosphorylated. Nevertheless the individual
mutation of each serine in alanine did not affect the receptor
phosphorylation. These results show that the phosphorylation of C5aR is a
hierarchical process and that serines in position 332 and 334 are
phosphorylated first. These mutant receptors were stably expressed in
promyelocytic HL60 cells and the influence of the mutations on the C5aRinduced signal transduction was tested. The unphosphorylated mutant
receptors induced a calcium mobilization which lasted 2-3 times longer than
the wild type receptor. This phenotype was also observed for MAP Kinase
and NADPH oxidase activities. These results indicate that the
unphosphorylated receptors have lost their ability to be desensitized.
Moreover, this loss has a clear influence on the respiratory burst, a distal
cellular response. Surprisingly. the phosphorylated SA338 mutant receptor
cxhibited the same phenotype as unphosphorylated receptors. This serine
could play a crucial role in the interaction of the receptor with an adapter
protein such as arrestin, leading to the uncoupling of the G protein.
These results assign for the first time a specific role for some serine residues
in the C5a receptor phosphorylation/desensitization process.
[1] Giannini and al., J.B.C.. 270 (1995) 19166. [2] Giaanini and aL, J.
Immuno., 154. (1995) 4055.

(Su/13.2/192)

Structure and binding of a killer cell lg-like receptor

K. Maenakaa, b, T. Juji b, J. Wyer c, G. Gao c, T. Maenaka a,
N. Zaecai a, P.A. van der Merwe d, D.I. Stuart a, E.Y. Jones a
aLaboratory of Molecular Biophysics, Clnstttute of Molecular Medwine,
John Radcliffe Hospital and aSir Wilham Dunn School of Pathology,
University of OxJbrd, Oxford U.K. bJapanese Red Cross Central Blood
Center. Shtbuya-ku, Tokyo 120. JAPAN.

Human natural killer (NK) cells express a repertoire of killer cell

tmmunoglobulin (Ig) receptors (KIRs) with two or three tandem Ig domains
m their extracellular regions. These KIRs acnvate or inhibit NK cell
cytotoxiclty following recognition of the MHC class I molecules on target
cells. Different two domain KIRs (KIR2Ds) recognise distinct subsets of
HLA C alleles. Here we use surface plasmon resonance to study the
interactmn of two KIR2D molecules, KIR2DLI and KIR2DL3, with a variety
of MHC class I molecules. Soluble recombinant KIRs and MHC class I
molecules were expressed in E. coll. K1R2DL3 bound to HLA-Cw7 with a
1'1 stoicbiometry and an affinity (Kd - 7 p.M at 250C) within the range of
values measured for other cell-cell recognition molecules, incIuding the T cell
receptor (TCR). More surprisingly, KIR2DLI also bound HLA-Cw7, albeit
with a ten fold lower affinity. Neither K1R2D molecule bound to HLA-A2, B35, -E, or -G. As with conventional cell-cell recognition molecules the low
affimties were a consequence of fast dissociation rate constants (k~, >1 s ~),
whereas the association rate constants were unremarkable (k -10 M'.s').
The binding of KIR2DL3 and KIR2DLI to HLA Cw7 was strongly
influenced by the nature of the peptide presented by HLA-Cw7. Recent
studies have shown that TCR/peptide-MHC interactions, unlike other cell-cell
recognition molecule interactions, are characterised by slow kinetic and
highly unfavourable entropic effects. In contrast, we show that the
KIR2DL3/peptide-HLA Cw7 interaction has fast kinetics and a favourable
binding entropy. Thus whereas the TCR and KIRs both show allele- and
peptide-specific MHC recognition, they bind with very different
thermodynamic and kinetic properties. Finally, we discuss the structurefunction relationship of KIR2Ds in the context of the recently solved crystal
structure of K1R2DL3 [ 1].
[1] Maenaka K. et al., Structure, in the press.

Abstracts FEBS'99

(Su/13.2/193)

s143

Gene expression and memory effects in T cells at low g

M.A. Meloni, P. Pippia, G. Cossu, F. Mannu, F. Turrini.,
1. Walther*, M. Schwarzenberg*, M. Cogoli*, A. Cogoli*.

(Su/13.2/194)

Dip. Scwnze Ft~tologiche. Biochtmiche e Cellulart; Vta Murom 25,

07100 Sassart, Italy. * Space Biology Group, ETH, Zurwh, Switzerland

Several investigations performed in space (Spacelab) and in simulated

microgravity have shown that in vitro activated T lymphocytes lose 80-90%
of their proliferating ability. It's very important to understand whether this
effect is due to a direct and specific interaction of the g forces with cellular
structures and functions or rather to metabolic effects arising from changes of
convection, sedimentation and other. Since upon interaction between
interleukm-2 (IL-2) and its receptor (IL-R) the full activation of T cells is
triggered, we have focused our effort on the gene expression of cytokines
and on "memory effects", using a new tridimensional clinostat (RPM) to
simulate micrograwty. The mRNA quantification of T cells was performed by
extraction in guanidinium tiocyanate and purification by a sylica based resin.
RT-PCR was performed according standard procedures. Amplified DNA
obtained from interleukins mRNA was referred to the amplified DNA
obtained from actin mRNA. We observed that simulated microgravity causes
a marked inhibition of genetic expression of IL-2 and alfa subunit of its
receptor (IL-2-R-c0 and an enhance of IL-I expression. The expression of
IL-2 and IL-2-R-Gt was delayed of 6 hours compared to lymphocytes control.
Levels of other mRNA such as actin, IL-2-R-]3 and IFN-~, were not affected
by simulated microgravity in&caring an apparent selective inhibitmn of IL-2
and IL-2-R-et.
Prehminary experiments on "memory effects" show that T lymphocyte
cultures pre-incubated without Con A for 24, 48 and 72 h at simulated
microgravity, followed by incubation for 72 h at 1 g in the presence of Con A,
have the same level of activation as their respective controls kept for 24, 48
and 72 h at 1 g without Con A, followed by incubation for 72 h at 1 g with
Con A. As expected, the activation was remarkably inhibited in a second set
of control cultures kept 24, 48 and 72 h in the RPM without Con A, followed
by incubation for 72 h in the RPM with Con A. These findings permit us to
conclude that despite prolonged exposure to simulated microgravity, T cells
do not lose their capacity to react to mitogens. The existence of "windows of
sensinvity" after delivery of the activation signal cannot be excluded,
however. The search for windows of sensitivity is now in progress in our
laboratories.
(Su/13.2/195) Binding between Aspergillus f u m i g a t u s
human Langerhans cells
F. Persat t, S Picot I, D Schmitt 2, C Vincent 2

eonidia and

ILaboratotre de Parasttologte, M vcologie m~dtcale et Pathologic

Exotique, Umversttb Lyon I. ~"INSEI~I Umt~ 346, Lyon France

Langerhans cells are immune cells present in human epithelia After

antigen contact, they differentiate and mxgrate towards lymph nodes in
order to activate naive T lymphocytes. A s p e r g t l l u s f u m i g a t u s is a fungus
responsible for different human diseases, such as invasive pulmonary
aspergillosis that is an increasing cause of morbidity and mortality in
immunosuppressed patients A. f u m t g a t u s conidia are inhaled and arrived
in contact of pulmonary epithelium where they can meet Langerhans
cells. The aim of this study was to analyze the binding of conidia on
Langerhans cells. Conidia were labeled with fluorescein isothiocyanate,
Langerhans cells were differentiated in vitro from human cord blood
CD34 progenitors upon 10-13 days of culture with GM-CSF, TNFe~ and
TGF]3. Differentiated cells contained 65-90 % of cells with the
characteristic Langerhans cell phenotype Binding of conidia on
Langerhans cells was analyzed by FACS and by direct fluorescent and
phase contrast microscopy. Binding was observed in a very short time
interval and increased with the contact time The percentage of labeled
cells and the amount of bound fluorescence per cell were directly related
to the ratio of conidia to cells. The binding was more important with
Langerhans type cells than with other cells of the culture. Evidence of
conidial fixation was confirmed by optical microscopy, conidia being
fixed on the cell body and also on dendrite extremity. The binding
specificity was studied by addition of various carbohydrates and
monoclonal antibodies against known receptors

Interactions of Leukotriene B4 with its

Detergent-solubilized Recombinant Receptor
J. Parello, J.-L. Ban~res. D. Durand, P. Hullot, L-P. Girard
ESA 5074 CNRS. 15 avenue Ch. Flahault, 34060 Montpellier, France

Leukotriene B4 (LTB4) is a potent chemoattractant that is primarily

involved in inflammation, immune response and host defense against
infection. Recently, the cloning of the complementary DNA encoding a
cell-surface receptor of LTB4 that is highly expressed in human leukocytes
has been reported [1]. The amino acid sequence of this protein (BLTR) is
suggestive of a G-protein-coupled receptor with seven transmemhrane
helices. We have cloned and expressed human BLTR in E. coll. This
allowed us to purify this 352-residue receptor as a detergent-solubilized
protein. This protein displays about 45% of c~-helical residues, as assessed
by circular dichro'fsm, in agreement with the presence of seven potential
seven transmembrane helices. A circular dichroism analysis of the
interaction between BLTR and its ligand demonstrates that the receptorbound LTB4 molecule adopts a skewed conjugated polyene geometry
within the 1:1 ligand - receptor complex. A fluorescence analysis with the
receptor containing two tryptophany[ residues shows that at least one of
these Trp residues interacts with the ligand through a hydrophobic contact.
A parallel study by circular dichroism and fluorescence of the interaction of
a synthetic LTB4 antagonist with the detergent solubilized receptor shows
similar results as those observed with the natural ligand LTB4 thus opening
the possibility to use the solubilized receptor as a suitable tool for screening
a vanet~ of synthetic antagonists and agonists in a pharmacological
perspectwe.
[1 ] Yokomizo, T., et al. Nature 387 (1997), 620-624.

(Su/13.2/196)

A new receptor from murine medullary thymocytes

identified with Amaranthus leucocarpus lectin
F. Porras',S. Martinezb, M.C. Jim~nez, M. Linares~, P. Hemfindez,~ and B.
Ortizc, aDep.BioquimicaFac. de Medicina UNAM, P O Box. 70159 M~xico
D F, bCentro MedicoNacional SXXIM~xico.CDep.Bioq. INER,SSA Mex.

Mucin-like molecules in the membrane of lymphocytes has been shown to

play a relevant role in cell communication and activation. In previous works
we have identified that the GalNAc specific lectin Amaranthus leucocarpus
(ALL) induces immunosuppression and recognizes routine and human
lymphocytes in the last stages of maturation. In this work we report the
characterization of the ALL receptor from murine medullary thymocytes,
aimed at identify the role of such receptor in maturation. The ALL thymocyte
receptor was purified using a complex with the biotin-labeled lectin and
avidin-agarose as the affinity matrix. The receptor is a 70 kDa glycoprotein
that contains 20% of sugar by weight. The receptor is mainly composed by
ASX, GLX, SER and PRO. In its glycannic portion is mainly composed by
Gal, GalNAc and NeuAc, as a typical O-glycosidically bonded glycoprotein.
The ALL-thymocyte receptor is composed by three isoforms, which possess
similar amino acid composition but shows slight differences in sugar
concentrations. The purified receptor as well as the purified isoforms
possesses the amino terminal blocked. The ALL receptor does not cross
react with specific antibodies against other lymphocyte markers which
contained O-glycans such as CD43. For this reason we considered that the
purified receptor should be considered, an specific marker for naive T cells.
ACKNOWLEDGEMENTS
This work was supported in part by CONACyT (26068-N), DGAPAUNAM (PAPIIT-IN224598) and ECOS Mexico-France (M97B05).

s 144

(Su/13.2/197)

Abstracts FEBS'99

Integrationof TeR V-REGION structural data into IMGT

M. Ruiz 1, G. Mennessier 2, V. Giudicelli l, D. Chaume i
and M,-P. Lefranc t
11MGT, UPR CNRS 1142, 1GH, 141 rue de la Cardonille, 34396
Montpellier Cedex 5, France, 2 UMR 5825 CNRS, Universit~ Montpellier 11

IMGT, the international lmMunoGeneTics database 0attp://imgt.enusc.fr:8104)

is an integrated database speeialising in Immunoglobulins (Ig), T cell
Receptors (TcR) and Major Histoenmpatibility Complex (MHC) o f all
vertebrate species, created in 1989 by Marie-Paule Lefranc, Montpellier II
University, CNRS, France. IMGT comprises alignment tables and expertly
annotated sequences. One o f the IMGT goals is to establish the first common
data access to all structural data concerning the TcR. The antigen binding site
o f the TeR is located in the variable regions (V-REGIONs). In a first approach,
we retrieved and analysed the TcR V - R E G I O N data from PDB, in order to
provide 2D and 3D representations according to IMGT standardization rules.
The identification o f the genes and alleles expressed in the TcR V-REGIONs
was performed using the multiple sequence alignment program CLUSTAL W
and the IMGT reference directory. These genes and alleles were described with
the IMGT gene and allele name nomenclature and according to the IMGT
unique numbering. This numbering allows a standardized delimitation o f the
loops involved in the antigen recognition (CDR or Complementarity
Determining Region) and facilitates comparison with other sequences whatever
the antigen receptor (Ig or TcR), the chain type, or the species. For each TcR
V-REGION, whose three-din'tensional structure has been determined, we
propose 2D representations designated as Collier de Perles. These
representations provide information on the amino acid positions in the 13strands and loops o f the variable domain and allow to quickly visualize amino
acids which are important for the structural configuration o f the V-REGION.
The IMGT unique numbering o f the amino acids has been applied to the 3D
data from PDB. R A S M O L 3D representations o f TcR V-REGIONs displayed
according to the IMGT numbering, are available from the IMGT repertoire at
the Marie-Paule page.
Our approach gives access to standardized descriptions o f exhaustive and
expertised data c t n c e r n i n g the TcR V-REGIONs. This standardization allows
to extract deduced knowledge from the IMGT database and to realize
sequence-to-structure studies. The coherence between different information
improves the data accuracy. This study is an advancement in the increase o f
interoperability between genomic and structural databases.

(Su/13.2/199)

Influence of whole body hyperthermia on

LPS-receptor expression in men
M. Zellner,N. Hergovich,A. Spittler,R. Oehler,B. Jilma,
E. Roth
Department of Surgery, Research Laboratories.
Department of Clinical Pharmacology,
University of Vienna, A-1090 Vienna, Austria

B A C K G R O U N D : Hyperthermia protects animals against the lethal effects of

pathogens and reduces significantly tumor necrosis factor-~ (TNF-e0 release
during endotoxemia. The C D I 4 molecule has high affinity to LPS and is
involved in responses of many other microbial pathogens. Recently CD62L, an
adhesion molecule which mediates the first step of extravasation, was also
reported as a signalling receptor for LPS. Here we investigated the influence of
whole body hyperthermia on LPS - receptor expression of human blood
leukocytes. METHODS: In a randomised, prospective cross over study six
healthy male and six healthy female volunteers were immersed in a hot water
bath (water temperature 39.5C) for 2h at 8:00 am, which increased body
temperature by 3C (-+ 0.5C). Blood samples were drawn just before,
immediately after and at 2h, at 6h and at 22h after the hot water bath. One week
later they served as their own controls. RESULTS: In the control group we
found significant diurnal variations of CD62L and CDI lb on monocytes with a
minimum of CD62L (P < 0.002) and a maximum of CDI lb (P < 0 . 0 2 )
expression at 1:00 p.m. CD14 on monocytes showed a tendency to peak at
noon. Immediately after the hot water bath C D I 4 and CD1 lb expression on
monocytes were elevated with a further increase at 6 h; (P < 0.001 ). In contrast
CD62L decreased after the hot water bath and a nadir was reached at 6h
(P < 0.001). CONCLUSION: These results show a significant influence of
heat on LPS - receptor expression of leukocytes. The different pattern of LPS receptor expression could be the reason for the reduced release of tumor necrosis
factor-a (TNF-a) after whole body hyperthermia. Furthermore the diurnal
variations stress the importance of sample time point standardization in clinical
studies.

(Su/13.2/198)

Prolactinsynthesisin humansalivaryglandsfrompatientswith
Sjilgren'ssyndrome
S. Steinfelda, Ch. Francoisb, S. Rommesb, D. Hoyauxb, R. K~ssb, R Pochetb
~Service de Rhumatologie, Hopital Erasme ,
hLaboratoire d'Histologie, Facult~ de M~decine U.L.B.. 1070
Brussels, Belgium

Sj/Sgren's syndrome (SS) is an autoimmune disorder characterized by lymphocytic

infiltration into salivary and lacrimal glands with concomitant tissue destruction.
Prolactm belongs to the cytokine superfamlly of the helix bundle peptide hormones and
its receptors are genetically a part of the cytokine-hematopotetin receptor superfamily.
Prolactm and prolactin-like pepades are able to stimulate humoral and cell mediated
immune responses and may have a role m the pathogenesis of various autolmmane
diseases [2]. Here we present evidence that prolactln-like proteins are synthensed in
labial salivary glands from panents with SS.
Immunohistochemistry was performed on sections prepared from human labial sahvary
glands biops,es fixed m buffered paraformaldehyde. Prolactin-tmmunoreactivity was
seen in minor salivary glands from SS but not m minor salivary glands from normal
subjects. Labelling was conspicuous in the cytoplasm from several mucous acinar cells.
Inununoprecipitation was performed after 8h incubation with 35S-methionine of fresh
human salivary glands obtained the same day from normal and SS patients. Human
tissue were further homogemzed and the 15,000g supernatants were incubated with antiprolactm. The inmaune complexes were precipitated with Protein G Sepharose and
electrophoresed on 10% SDS-PAGE acrylamide gels.
Autoradiography of the
electrophoresed immune proteins from SS salivary glands revealed a major band of
proteins around 60 Kda and another band at 16 Kda whereas barely detectable
radioactive bands could be seen in lmmunoprecipitates from normal patients although
the radioactwlty incorporated into the total protems of normal and SS glands was
similar. The 60 kDa protein may correspond to the big prolactin described by Shoupe
et al. [3] and the 16 kDa to the N-terminal fragment of prolactin also called anuangiogenic factor [1]. This result demonstrates that in salivary glands from SS patients.
prolactins-like pepudes are mdeed synthetized and provide further evidence for a
potential role of prolactins in thin aotoimmune disease. Interestingly, in SS salivary
glands we have also detected by immunohistochemistry PRL receptors and S-100A6
protein (a PRL receptor binding protein). Both were not detected m normal human
salivary gland.
Thts work was financially supported by a grant from FRSM.
References
[1] Clapp C et al., Endocrinology 133, (1993) 1292.
[2] Neidhart M, Proc Soc Exp Biol Med 217, (1998) 408.
[3] Shoupe D et al., Am J Obstet Gynecol 147. (1983) 482.

Abstracts FEBS'99

s145

16.2 Metal-assisted catalysis

(Su/16.2/200) Zinc dependence of a recombinant alcohol dehydrogenase
Glynis A. Robinson & Christopher J. Bailey

(Su/16.2/201)

Department of Biochemistry , Trinity College, Dublin 2, Ireland

First evidence for the presence of a hydrogenase in the

sulphur-reducing bacterium Desulfuromonasacetoxidans
M. Brugna t, R. Toci2, W. Nitschke I, M. Bruschi1& M.-T.
Giudici-Orficonil,tBIP, 2LCB, LB.S.M.-C.N.R.S., 31 chemin J.
Aigu~er, 13402 Marseille cedex 20, FRANCE

The adh gene of Bacillus stearothermophilus DSM 2334 has been

cloned and sequenced [1]. A gene for expression in E. colt was
synthesised by PCR amplification. The lkb BspHI - Xhol gene contains
an ATG codon for the initiator methionine and a suitable cloning site at
the 3' end of the structural gene. The gene was cloned into a plasmid
pET21d [2] that had been restricted with NcoI and Xhol, and expressed
in E. colt BL21(DE3)pLysS. Alcohol dehydrogenase activity was
produced in the cells in an IPTG-dependent manner. The ADH
polypeptide was the most abundant protein in the cells at optimum
expression. The identity of rADH and native protein was confirmed by
enzyme assay, amino acid sequence and gel electrophoresis.
However the specific activity of the protein was lower than expected.
Purified rADH contained about 1 Zn2+ per mol ADH and had a specific
activity of about 0.6 U mg-1. Preparations were notably unstable. In
vitro addition of zinc did not increase the specific activity.
Further investigation demonstrated that zinc supplementation of the
cultures increased ADH activity by up to four-fold. After optimal
supplementation of the culture medium with zinc, pure, stable rADH
contained about 2 Zn2+per mol ADH and had a specific activity of 2.3 U
mg-1, slightly higher than the value of 2.1 U mg-1 recorded for the
native enzyme[3].
It is concluded that overexpression of this protein can overwhelm the
ability of E. colt to provide sufficient zinc co-factor, unless culture
conditions are suitably adjusted.
1

G.A.Robinson et al. Biochim. biophys. Aeta 1218,( 1994),432.

F.W. Studier et al. Methods in Enzymology 185, (1990), 60.

M. Sheehan et al. Biochemical Journal, 252, (19gg), 661.

Hydrogen metabolism plays a central role in the energy generating

mechanisms of various micro-organisms and hydrogenases have been
characterized in a wide range of anaerobic and aerobic organisms.
Despite the fact that hydrogenases, ubiquitous in sulphate reducing
bacteria, were thought to be absent in the strict anaerobe sulphur respiting
bacterium Desulfuromonas acetoxidans, we have detected and purified this
enzyme for the first time in this micro-organism. The enzyme, composed of
two subunits of 60 kDa and 30 kDa, requires activation by hydrogen and
dithionite in order to express its activity. The characterization of the enzyme
(by enzymatic assays and EPR spectroscopy) strongly suggests that it belongs
to the class ofNi-hydrogenases [ 1].
In the sulphate-reducing bacteria belonging to the Desulfovlbrio
species, a model for periplasmic electron transfer from oxidation of molecular
hydrogen by hydrogenase to the cytoplasmic membrane electron transport
system has been recently proposed via low-potential, multiheme, c-type
cytochromes [2,3]. D. acetoxtclans possesses several multiheme and
periplasmic cytochromes, specialy the triheme cytochrome c7 which presents
homologies with the cytochrome c~ from Desulfovibrio. In order to
determined the role of the hydrogenase in the metabolism of D. acetoxidans,
its reactivity with polyheme cytochromes has been examined. The first results
show that the reduction of the cytochrome c (Mr 50000), that we isolated
from the same bacterium, is very inefficient compared to the reduction of the
cytochrome c7. Experiments are in progress to elucidate the electron
transport patways involving the hydrogenase and the role of this enzyme in
the metabolism of this sulphur-reducing bacterium.
[1] Brugna et at., submitted for publication (1999).
[2] Pereira et at., J.B.I.C., 3, (1998) 494.
[3] Aubert et at., submitted for publication (1999).

(Su/16.2/202)

Metal ion dependent conformational changes of

alkaline phosphatase
V. Bu~,evid-Popovid, S. Orhanovid, M. Pavela-Vran~i6
Faculty of Natural Sciences, Mathemattcs and Education, Teshna 12,
Croatia

Alkaline phosphatase (PhoA) is a multimeric metalloprotein catalyzing

phosphohydrolytic and phosphoryl transfer reactions. It is present in the
periplasmatic space of prokaryotes or associated with the outer side of the
plasmatic membrane in eukaryotes, suggesting its involvement in phosphate
transport as a possible biological role of alkaline phosphatases. According
to the crystallographic study, PhoA from E. colt is composed of two
apparently identical subunits. On the other hand, a vast amount of data
exists indicating a non-equivalence of the PhoA subunits, including Pi
.
.
.
.
.
.
.
. saturation wtth M g2+,
binding
wtth
strong
negative
cooperattvtty,
unequal
biphasic thermal inactivation and deviations from Michaelis-Menten
kinetics substantiated by enzyme asymmetry. These data suggest
conformational heterogeneity between the subunits. It has been reported
that PhoA from E. coli is not fully saturated with metal ions. Unequal
saturation of the subunits could induce the observed enzyme asymmetry.
The dependence of the kinetic constants and the deviations from MichaelisMenten kinetics on the Mg2+-ion concentration has been investigated. We
have examined the possible structural differences between the subunits
using limited proteolysis with trypsin as a probe of conformational changes.
Susceptibility to proteolysis was studied in solutions containing different
concentrations of metal ions and a metal-complexing agent. A variable
kinetic behaviour of PhoA was observed. The progress of the proteolytic
reaction was monitored by SDS-PAGE and the PhoA activity assay. The
proteolytic pattern and the time course of tryptric digestion were dependent
on the concentration of metal ions in the reaction solution. We conclude
that binding of metal ions to PhoA induces conforraational changes in the
protein that alter its susceptibility to proteolysis and influence its enzymatic
activity.

(Su/16.2/203)

The HELLGH Motif in Rat Dipeptidyl Peptidase I11 is

Involved in Zinc Coordination and Catalytic Activity
K M Fukasawa a, K Fukasawa a, M Harada a
H lwamotob, J lq~roseb
aDep. of(hal Bu~chemt.stl~ 3[atsumoto ])ental ~ m~ Shtojtrl .lap~m
bDep of.4pphed Btologrcal Sctence Faculty of l~ngtnteerm~z
Fukuvama (my l'ukuvama. Japan

The role of the HELLGH (450-455) motif m the sequence of rat d~peptidyl
pept~dase ill (EC 3 4 14 4) was investigated by replacing Glu with Ala and
His with Tyr by site-directed mutagenesis
The cDNA were expressed in
Escherichia colt, and the resulting recombinant proteins named H450Y,
E451A and H455Y were purified to apparent homogeneity
Non~ of the
expressed mutated proteins showed peptldase actwity
Howevt~r, the
E451A contained one tool of zinc per tool of protein, and the H450Y and
H455Y, did not contain significant amounts of zinc, as determined by
atomic absorpUon spectrometry
These results prowde evidence that the
two histxdines in the HELLGH motif are zinc binding resadues and the
glutamtc acxd is essential for the en~me activity
The Leu453-deleted mutant which are same as "zmcin" motif conserved
among the M1 famxly of metallopeptidases had almost the same order of
binding affimty (for Arg-Arg-2-naphtylam~de) as the wild-type enzyme,
but the specificity constant was at about l0 %
By a search of the NBRFPlR protein sequence data base. three kinds of monooxygenases (tyrosine.
phenylalanine, and tryptophan hydrooxylases) had m common the
HExxGH motif, whmh was nearly the same as that m rat DPP II1
(HELLGH) In the case of HELLGH motif m phenylalanine hydrox2lase,
two histidine resxdues and a remote glutamtc aod (His285. His 290 and
Glu330) were identified as iron-binding hgands to the acnve-site aron by the
s~te-d~rected mutagenesls and X-ray crystallography
The actxve site
res,dues known by crystallographic analysis in thermolysme (HExxH)
superimposed onto these m tyrosme hydrooxylase (HExxxH)
In spne of
the changing of the motffofthe zinc binding site IHExxtf) to that of the ~ron
binding site (HExxxI-t), the arrangements of the residues m both acnve sites
were ver~ s~mdar Therefore, the tertiary structure of the HELLGH motif
m DPP Ill may be similar to that of the "zmcm" mot:f

s 146

(Su/16.2/204)

Abstracts FEBS'99

Pteridine derivatives: their function as catalysts

cleaving the porphyrin
R. Horejsi, G. Reibnegger
Medical Chemical Institute and Pregl- Laboratory. University
Graz. Harrachgasse 21/11. 8010 Graz, Austria

Pteridines occur ubiquitously in many cell types, and their

biological functions are manifold and not completely understood.
Different features in pteridine structures correlate with their modulating
efficacy on processes induced by radicals in several experimental
designs: aromatic pteridines like neopterin and biopterin enhance
radical-induced processes, while reduced derivatives such as
dihydroneopterin or tetrahydrobiopterin were found to be scavengers of
nascent radicals [1, 2].
Reduced pterins, especially the tetrahydro-compounds, act as
electron donors to the porphyrin system in hemoglobin, myoglobin,
cytochrome c and cytochrome P450. Moreover, degradation of the
porphyrin ring by hydrogen peroxide was observed to be partially
prevented by tetra- and dihydropteridines, while aromatic pterins
showed no influence on radical-induced porphyrin degradation.
However, the incubation of the porphyrins with reduced pterins
alone caused the cleavage of the porphyrin ring system yielding
subsequently carbon monoxide. This result is explainable by the most
recently detected generation of hydroxyl radicals via superoxide anion
radicals, catalysed by reduced pteridines in air-saturated solutions. Our
observations may be relevant for explaining anemia in chronic immune
stimulation, because neopterin and 7,8-dihydroneopterin are produced
and secreted by human macrophages activated by interferon-'/.
References:
[1] R. Horejsi et al, Free Radic Biol Med 21(1996), 133
[2] G. Reibnegger et al, Free Radic Biol Med 18(1995), 515

(Su/16.2/206)

Complex between Acetohydroxyacid Isomeroreductase,

ADP-l~bose, reaction product and various divalent cations,
K. Thomazeau ~, R. Dumas ~, V. Biou ~
+Institut de Biologic Structurale, Grenoble, France.
Unit~ Mixte CNRS- Rhrne Poulenc. Lyon. France.

Acetohydroxyacid isomeroreductase catalyses a 2-step reaction composed of

an alkyl migration followed by an NADPH-dependent reduction. Both steps
require a divalent cation, and the first step has a strong preference for
magnesium. In order to observe the structure of the enzyme complexed with
its substrate, we tried to crystallise it in an inactive form. Manganese ions arc
highly unfavourable to the reaction, only 3% residual activity is observed in
the presence of this cation. Therefore, we crystallised acetohydroxyacid
isomeroreductase with its substrate, 2-aceto-2-hydroxybutyrate (AHB), Mn ~*
and NADPH.
The 1.6A resolution electron density map showed the reaction product (2,3dihydroxy-3-methylvalerate, DMV) and a density corresponding to ADPribose instead of the whole NADP +. The structure of this complex was refined
to an R-factor of 19.3% and an R-free of 22.5%. The overall structure of the
enzyme is very similar to that of the complex with competitive inhibitor
IpOHA. However, the active site shows some differences: the nicotinamide is
cleaved and the surrounding ammoacids have rearranged accordingly.
Kinetic studies were carried out with other cations such as Ni + and Zn** and
showed very low enzyme activity (0.02%) with respect to Mg 2+. Crystals
were obtained with these cations, but the collected data showed that the
reaction had ocurred. As with the Mn, the NADPH was dived and only ADPRibose was present in the active site. Mass Spectrometry measurements
confirmed nicotinamide cycle clivage [1 ].
Even wtth a very low residual acttvity, the time needed for crystallisation was
long enough to allow the reaction to be complete in the crystal.
[ 1] Thomazeau et al., submitted.

(Su/16.2/205)

Formation of oxygen radicals in solutions of

7-8-dihydroneopterin
K: Oetth T: Grammer, G. Reibnegger
Medical Chemwal ln.~tttute and Pregl Laboratorl
Umverst0 G'raz, Harrachgasse 21/]1 A-801O Gra:. Au~trta

Neopterin and 7,8-dihydroneopterin, two compounds

which are secreted by activated macrophages, have been shown to
interfere with radicals generated by cellular and different chemical
systems. Reduced pterins were reported to scavenge, whereas
aromatic neopterin was reported to enhance radical mediated
reactions. However, recently it was found that high concentrations
of
7,8-dihydroneopterin
promote
luminol
dependent
chemiluminescence and T-cell apoptosis, suggesting an
enhancement of free radical formation.
In our study aromatic hydroxylation was used for detection
of hydroxyl radicals. It is shown that in solutions of 7,8dihydroneopterin hydroxyl radicals were formed in the absence of
any radical source. The presence of EDTA chelated iron
enhanced, and addition of deferoxamine inhibited hydroxyl
radicaI formation. While additon of iron accelerated the
hydroxylation reaction, 7,8-dihydroneopterin was responsible for
the degree of hydroxylation without altering the kinetics. In the
presence of superoxide dismutase or catalase as well as by helium
purging hydroxylation was inhibited.
Our data suggest that in solutions of 7,8-dihydroneopterin
superoxide radicals are generated which are converted to hydroxyl
radicals by Fenton or Haber-Weiss type reactions. The detailed
effect of 7,8-dihydroneopterin is currently under investigation.

Abstracts FEBS'99

s147

Others, including Metabolism

(Su/oth/207)

Structure and regulation of the ~auman GGT gene

M. J. Accaoui-, A. Pawlak', N. Haddy', H. Leh I~. G.
Guellaen 2. M. Wellman 1, A. Visvikis 1.
Centre du M~dicament, 54000 Nancy, France. z Unit~ INSERM U99,
94010 Cr6tefl. France. ~: lnslitutGuslave Raussy,94009 Viltejuif, France

In human, five distinct mRNAs code for y-glutamyltransferase (GGT). Their

coding regions are identical but their 5' untranslated regions exhibit common
as well as mRNA type specific sequences. To elucidate the mecanisms that
generate these different mRNAs, we cloned the 5' region of the human GGT
gene and deduced its structure. By comparison to the GGT mRNA sequences,
the common regions of the 5' UTR can be devided in 5 exons, which in the
gene are localized within a 2.4 kb fragment. Four of the presumed exons are
not separated by introns but they are beginning or ending either with intron
donor or intron acceptor sites. According to this particular organization of
these exons we propose mecanisms of tissue specific splicing responsible for
the generation of these 5'UTRs. In the case of the major GGT mRNA in
HepG2 cells the specific splicing leads to the formation of a hairpin and loop
structure containing translational enhancers.
We then isolated a genomic fragment containing the most 5' sequences of the
major GGT mRNA in HepG2 cells. Primer extension analysis revealed one
major and three minor transcription start sites. Upstream the maior
transcription start site neither classical TATA nor CAAT boxes were tbund.
Instead, sites for AP1, AP2 NF1 and SP1 transcription factors were
identified. Chimeric plasmids, including the GGT promoter and the luciferase
gene were then transiently expressed in several established human cell lines.
Significant promoter activities were detected indicating that transcription
starts within this region. We also show that this promoter is regulated by API m conditions of oxidative stress.

(Su/oth/209)

CYCLIC NUCLEOTIDES IN CULTURED BPH SMC'S

P.I Adolfsson and S.P.S. Svensson
Division of Pharmacology,Departmentof Medicine and Care, Faculty of
Health Science, LinkSpingSwedenSE-581 85

Introduction
With a diagnostic level of 50%, benign prostatic hyperplasia (BPH) is
probably the most common affliction among men beyond 60 years of age.
Although cAMP is an established inhibitor of proliferation regarding both
airway- and vascular smooth muscle cells (smc's) only a few studies of
cAMP in cultured BPH smc's have been published. This investigation
mainly consider the effects of cAMP/cGMP on cultured BPH smc
proliferation.
Materials and methods
Prostatic tissue was obtained from transuretheral resection for BPH The
tissue was minced into lmm 3 explants and incubated in DMEM (5% FCS)
in 1130mm dishes. Before the beginning of an experiment the cells were
placed in serum-free medium for 24h. Proliferation was measured with or
without drugs using [3H]-thymidine incorporation during 24h incubation.
Results
The cAMP/cGMP levels were controlled by adenylyl cyclase (AC)
stimulation with forskolin and/or phosphodiesterase (PDE) inhibition with
IBMX or Zaprinast. Both Forskolin and IBMX produced by them self
significant inhibitory effects on the proliferation. However there was a
stronger effect when combining the drugs. When incubating cells with
Zaprinast, a selective PDE V inhibitor used to increase the cGMP level, an
inhibitory effect on [3H]-thymidine incorporation was received. Preliminary
results also suggest that there is a synergistic antiproliferative effect when
increasing both cAMP and cGMP levels.
Conclusion
Although Cq-adrenergic antagonists are known inhibitors of BPH growth, so
far there is no successful pharmacological treatment replacing transuretheral
resection. Our results show that pharmacological tools as PDE inhibitors
which increase cAMP/cGMP levels inhibit BPH smc proliferation, These
findings could offer a new pharmacological target to treat this affliction
among aging men.

(Su/oth/208)

The Effects of Different Foods and Nutrients on Super Oxide

Dismutase (SOD) Activity, Blood Glucose and Plasma Lipids
F Akurt. G, Biringen & M. LOker
TUBITAKAtRC, FSTRL p.O.Box 2L 41470Gebze. KocaelL Turkey

The effects of dietary factors on SOD activity was investigated on 28

healthy subjects (13 men and 15 women) between 20 to 48 years of age.
Vitamin E supplement, hazelnut, carrot and pure royal jelly were supplemented
to their usual daily diets for a period of 75 days. Blood samples were taken
from subjects both at the begining and the end of the study and analyzed for
Hb, SOD, total cholesterol, triglycerid, HDL, LDL, VLDL cholesterol and
fasting blood glucose levels. During the study, dietary records of the subjects
were recorded at 2 different times and energy and nutrient composition of the
diets were determined. The results showed that SOD activity was increased
significantly (p<0.05) for the subjects supplemented only with carrot. Although
there was no significant change of SOD activity found with the subjects treated
with the other supplements, the SOD activity of the male subjects supplemented
with vitamin E were significantly increased (p<0.05). There was no correlation
found between SOD, blood lipid parameters and glucose levels. A significant
positive relationship (p<0.05) was found between SOD activity and dietary
iron, calcium, zinc, vitamin B groups (Bj, B:, niacin, B6, Bl2, folio acid), 13carotene, vitamin A, E and C.

(Su/oth/210) Cell-cell interactions effect on chemosensitivity and invasive

properties of sensitive and MDR MCF-7 cells in spheroids.
M Affoue, J,C. Jardillier
Laboratoire de 131ochtmm. IFR 53. UFR Pharmacm. 51096 Reims. France

The occurrence of solid tumours spreading through the body is a major

concern for the clinicians Moreover, in numerous cases, metastases exhibit
a multidrug resistant (MDR) pattern This dual characteristic still remains
supported by few biological explanations. The purpose of our study was,
first, to compare drag sensitivity and invasive properties of sensitive and
MDR MCF-7 cells. Spheroids were chosen as an experimental model, since
they exhibit a number of characteristics (i.e. three-dimensional structure)
close to the growth of an in vivo tumour [1] Then to be respectful with the
tumoral structure we have to take into account the heterogeneity of tumoral
cell subpopulations [2]. Therefore, mixed spheroids, made from sensitive
and MDR cells in various proportions, were studied. Surviving fractions
after doxorubicin treatment and invasive properties of such spheroids were
greater than expected. This might be due to the cell-cell interactions that
facilitate molecular exchanges and are linked to muhicellular resistance The
decrease of those phenomenons with Rp-cAMP, an antagonist of cAMP
confirmed the importance of intercellular communications in tumoral
progression.
[1] : A Leoniet al., Int. J. Exp Path., 79, (1998) 1.
[2] G H. Heppner etal., Semin. Oncol., 16, (1989) 91.

s148

Abstracts FEBS'99

(Su/otld211) The Effect ofOestrogen Theraphy on Lipid Peroxide and

Antioxidative System in Post-menopausal Women.
Ak~a~ T. DmqcrY. ColgarU. Oral E

A Short term evaluation of 6 months oestrogen theraphy in 38 postmenopausal women was conducted The level of serum lipid
peroxidation and the status ofglutathione (GSH) and glutathionerelated enzymes were evaluated before and after 6 months oestrogen
treatment
At 6 months evaluation, oestrogen treated post-menopausal women
showed a significantly increased concentration ofmalondialdehyde
(p<0 025), an end product of lipid peroxidation and remarkably
increased levels ofGlutathione peroxidase (GSH-PX) (p<0 025) but
glutathione reductase (GSSG-R) activity was low (p<0.025), despite
the plasma GSH levels were not different
The levels of a lipid peroxide and glutathione-related enzymes in
blood are probably influenced by oestrogen action

((Su/oth/212) Conversion of Cheese Whey and Vinasse to Single Cell Oil

and Protein
Necla Aran,
istanbul Technical University, Department of Food Engineering
80626 Maslak, istanbul, Turkey

Since ancient times microorganisms have been used to produce foods,

They have advantages over plants and animals as being able to be
produced in large quantities. Also, productivity and variety of the
metabolites of microorganisms may be increased by using improved
biotechnological techniques and altering growth conditions. The use of
waste material and by products of industry as production medm provides
economical and environmental advantages for large scale processes.
Production of biomass, fermented foods, microbial enzymes and
metabolites are the major groups of commercially important microbial
processes. Biomass has been produced mainly to be consumed as protein
source (Single Cell Protein) except that of bakers' yeast. However,
microbial cells may be valuable sources of other food comp,.~,ents such
as oil and vitamins.
This study was carried out to investigate production of oil and protein
by Aspergitlus ustus CBS 10725 grown on cheese whey arid vinasse
obtained from a rakl factory that produces ethanol from raisins, and
Rhodotorula glutinis NCYC 377 grown on vinasse. Culture media were
supplemented with nitrogen and phosphorus sources at different
concentrations. Biomas yields, when cultivated in batch eult~re, were
l 1 and 20 g/l forAspergillus ustus depending on the substrate and 10 g/l
for Rhodotorula glutinis. Oil and protein contents of the microorganisms
ranged from 19.5 to 25% and 13 to 21% respectively. Olelc (18:1),
palmitic (16:0), linoleic (18:2) acids and stearic acid (18:0A were the
predominant fatty acids in the extracted oils. Amino acid composition
and riboflavin contents of the microorganisms were also determined,
only methionine and cystein contents of the test organisms were found to
be low as observed in other microbial sources of proteins.

(Su/oth/213)

Intestinal epithelial cell proliferation is inhibited in vitro by

cAMP and pentoxifylline (PTX) via differential regulation of
transforming growth factors expression.
M. Assef (1). JM. Reimund (1,2), J. Ezenfis (l) B Duclos (l 2) M
Kedinger (l), C Foltzer-Jourdainne(1). (1) INSEI~.I [7381. 3 av Moh~re, et
(2) Servwe d'H~pato-Gastroent~rologie, CHU de Hautepierre, Stmsbourg

Failure of PTX to improve TNBS-induced colitis in rats, despite decreasing

mucosal TNF-~ expression (1) led us to study the effects of cAMP and PTX
on the proliferation of rat intestinal epithelial cells and to examine their effect
on TGFs mRNA expression.
Methods: The effect of dibutyryl cAMP (DBcAMP) (0.1-2.5 p.M) and PTX

(10, 100 tag/ml) on cell proliferation (cell counts) and TGF-ct, -131 and -132
mRNA expression (RT-PCR) was examined after 3 and 9 days of culture. The
IEC18 cell line was analyzed in basal conditions or after TNFct addition (1
ng/ml stimulates and 100 ng/ml inhibits IECI8 proliferation). In experiments
with PTX, intracellular cAMP was measured (ELISA).
Results: After 3 days of culture, PTX and DBcAMP inhibited in a dose-

dependent manner basal, TNTo~-stimulated or -inhibited IECI8 proliferation.

The effect of PTX was associated with an intracellular dose-related increase
in DBcAMP, concomitantly a decrease in TGF-cc and an increase in TGF-B2
expression occurred. The effect of DBcAMP was associated only with a
decrease in TGF-cx mRNA. After 9 days, the effects of PTX on basal
proliferation and on TGFs expression were no more detected whereas
DBcAMP effects were maintained TGF-BI expression was never modified.
Conclusion: The phosphodiesterase (PDE) inhibitor PTX inhibited IECI8
proliferation via differential modulation of TGF-~x and -132 expression. The
decrease in TGF-c~ is related to the increase in intracellular cAMP whereas
the effect upon TGF-132 appears cAMP-independent. These results may partly
explain the failure of PTX to improve "l'Nl3S-induced colitis. Development of
PDEs inhibitors without effect upon cell proliferation and TGFs expression
represent a potential therapeutic approach in IBD

(1) Ezenfis J. Application therapeutique d'un module experimental d'inflammation du tube digestif chez le rat. Effet sur les cytokines. DEA. ULP de
Strasbourg 1996

(Su/oth/214)

CrystamzJon of Biological IVllgmmolecules under ~m Extelrld

EleeWle Field by/~ AutO, M T~t, J.P. lvhng~, C Didi~jem', C
Jetsd~atqdB. Cap~. Yi~mupe~
I_CM31~ESA 7036 UI~ Na,t'y t
BP23~,MSO6V ~ t e 4 ~ Nat-y C.a~, bLMCP,Urlva~itJP & M Cu~ieaarel lS, 4 pla~
Jus~a~ 75252Pai~CatvcO~

Crystallization of macromolecules, like any crystallization, is a multiparametrie

process involving the three classical steps of nucleation, growth, and cessation
of growth, What makes crystal growth of biological macromolecules different is,
firstly, the much larger number of parameters than those involved in small
molecule crystal growth and, secondly, the peculiar physico-chemical properties
of these compounds. Some of these particularities will be discussed in regard to
the solubility curve.
Among the different methods to erystallyse biological macromolecules, the
vapour diffusion method will be used together with the hanging drop and the
sitting drops techniques.
To obtain crystals, the biological macromolecules are first dissolved in a 'mother
liquor' containing some salts and some precipitant agents at define pH values.
Proteins and peptides molecules are polymers of amino acids containing dipoles
and ionic groups and the net charge of such molecules depend on the pH value
of the solution, Furthermore, it is possible U)obtain migration of these molecules
in the solvent when an electric field is applied. In this way, it could be possible
to create some supersaturated areas in the cristallizing medium leading to
crystals.
We crystallized very pure lysozyme at pH 4.5 with sodium chloride as
crystallizing agent. Experiments were carried out with and without the
superposition of an electric field. The mosaicity of the crystals was measured at
LURE, for both kind of crystals, by direct recording of Bragg reflection profiles
with a quasi-planar X-ray wave [1]. The statistical results showed a better
homogeneity of the crystal quality when an electric field is applied during the
crystallization. In order to improve this study, we have built a special
experimental device giving very interesting results which can be correlated with
theoretical calculations. In the one hand, fewer crystals were found in drops
under an external electric fidd and in the other hand these crystals were always
located on the surface of the drop, near the cathode side. Details of the new
experimental device and of the results will be presented together with some more
recently results coming from experiments actually in progress.
1l] R. Fourme et AI., J. SynchrotronRad. , 2, 1995, 136

Abstracts FEBS'99

(Su/oth/215)

Tubulin binding-induced chirality of ethyl 5-amino-i,2dihydro-3-phenylpyrido [3,4-b] pyrazin-7-yl carbamate

s149

(Su/oth/216)

I. Barlier', C. BellinP and V. Santoni:

P Barbicra, V. Peyrota. M Sarrazin~. C Briand'~and J.M Andreub.

a i 7~t~:5~,;-(WRS6)32. pk~c'ultd de Pharmacte. 27 bd J. ~loulm. 13005 ~larsellle
h ('entro de Investtgactones Baotogtca& (" S.J C.. I'elaztmez 144. 2~'006 Ala~b'td

Tubulin, an heterodimeric GTP binding protein of the cytoskeleton, is the target

of numerous antimitotic drags. The consequences of the binding on tubulin of
two chiral isomers (R)-(+) and (S)-(-) of ethyl 5-amino-2-methyl-1,2-dihydro-3phenylpyrido[3-4b]pyrazin-7-yl carbamate [1;2] and three achiral analogs NSC
330770, NSC 337238 and C179 [3], have been intensively studied. Here, we
investigate the spectroscopic ligand changes induced by protein binding.
Comparison of the fluorescence emission properties of the five ligands in neutral
buffer and bound to tubulin indicates that the tubulin site is a chiral locus which
differenciates between the R-and S-isomers For all ligands a blue shift occures
upon binding. This may be interpreted as a reduced polarity of this locus with
respect to the aqueous phase. By circular dichroism (CD), we demonstrate that,
when they are bound to tubulin, the near UV circular band of the R-isomer is
largely reduced whereas the one & t h e S-isomer is increased. CD signal due to
the binding of the achiral NSC 330770 and NSC 337238 compounds confirms a
chiral binding locus and ligands conformational changes. CD signal is reversible
and highly specific of the interaction The absorption and fluorescence emission
of ligand is analyzed in various solvents. We observe a specific excess Stokes'
shift effect on water for R-and S-isomers and NSC 330770, absent for
NSC337238 and C179, suggesting the formation of an hydrogen bond between
N1H group and water This hydrogen binding in the tubulin site could be
modified and theretbre contribute to the observed CD changes No specific
solvent effect is found with other hydrogen bond makers like alcohol In
conclusion, the absorption and fluorescence changes and the binding-induced
CD effects pinpoints the structural features of ligand recognition by tubulin,i e,
freezing of the intramolecular rotation between the pyridopyrazin and phenyl
rings of these compounds

Functional analysis of a plasma membrane protein

from A. t h a l i a n a .
t INRA, Versailles, France
21NRA/ENSA-M/CNRS URA 2133, Montpelher, France

The plasma membrane is a source of a large number of unidentified proteins

which might be involved in material and/or information exchange. A
systematic analysis of plasma membrane proteins from A r a b i d o p s i s thaliana
has been undertaken at the EEC level. Informations on the expression and
function of these proteins, the analysis of the corresponding genomic and
EST sequences and finally the characterisation of the corresponding mutants
are being used to build a specific database (http://sphinx.rug.ac.be:8080/).
A large scale analysis of plasma membrane proteins by two dimensional gel
electrophoresis and micro-sequencing was undertaken. Based on these
results, we isolated and characterised a mutant corresponding to one of the
major proteins (PI 91), by screening the Versailles T-DNA collection using a
reverse genetic approach.
The P 1 9 1 gene appears to be single copy in A r a b i d o p s i s . This gene is
expressed in leaves, stems, flowers and siliques and throughout the
development. In the mutant, we could not detect any P 1 9 1 transcript or
protein. However, in our culture conditions, no phenotype has been found for
the homozygous mutant. Different culture conditions as well as various
stresses are being tested to determine whether PI91 loss of function affects
plant development.

[1]Leynadier et al, Biochemistry, 32, (1993) 10675

[2] Barbier et al., Biochemistry, 34, (1995) 16821
[3] Barbier et al., Biochemistry, 35, (1996). 2008

(Su/oth/217) Thermogenic effect of glucagon in perfused liver

of cold acclimated ducklings (Cairina moschata)
E. Bedu, F. Cohen-adad, HBarre et C Duchamp
UMR 5578, 43 Bd du 11 novembre 1918, 69622 VilleurOanne
cedex, France

Despite the lack of brown adipose tissue (BAT), the specialized

thermogenic nssue of small mammals, cold-acclimated btrds can develop
nonshivering thermogenesis (NST). Contrary to mammals, avian NST is
mainly of muscle origin. In vivo and in vitro studies have suggested that
this non-BAT NST may be controlled by gtucagon and noradrenatine
However, skeletal muscle does not account for all the cold-induced or
glucagon-induced avian NST indicating that other organs should be
involved. Among these, the liver may play an important role
We set up an m mtu perfused liver preparation enabling us to monitor
both the oxygen uptake of the liver (LVO2) and its glucose output. Two
groups of 5-wk-old duckhngs ( ( ' a i r m a
moschata)
reared at
thermoneutrahty (25C, TN) or cold-acclimated (reared at 4C for 4 wks,
CA), were used after 24h of fasting. The effects of increasing doses of
glucagon and noradrenaline on hepatic energetic metabolism were
investigated in the presence of neoglucogenic substrates (lactate 2raM).
1) m s l t u perfused ducklings li~er was viable. 2) Glucagon and
noradrenaline increased LVO2 m a dose-dependent manner in association
with an increased hepatic glucose production. 3 ) Cold acclimation ~as
not assocmted with an improved thermogenic response of perfused livers
to the hormones tested. By contrast, an increased hepatic sensitivity to
glucagon was noticed in CA ducklings.
These results show that the liver may be a s~te of the m v t v o glueagoninduced thermogenesis observed m duckhngs, although It cannot account
for the increased thermogenic effect of the hormone observed after coldacclimation.

(Su/oth/218)

Primary sequence requirement for autocatalytic

palmitoylafion of peptides derived from 13radrenergic receptor
C Belanger, H Ansanay, R Qanbar and M Bouvier
Dopt de hlochmue el GR,S~\~4 ~ )m'er~tte de ~mtrOaL Quebec ( "cmada

Palmitoylation is a reversible and dynamic post-translational

modification that occurs on cysteine residues of many proteins, including
G protein-coupled-receptors (GPCR) The present study was undertaken
to determine if autocatalytic palmitoylation could occur for GPCR and if
so, to assess specificity determinants that allow palmitoylation of certain
but not all cysteine residues. A high proportion of basic and hydrophobic
residues was found near the palmitoylated cysteine of many GPCR To
test the hypothesis that those residues would favor palmitoylation, short
peptides derived from the [3z-adrenergic wild type sequence bearing
different proportions of basic, acidic and hydrophobic residues were
tested in an m wtro acylation assay Incubation of those peptides with
[~H]-palmitoyl-CoA led to a time dependent incorporation of [-aH]palmitate that could be detected by autoradiography, following separation
by TLC The covalent nature of the [3H]-palmitate incorporation was
revealed by its sensitivity to neutral hydroxylamine treatment that
confirmed the presence of a thio-esterified cysteine Basic residues
flanking the cysteine proved to he essential for autoacylation in vitro.
whereas hydrophobic residues greatly enhanced it. Acidic residues, on
the other hand, were found to be detrimental for the reaction Moreover,
basic pH was found to favor the autocatalytic aeylation while acidic pH
inhibited it Also, we found that the thioester bond formed during autoacylation was stable and almost irreversible because peptide bound [3H]palmitate could not be displaced upon incubation with a hundred times
more
non-radioactive
palmitoyl-CoA,
suggesting
that
the
depalmitoylation reaction could require a thio-esterase to proceed
Whether the primary sequences flanking palmitoylated cysteine have a
similar effect on the palmitoylation reaction in vivo remains to be
investigated

s 150

(Su/oth/219)

Abstracts FEBS'99

Expression of anti-oxydant proteins in ciofibric

acld-treated HepG2 human cells
A. Bianchi, P. Becuwe, J.M. Keller, M. Dau~a

((Su/oth/220)

G Btringen, F.A~kurt & M.LOker

TUBITA K AIRC, FSTRI, P. 0 Box 21, 41470 Gebze, Kocaeh, Turkey

EA.2402. Prolif~rateurs de Peroxysoomes, UPH Nancy 1, Facult~ des

Sciences, 54506 Vandoeuvre-les-Nancy Cedex, France

Clofibric acid (CA), a peroxisome proliferator (PP), has been

shown to promote an oxidative stress in rodent hepatocytesvia the
induction of peroxisomal hydrogen peroxide-producing enzymes and the
repression of antioxidant proteins, such as superoxide dismutases (SOD)
and metallothioneins (MT). This effect has not been yet investigated in
human ceils. In the present study, the rate of production of superoxide
anion and lipid peroxidation-defived products, and the expression of
superoxide dismutases and metallothionein IIA have been investigated in
human HepG2 hepatoma cells treated with 0.25, 0.50 and 0.75raM CA.
The rate of superoxide radical production was enhanced only for a 5 daytreatment with CA but this effect was not accompanied by an increase in
the lipid peroxidafion processes. The manganese dependent SOD mRNA
and protein levels were increased in HepG2 cells treated with different
concentrations of CA for 1 and 5 days. This induction of MnSOD gene
expression was correlated with the activation of AP-1, a transcription
factor capable to be involved in its regulation. The PP had not effect on
copper-zinc dependent SOD. On the other hand, the MT IJ.A gene was
repressed for the durations of treatment with the concentrations of CA. In
contrast to rodent ceils, these results show an induction of MnSOD and a
repression of MT IIA in the CA-treated human hepatoma cells which are
not related to an induced oxidative stress.

(Su/oth/221)

Effect of ciprofibrate on cholesterol metabolism

in rat cultured hepatocytes
P. Boumot, S. Duclos, L.C. Ramlrez
LBMC, UniversitJ de Bourgogne, 21000 Doon, F r a n c e

Fibrates are used to lower plasma triglycerides and cholesterol levels

in certain types of hyperlipoproteinemia ; they are also known to produce
peroxisome proliferation and to induce peroxisomal enzymes in rats and
mice. Peroxisomes play an essential role in cholesterol biosynthesis (1), and
in its conversion to bile acids (2,3). To clarify the hypolipidemic effect of
ciprofibrate, we examined its effect on hepatic cholesterol metabolism.
Studies were performed in adult rat cultured hepatocytes maintained in
Leibovitz L15 culture medium supplemented with delipidated calf serum,
during the first 24 h or 48 h following the cell attachment period (18 h).
Treatment with ciprofibrate had no significant effect on bile acid production,
as shown by their quantification using capillary gas chromatography. In
contrast, ciprofibrate reduced ~4C-bile acid synthesis from 14C-cholesterol by
about 40% after 24 h and 20% after 48 h. No change in biosynthesis was
observed using culture medium supplemented with foetal calf serum.
Ciprofibrate modified bile acid composition, increasing the ratio between
cholic acid and [3-muricholic acid, the major bile acids, from 1.7 to 2.6 after
24 h of treatment in the presence of delipidated serum. Therefore,
ciprofibrate favoured the synthesis of bile acids by the neutral pathway
relative to the acidic pathway. Although it is generally assumed that fibrates
produce inhibitory effects on HMG-CoA reductase and cholesterol 7~hydroxyIase, our observations in cultured hepatocytes do not clearly sustain
this assumption. Studies are actually under way to elucidate the relationship
between bile acid synthesis and cholesterol availability which in the absence
of serum lipids, depends on cholesterol synthesis and secretion of this newly
synthesised cholesterol.
(1) L. Biardi and S.K. Krisans (1996) J. Biol. Chem. 271 : 1784-1788.
(2) I. Bj6rkhem (1992) J. Lipid Res. 33 : 455-471.
(3) P. Van Veldhoven et al. (1996) Biochem J. 320, 115-121.
LBMC, Laboratoire de Biologic MolPculaire et Cellulaire

The Effects of Different Foods and Nutrients on Super

Oxide Dismutase (SOD) Activity

The effects of dietary factors on SOD activity were investigated. 28

healthy volunteers between 17 and 47 years of age were joined to the study
They were supplemented to their usual daily diet with algae (Chlorella
pyrenoidosa), fish oil, vitamin B complex, vitamin C and natural food mix
(hazelnut, apricot, lentil, boiled grape juice, tahini paste, milk powder,
cocoa) for 40 to 80 days. At the initial and at the final of the study blood
samples were taken from the volunteers in order to determine the levels of
hemoglobin, total cholesterol, triglycerid, HDL, LDL and VLDL
cholesterol. Otherwise during the study, the height and the weight of the
volunteers were recorded and the Body Mass Index (BMI) values were
calculated A significant increase was observed in the mean of the
hemoglobin levels in the group 2 (fish oil supplemented group) and in the
group 4 (vitamin C supplemented group). It was also a significant increase
observed on the SOD and SOD/Hb levels in the group 4 (vitamin C
supplemented group) and in the group 5 (natural food mix supplemented
group) However, there were no significant change observed on the blood
lipid parameters in the groups during the study.

(Su/oth/222)

Amino acids as modulators of

polyamine homeostasis regulatory pathways
P. Brachet, H. Chabanon, C. Aubel, S. Blinet, M. Ferrara
UR INRA 238 Nutrition Cellulaire et Mol~culaire, 63122 Theix. France

Abnormal amino acid (AA) metabolism, characterized by differential

alterations in the AA tissue levels, occurs in numerous diseases. While
supplementation with AA is used clinically for improving the nutritional
status of patients, AA restriction has only been examined experimentally,
mostly with the purpose of AA imbalance therapy for cancer. Since
polyamines (putrescine, PUT; spermidine, SPD; and spermine, SPM) are
important in cell growth and functions, we hypothesised that their regulatory
pathways can respond to changes in the AA availability in cultured human
cell lines. First, activity of ornithine decarboxylase (ODC), a key enzyme of
polyamine biosynthesis, was found to increase transiently 10 to 15-fold
following supplementation with any one of a variety of Aas; glycine, Lasparagine and L-serine being by far the most effective. Western and
Northern blot analyses showed that the level of ODC protein, unlike ODC
mRNA, increased markedly in AA-supplemented cells. The increase was
found to be due to increased ODC stability and biosynthesis efficiency. The
5'- and/or 3'-UTRs of transcripts might play a role in the regulation of ODC
translation efficiency by the AAs. Second, in contrast with ODC activity that
decreased or did not vary, SPD transport augmented markedly in response to
AA starvation. Deprivation for a single essential AA led to a higher increase
in SPD transport (3-fold increase in Vmax) than deprivation for a
nonessential AA. This apparent increase in the number of active SPD
transporters was concurrent with a decrease in the amount of antizyme (AZ),
a protein that both downregulates polyamine transport and inactivates ODC,
in AA-deprived cells. However, exposure of cells to limited amounts of AA
could also increase transport without altering protein synthesis and AZ
levels. The nature of the AZ-independent mechanism(s) is currently under
investigation. Third, the activity and mRNA levels of the key enzyme of
pulyamine degradation, SPD/SPM N~-acetyltransferase (SSAT), were
additionally found to increase 3-4 fold following deprivation of cells for one
essential AA. Due to the high stability of SSAT mRNA, the increase might
be due to increased expression of the SSAT gene. Overall, our data indicate
that important regulatory steps of polyamine homeostasis can respond at
various expression levels to changes in the AA sufficiency.

Abstracts FEBS' 99

(Su/oth/223)

The antioxidant enzyme employed by vitamins in the

protection of the isehemic myocardium
C But'tea. M Mardare. C T Dragom~r
I )c tot Babe~ ]llsllllllc, ,t;p/ ]Ilde/lelldelllel 99 tO/. l~uc hare~l, tmftama

Oxygen free radicals play a major role in the pathogenesis of postischemic injury
in the heart Antioxidative defense mechanisms ( i e nonenzymatic and
enzymatic) protect cells from the harmful effects of reactive oxygen metabolites
[1] The enzymatic antioxidant system has been proposed by our experiment as a
possible mechanism employed by vitamins in the protection of the ischemic
myocardium Wistar rats (males and females) were treated with isoproterenol
associated or not with vitamins A, E, or C. The hearts were isolated and
ventricular tissue was homogenized and assayed for enzymatic activity of
catalase, glutathione S-transferase (GST), glutathione reductase (GRID) and 7glutamil transferase (;~-GT) Lipid peroxide content (LPC) was determined by
quantifying the thiobarbituric acid reactive substances Vitamins did not
significantly reduced the level of hypertrophy induced by Is on myocardium,
which was generally higher in females than in males The activity of catalase was
intensified both in males and females treated with Is, while of GRD was elevated
in female groups, GST and y-GT were depressed in males Vitamins A and E
restored the control level of catalase, 3,-GT and GST in male groups, while
vitamin C had the same effect on catalase The following situation was observed
in female groups. (i) vitamin A enhanced the activity of catalase and 7-GT,
normalized GRD, and depressed GST, (ii) vitamin E elevated the activity of 7GT, and normalized the ones of GST and GRD, (c) vitamin C induced an
increase of ,v-GT activity, decreased the activity of catalase and GST, and
brought to control level the one of GRD The LPC of myocardium was
increased by ls only in females, and depressed by treatment with vitamins
Therefore. the biochemical feature of myocardial hypertrophy was gender
specific, meaning that antioxidant enzymes had a higher activity in females
probably as a consequence of the increased LPC Our results suggest that
catalase should be an auxiliary antioxidant mechanism of vitamin A in females'
myocardium, imposed by the oxidative stress Our experiment seems to highlight
7-GT as a constituent of antloxidant vitamins" pathway of action
[I] J T Flahert3. Am J Med. 91 (sup# 3C), (1991). 79S

(Su/oth/225)

Multiple resistance mechanisms to azole antifungals in

obligate biotrophic fungus (Uncinula necator).
M-F Corio-Costet, C. Dtlye, C. Stievenard
U.R. en Saat6 Vtg6tale, INRA-, BP 81, 33883 Vfllenaved'Omon, France

s 151

(Su/oth/224)

B.Raf mediates the cAMP activation of MAPK in B16 melanoma

cells
R. Buscha, P. Abbea, Frtderlc Mantouxa, AlainEych~neb, Jean-PaulOrtonnea
and RobertBalloma
a INSERM U385, Av. Valombrose 06107, NLce, France
b CNRS U.M.R 146, lnstitut curie. 91405 Orsay, France.

Melanocytes are neural crest-derived cells specialized in the synthesis

of melanin pigments. In melanocytes and melanoma ceils, cAMP, the
most potent melanic agent, activates MAP kinases (MAPK), event that
has only been observed in some neural models, thus suggesting its
tissue-specific nature.
The aim of our work was to elucidate the mechanisms of MAPK
activation by cAMP in the melanocyte cell system taking as a model
the B16 mouse melanoma cell line.
Previous results have indicated that the MAPK kinase (MEK) is also
activated by cAMP, but not Raf-1, the kinase upstream of MEK.
Considering the neural origin of melanocytes we have focused our
attention on B-Raf, a neural-cell expressed Raf isoform . We have
observed that B-Raf which is expressed to a high level in B 16 cells, is
activated by cAMP, Furthermore, a dominant negative mutant of B-raf
but not Raf-I is able to block the cAMP-induced activation of MAPK
meaning that B-raf is the MEK upstream regulator mediating this
cAMP effect. In order to dissect molecular events between B-Raf and
cAMP, we used the Clostridium sordelii Lethal toxin (LT) which
inhibits the small GTPases Rap-l, Ras and Rac. Very low
concentrations of LT completely block the cAMP activation of MAPK
indicating that either Rap-l, Ras or Rac mediate the cAMP effect. The
overexpression of Rap-1 mutants in B 16 cells does not affect MAPK
activation or Elk transactivation meaning that Rap-I does not play a
role in the MAPK activation by cAMP in our cell system. In addition,
we have observed in the melanocyte cell system that cAMP is able to
inhibit the growth factor or phorbol ester stimulation of Raf-1 but not
that of B-Raf meaning that, independently of the upstream activators,
cAMP is able to inhibit specifically Raf-1. Taken together, our results
propose a cell specific mechanism of MAPK activation by cAMP
involving B-Raf and Ras or Rac related small GTPase.

(Su/oth/226)

Mannose therapy partly corrects glycosylation

defects observed in CDGS type Ib (PMI deficiency)
M. Cuera, P. de Lonlayb, G. Beaunea, M. Kretzc, G. Dur'dnda,
IM. Saadubrayb, N. Setaa ; aLab. Biochimie A, h@. Bichat, bpddiatrie
Gdn~tique, hOp."Necker, Pans, Cpt~dxatrte,. " hOp. Civils, Colmar, France.

The intensive use of antifungal agents, especially the azole class, has led to
the development of resistance in a number of fungi like grape powdery
mildew[l]. The mechanisms of resistance in field populations of DMIresistant fungi have been difficult to ascertain especially in the obligate
powdery mildew parasites. Currently there are no satisfactory explanations of
the resistance mecanisms to DMI fungicides in tolerant field isolates.
In triadimenol resistant field isolates, we showed that a high resistance level
to triadimenol was correlated to a single point mutation in the CYP51 gene
[2]. However DMI resistance seems to be polygenically governed in many
fungi, including U n c i n u l a n e c a t o r [3].
To understand which other resistance mechanims could be involved, we
crossed triadimenol-sensitive and resistant strains of grape powdery mildew.
The characterisation of progeny displayed five major phenotypes. One was
resistant to four DMIs (triadimenol, triadimefon, cyproconazole and
fenarimol), but its CYP51 gene was a sensitive gene without any resistant
mutation. In this case we expect an energy-dependent efflux resistance
system, Other resistant isolates, displaying single mutation at codon 136 in
~he CYP51 gone, showed an abnormal sterol distribution in mycelium and
conidia without fungicide treatment. Analysis of the sterol composition of
azole-resistant powdery mildew has provided several hypotheses on specific
alterations of enzyme and/or regulation involved in the complex sterol
biosynthesic pathway.
Our results show that there are multiple mechanisms of DMI resistance in
field isolates. We suggest that near the major point mutations in the CYP51
gene, other genes are involved in the regulation of sterol biosynthesis and
DMI
resistance and together may
control several resistance
mechanisms.Unserstanding resistance mechanisms is of value not only for the
design of new antifungal agents but also the developement of strategies of
overcome or delay the emergence of resistance.
[1] Adams D.J, I997, [n : Mo|ecular genetics of drug resistance, eds Hayes JH, WolfC R.,
Harwood Acad Publ. Netherlands.pp30-80.
[2] Delye Cet al., 1997, Appl. Environ. Microbiol.,63 : 2966-2970.
[3] Cono-Costet MF etal, 1998, 4t~ Int. Syrup. On P4~0Biodlversztyand Bmtechnology,
(July 14-16, Strasbourg, France).

We report the case of a patient who developed hyperinsulinemic

hypoglycemia at 3 months of age associated with enteropathy and
hepatopathy. Routine blood tests revealed hepatic cytolysis and coagulation
factor deficiencies, suggestive of CDGS. Abnormal glycosylation of serum
transferrin which was evidenced by two methods : Western blotting [1] and
isoelectric focusing, was typical of the CDGS type I. Western blotting
revealed 3 bands corresponding to transferrin with 2, 1 and 0 glycan chains.
Isoelectric focusing showed a shift toward desialylated isoforms (asialo 4%,
monosialo 6%, disialo 40%). The enzymes involved in the conversion of
glucose to Man 1P were measured in leucocyte extracts [2].
Phosphomannomutase (PMM) activity was normal but phosphomannose
isomerase (PMI) activity was undetectable. PMI deficiency (CDGS type Ib)
block interconversion pathway (Fuc 6P-->Man 6P), but GDP-Mannose can
still be synthesized from exogenous mannose via hexokinase (Man 6P) and
PMM (Man 1P). So, oral mannose supplementation was initiated with 0.10
g/kg 4 times a day and carried up to 0.17 g/kg 6 times a day. Plasma
mannose levels were determined by an enzymatic method [3] before and after
each increase in mannose posology. Before treatment, plasma mannose level
(12 lamol/1) was much lower than infant control values (455:7 tarnol/1; n=9;
age: 25:1.7 months). After 3 months of mannose therapy, basal plasma levels
stabilized between 102 and 110 I.tmol/1. Maximum mannose level
(157 lamol/l) was obtained 2 hours following ingestion. An important urinary
elimination (1.3 mmolfl) reduced the mannose ha/f-life to about 2.5 hours. A
significant clinical improvement was observed after 1 month of oral marmose
administration. After 3 months, hepatic cytolysis was reduced and clotting
factor levels returned to near-normal values. We observed an improvement in
transferrin glycosylation pattern as well by Western blotting (disappearance of
the lighter band) as by isoelectric focusing (disappearance of asialo and
monosialo isoforms). Oral mannose therapy of this patient is considered
successfull after 11 months of follow-up. Regular control of mannosemia and
mannosuria allows permanent adaptation of oral mannose posology to
maturation of small intestine and kidney.
[1] Seta et at., Clin. Chem. Acta, 254, (1996), 131. [2] Van Schaftingen E.,
Jaeken J., FEBS Lett, 377, (1995), 318. [3] Etchison JR, Freeze HH., Clin.
Chore, 43 (1997), 533.

s152

(Su/oth/227)

Abstracts FEBS'99

Sulfate transport and concentration in porcine thyroid

cells. Regulation by thyrotropin and iodide.
D. Cauvi a, MC. Nlend a, N. Venot a C. Penel b, O. Chabaud a
~INSERM U38, Facultd de mrdecine 13385 Marsedle, France
Canc&ologie expOrimentale. Facult3 ~]em~decme, 13916 Marseille

Sulfation of thyroglobulin (Tg), the precursor of thyroid hormones, is

regulated by thyrotropin (TSH), the major effector of thyroid cells. Tg
secreted by porcine thyroid cells bore 1.5 fold less sulfated residues under
TSH stimulation [ 1].
The aim of this work was to observe the steps of sulfation process that are
regulated by TSH and iodide (KI). We studied their effects on the transport
and on the concentration of intracellular inorganic sulfate (iSO42-) which is
the first substrate of the sulfation process. Experiments were carried out in
thyrocytes cultured as monolayers on collagen-coated filters.
The amount of iSOaz', determined by capillary electrophoresis, was similar
(about 3 fmoles/cell) in the absence or in the presence of TSH (+TSH) and
without or with KI (+KI). The volume of TSH-stimulated cells, determined
on living cells by confocal microscopy, was 1.7 fold more important
(+TSH=5.06+0.91 pL; -TSH=3.02+0.56 pL). This effect was cAMPmediated. Only a weak variation was observed in the presence of KI. The
volume of cytosol was then measured, its proportion was different in each
culture condition. Values determined in pL (+TSH-KI=2.74+0.50;
+TSH+KI=2.49+0.21; -TSH-KI=l.98_+0.22; -TSH+KI=2.29-~'_0.22) were
used to calculate the iSO42" concentration in mM (+TSH-KI=I.12;
+TSH+KI=l.26; -TSH-KI=1.54; -TSH+KI-1.29).
Kinetic studies showed that 35SO42" influx was accelerated in the TSHstimulated cells and was not modified by iodide. Only apical effiux of
35SOa2" was controlled by TSH and iodide, while basal effiux was not
changed.
These results show that thyroglobulin sulfation is regulated by TSH and
iodide, in part through the control of sulfate transport and intracellular
concentration.

((Su/oth/228) Is membrane Helix IV of the melibiose permease of E. Coil

a hinge between the ionic and sugar binding sites?
E. Cordat, G. Leblanc and I. Mus-veteau
CEA/LRC16V, LPMC, Univ. Nice/SophiaAntipolis, CNRS, BP68, 06230
Villefrances/Mer,France

The melibiose permease of ECoti (MelB) is a membrane protein of the

Na+/solute symporter family. It catalyzes the cotransport of a and 13galactosides by a cation (Na, Li or H) -sugar symport mechanism.
Previous site-directed mutagenesis of MelB tryptophans (Yrp) and
intrinsic fluorescence spectroscopy studies on the two C-terminal Trp
suggest a localization of the cation binding site in the N-terminal half of the
protein, and of the sugar binding site in the C-terminal half one. It was
observed that I) Na+ or Ll+ quenches MelB fluorescence; 2) a-galactosides
enhances only the fluorescence of the two C-terminal Trp whereas
galactosides perturb both N and C-terminal Trp fluorescence signal. To
further delineate these binding s~tes, the N-terminal Trps were
systematically replaced by phenylalanine. Only the mutation of Trp 116
and 128 ( in or close to helix IV), significantly alters MelB transport and in
pamcular the sugar binding properties. These mutants were too unstable to
be stu&ed in purified state. In contrast, purified W54F, W64F, W73F and
W79F were stable, and the effect of ions and sugar on their intrinsic
fluorescence was analyzed. In line with a N-terminal localization of the
canons binding site, the data indicate that sodium quenches Trp 54
emission and suggest by deduction i) that these ions or b-galactosides
enhance Trp 116 and Trp128 emission. Finally, mutants WII6F and
W 128F and single point mutation of other residues of helix 1V share similar
functional defect Altogether , the concomitant effect of ions and ~galaetosides on Trp 116 and Trp 128 fluorescence and the observation that
more than more that one mutation in Helix IV introduce similar defect in
MelB funcnon suggest that helix IV may act as a couphng hinge between
the sugar and cation binding of MelB

[lr : Nlend et al., J. EndocrmoL Invest. 19(6): 38(abs 76)

(Su/oth/229)

Study ofA. thaliana mevalooate-PP deearboxylase

H Cordier, F Karst and T Berges
IBA'IIG, Universttb de Poitiers, 86022 Poitters c'~dex, France.

(Su/oth/230)

In vitro differentiation of immature hepatic cells into the

biliary lineage
D. Couchie, M.N. Chobert and Y. Laperche
INSERM U99, HOpital Henri Mender. 94010 Crdtei/. France

The mevalonate pathway in eukaryotic cells produces sterols,

mainly involved in plasma membrane structure, and isoprenoids involved
in many cellular functions, such as respiration, protein regulation or signal
transduction In addition, isoprenoids in higher plants lead to the synthesis
of a large number of various molecules The mevalonate diphosphate
decarboxylase (MVD) catalyses the first step of isoprenoids and sterols
biosynthesis, ie the decarboxylation of mevalonate diphosphate into
isopentenyl diphosphate (1PP). Recently, a non-mevalonate pathway for
the synthesis of IPP has been described in plants as taking place in the
plastid compartment, addressing the question of the relative
compartmentalization of the two pathways. Therefore, isolation of a plant
MVD eDNA could provide an interesting tool to study the cellular
localisation of the mevalonate pathway
The eDNA encoding A. thaliana M V D ( A t M V D I ) was isolated
from an EST clone identified by sequence comparison with the yeast
MVD Analysis of the mRNA by northern blot on various tissues of A.
thahana shows a preferential expression of dtMVD1 in the roots. The
plant eDNA A t M V D 1 complements yeast mutant strains defective for
MVD activity and restores the sterol biosynthetic pathway, although the
amount of sterols produced does not reach that of the wild-type strain.
Oligomerization of MVD was studied by two-hybrid assay. It was shown
that both yeast and plant MVD form homodimers, as well as heterndimers
m rive while the L79P single mutation of the yeast mutant strain M N 1 9 34 impairs the dimerization of the enzyme A. thaliana genomic DNA was
hybridized with A t M V D 1 specific probe and the results suggest the
presence of a second gene Isolation of AtMVD1 regulatory regions and
cloning of the gene encoding the putative isoform are in progress.

We investigate the mechanisms involved in the differentiation of

hepatic precursor cells (hepatoblasts) into the biliary lineage, using the
WB-F344 rat liver epithelial cells as a model. These established diplo'id
cells display phenotypic characteristics of immature hepatic cells [1] and
in rive possess the capability to express either hepatocytic or biliary
differentiation [2]. We report here an in vitro system promoting
differentiation of these hepatoblast-like cells into the biliary lineage.
Ceils are grown on matrigel coated dishes in Ham F-12 medium
supplemented with EGF, IGF I1 and insulin which are essential lbr cell
growth. One day after plating, cells become elongated and organized in a
network. From the fourth day of culture, most of the cells are tighly
associated and have the tendency to rise above the matrix; occasionally
few cells are arranged around a lumen. Inside the network, we observe
islands of cells at a lower density with the polygonal appearance of the
WB-F344 cells grown on plastic.
Biochemical evidences of biliary differentiation are investigated in
these cultures. Cytochemistry analysis shows an expression of
cytokeratin 19 and gamma-glutamyltranspeptidase in the cells organized in
network; in contrast, cells in the spaces of the network remain negative tbr
these two biliary markers. Analysis of mRNA expression by RT-PCR
reveals the induction of the GGT IV and CFTR mRNAs, two
characteristics of biliary cells. No mRNA coding for albumin, catalase or
tyrosine amino-transferase was detected. These phenotypic characteristics
are consistent with a differentiation of the WB-F344 cells along the biliary
lineage.

[1] J.W. Grisham et al., Prec. Soc. Exp. Biol. Med., 204, (1993) 270.
[2] M.J. Hooth et al., Am. J. Pathol., 153, (1999) 1913.

Abstracts FEBS'99

(Su/oth/231)

Regulation of protease activities by soluble factors in

osteogenic sarcoma cells (MG-63 and SaOS2).
Damiens C.~ Rousselle A.V., Grimaud E., Fortun Y., Heymann
D., Padrines M.. UPRES EA 2159. CJroupede Physiopathologie de
la R~oorplion Ossease, Nantes, France.

The present study investigated the ability of human osteesarcoma cells : MG63 and SaOS2 (different differentiation states) to produce enzymes involved in
bone resorption (cystein-proteases and metaUoproteases) and their regulation
by interleukin-ll3 (hlL-11~), intedeukin 6 (hlL-6), insulin growth factor-1
(hlGF-1), oncostatin M (hOSM), leukemia inhibitory factor (hLIF) and
growth hormone (hGH). Osteosarcoma cells in culture released enzyme
activities. Cathepsin activity was detected using a fluorescent synthetic
substrate, 7-N benzyloxycarbonyI-L-phenyl-L-arginylamide-4methylcoumarin
and its specificity was checked with E-64, a specific inhibitor of cysteinprotease
and
by
7-N
benzyloxyearbonyl-L-phenylalanyl-L-tyrosyl(lertiobutyrate)-diazomtthylcttone, a specific and irreversible inhibitor of
cathepsin L. The metalloprotease activities were determited by a zymography
technique and a immunochemistry method. No collagenase activity was
underlined in SaOS2 cell culture The cystein-protease activities of both cell
lines especially cathepsin L activity were increased significantly in presence
ofhlL-l[3, hIL-6 and hOSM. The collagenolytic activities in MG-63 cells were
upmodulated by the same cytokines excepted by hOSM In contrast hIGF-1
and hGH decreased the type I collagenolytic activities and no effect was
detectable in presence of hLIF. In conclusion, the present paper show that in
increasing collagen type I degradation, hlL-ll3, hIL-6 and hOSM could be
involved in bone resorption. Whereas inhibiting action of hIGF-I and hGH on
collagen type I degradation could imply this factor in bone formation.

s153

(Su/oth/232) Porcine pancreatic preprocarboxypeptidase AI: Molecular

cloning and functional expression in S. cerevisiae.
S. Damis', N. Juge', C. Marino~, F.X. Aviltsb, A. Puigserver',
J-C. Chaix' and X-J. Guo'
LBBN, CNRS.ESA 6033, Marseille, France.bDBBM, UC-IBF,Bellaterra, Spare.

A full-length eDNA clone coding for porcine pancreatic

preprocarboxypeptidase AI (PCPA1) was isolated from a cDNA library
[1]. The open reading frame (ORF) of the nucleotide sequence was 1260 nt
in length and encoded a protein of 419 amino acids (aa). The eDNA
included a short signal peptide of 16 aa and a 94 aa-long activation segment.
The calculated molecular mass of the mature proenzyme was 45561 Da, in
accordance with that of the purified porcine pancreatic PCPA1. The
deduced aa sequence of the corresponding enzyme differed from that
predicted by the 3-D structure by 40 aa, and showed 85 % identity and 55
% identity to that of procarboxypeptidases A1 and A2, respectively.
Moreover the sequence was identical to that of several independent eDNA
clones, suggesting that it is the major transcribed gene. No evidence for a
second variant was observed in the e D N A library and PCPA2 is
apparently absent from the porcine pancreas. The eDNA was expressed in
Saccharomyces cerevisiae under the control of the yeast triose phosphate
isomerase promoter. The signal peptide of the PCPA protein efficiently
directed its secretion into the culture medium (1.5 rag/l) as a protein of the
predicted size. The recombinant proenzyme was analyzed by
immunological and enzymological methods. Its activation behaviour was
comparable to that of the native form and led to a 35 kDa active enzyme.

[1] Darnis et at., Eur. J. Biochem. 259, (1999) 719

(Su/oth/233)

Deletion of the knee operon in Desulfovibrio vulgarJs

Hildenborough alters the hydrogen metabolism
A. Della *, M. Bruschi ~and G. Voordouw2.~BlP, 1BSM-CNRS,

(Su]ofll]234)

LN.S.E.R.M. Unit~ 38, Facult6 de m~decine Timone, Universit6 de la

M~diterran~e, Marseille, France.

Marseille, France and 2Department of Bmlogical Sciences, The

University of Calgary. Calgary, Canada.

Sulfate reducing bacteria of the genus Desulfovibrio can generally use

hydrogen and organic acids as electron donors for sulfate reduction. Sulfate
reduction and hydrogen oxidation are spatially separated in the cytoplasm and
the periplasm, respectively. The Hmc complex consists of six different
proteins (HmcA to HmcF); HmcA is a periplasmic cytochrome with 16 c-type
heroes organized in cytochrome c3-type units. HmcB is an iron-sulfur
containing protein with the iron-sulfur dusters residing in the periplasm.
HmcC, HmcD and HmcE are integral membrane proteins that bind b-type
heine whereas HmcF is a membrane-associated, cytoplasmic iron-sulfur
protein. All these proteins would be organized in a transmembrane complex
which has been proposed to be the electronic link between the periplasmic
hydrogen oxidation and the cytoplasmic sulfate reduction [1].
To elucidate the physiological function of the Hmc complex, we
generated a mutant with the deletion of the whole Hmc operon with the SacB
mutagenesis method [2].
The mutant, in which no lime complex is expressed anymore, does
not seem to be affected when it is grown in lactate or pyruvate / sulfate
medium. On the other hand, the growth was inhibited when hydrogen was the
used as the only electron donor for sulfate respiration. Moreover, a significant
growth difference on both liquid media depending thus on the inoculum sizes
and on solid media suggests that the Hmc complex might have also a function
in the niche development of the bacteria.
[1] Rossi at al, J. bacteriol. 175, (1993), 4699-4711.
[2] Fu and Voordouw, Microbiology, 143, (1997), 1815-1827.

Production of a Thyroglobulin C-terminal Fragment during

Hormonesynthesis. Implication for Thyroid Pathogenesis.
C.Duthoit,V.Estienne,F.Delom,J.Durand,B.Mallet,P.Carayon,J.Ruf.

Deciphering the mechanisms by which proteins play a role in

physiological and pathological events asks for structure-function studies of their
different domains. This approach is particularly suited to understand the
involvement of specific proteins in the molecular mechanisms of autoimmunity
and, more precisely, to clarify the restricted induction by B-lymphocytes of
autoantibodies directed to few of the protein epitopes. In the thyroid model of
organ-specific autoimmunity, two proteins, thyroglobulin (Tg) and
thyroperoxidase (TPO) are major autoantigens (aAg), Interestingly, these
proteins are funetionnaly linked since TPO is a specific membrane bound
enzyme that catalyse the iodination and coupling of unique tyrosine residues
within the Tg, to form the thyroid hormones, triiodothyronine (T3), and
thyroxine (T4).
Regarding Tg, a 660 kDa glycoprotein, only a restricted set of
autoepitopes belonging to a unique immune-dominant region on the molecule
has been ~..ported. However, little is known about the molecular characteristics
of the varrous autoepitopes. To further localize them, we search for their
presence into smaller Tg fragments. Hormone synthesis is an oxidative process
involving H202 production. In this study, we have observed the fragmentation of
Tg by in vitro iodination and coupling, a process involving iodide, TPO, and
glucose-glucose oxydase as H20~ generating system. This protocol led to the
production of four peptides ranging from 80 to 40 kDa. The smallest peptide (40
kDa) obtained was immunoreactive with Tg aAb from patients with autoimmune
thyroid diseases, and with monoclonal antibodies (mAb) directed to the
immunodominant region of Tg. This fragment contained a T4-bound
hormonogenic site. It was localized in the C-terminal part of the Tg molecule,
which shares homology with the acetylcholinesterase and contains the second
hormonogenie site (tyr 2553) which is also the earliest iodinated site. Since
reactive oxygen species (ROS) can induce the oxidative modification of proteins,
including fragmentation, we used a metal-catalysed Fenton reaction to generate
ROS and addressed the'question whether hormonosynthesis might induce ROSmediated Tg fragmentation. Effectively, a 40 kDa peptide reactive with aAb and
mAb was thus obtained by Cu-mediated oxidation of Tg, in the presence of

H202.

We suggest that during hormonosynthesis, ROS which are produced

by oxidative stress induce the production of autoreactive Tg fragments. Since
the 40 kDa peptide contains B-cell autoepitopes, co-localized immunocryptic Tcell epitopes would thus be demasked, breaking the self-tolerance and leading to
autoimmunity.

s 154

Abstracts FEB S'99

(Su/oth/235) Structure of a human TPO peptide containing a eonformational

B-cell epitope specific for Hashimoto's thyroiditis.
V. Estienne, C. Blanchet, P. Niccoli, C. Duthoit, J. Durand, D. Baty,
G. Deleage, P. Carayon, J. Ruf.

((Su/oth/236)

Deparonen~ o f Food Engineering

]stanbul Techmcal L)m er.slr~, 80626 Istanhu[. Tur~e~

Unitd 38 I.N.S.E.R.M. Faoultd de mddecine Timone, Universit~ de la

M~diierranle, Marseille, France.

Deciphering the mechanisms by which proteins play a role in

physiological and pathological events asks for structure-function studies of
their different domains. This approach is particularly suited to understand the
involvement of specific proteins in the molecular mechanisms of
autoimmunity and, more precisely, to clarify the restricted induction by B!ymphocytes of autoantibodies directed to few of the protein epitopes. In the
thyroid model of organ-specific autoimmunity, two proteins, thyroglobulin
(Tg) and thyroperoxidase (TPO) are major autoantigens (aAg). Interestingly,
these proteins are functionnaly linked since TPO is a specific membrane
bound enzyme that catalyses the iodination and coupling of unique tyrosine
residues within the Tg, to form the thyroid hormones.
Regarding TPO, epitope mapping, using monoclonal antibodies,
disclosed two autoimmune domains on the surface of the protein. As
identification of these domains by molecular biology techniques failed, we
investigated the B-cell autoimmune epitopes on human TPO by a biochemical
approach generating proteolytic peptides by enzymatic hydrolysis of TPO in
non-denaturing and non-reducing conditions.The hydrolysate was
chromatographed on a reverse phase column. We eluted a material
immunoreactive with TPO antibodies from patients.By Western-blot, a 20
kDa peptide was found to contain a eonformationnal B-cell epitope; and after
NHrterminal sequencing we localized the peptide at the C-terminal end of the
extracellular part of TPO, within amino-acids 742-848. At least one tyrosine
residue of the peptide was found to be involved in the epitope. The 20 kDa
peptide was thus produced in eucaryotic system. By sequence alignment, the
peptide was found to contain a sushi-like domain followed by an EGF-like
domain. These domains stood into a flexible arrangement which was
stabilized by calcium binding. We proposed a structural model for this peptide
which point out to the tyrosine 772 in the interdomain for epitopic
localization. This peptide was mainly recognized by Hashimoto's but not
Graves'disease patients, thus representing the first disease-related B-cell
autoepitope to be reported.
In conclusion,we propose a structural model for the C-terminal part of
TPO that contains a eonformationnal B-cell epitope specific for Hashimoto's
thyroiditis. This model may allow to a more precise delimitation of specific
auto-epitopes, with would open to targeted immunotherapy and prevention of
autoimmune disorders.

(Su/oth/237)

Characterization of a high-affinity nitrate t r a n s p o r t e r

in Arabidospis thaliana
S. Filleur and F. Daniel-Vedele
INRA-Biologze Cellula.-e, Versailles, Fram e

In an attempt to isolate genes specifically expressed on nitrate, we have

used lhe "differential display" method. DDRT-PCR was performed on total
RNA extracted from roots ofArabidopsis thaliana grown m liquid medium
containing either nitrate or glutamine as sole nitrogen source. This led to
the ~solation of a eDNA that is only present in medmm containing mlrate.
This positive cDNA was cloned and sequenced. The deduced aminoacid ~equence shows 78% identity and 83% similarity with NRT2:INp
prolcin [1]. involved in high-affinity nitrate uptake in Nitwtiana
phtmh,~,inifidia. This suggests that this A. thalicma protein named
NRT2:I At belongs to the high-affinity nitrate transporter family related to
~hc CRNA protein [2].
We have further characterized molecularly this eDNA, First, southern
mmlys~s reveals that at least two copies of NRT2.'IAt gene are present in
gcnome of A. thallana. Only one chromosomal localization was tbund on
the top of chromosome 1. We have undertaken too a physiological study of
NRT2:IAt expression. An increase in Nrt2.'lA! mRNA levels was
ob~,crved in roots 2 hours after Iransfert from glutamine medium to 50ram
or lOmM nitrate medium. Similar results were obtained with the CHL/
~cnc (aF,o named Nrt]:/At), encoding a putative low-affinity nitrate
transporter in A. thalkma [31. In parallel we analysed the NR72:IAt
cxpre,,,,iou in the G'4-3 strain, a A. thaliana nitrate reductase deticient
mub:lnl . The mRNA levels are more important in the mutant than in the
wild lypc. but the regulation of the NRT2:IAt expression is Ihc ~,amc in
both gcnotypes f4].
Fulurc work include the search for an insertional mutant disrupted m the
NRT2:IAt gene. Two oligonucleotides has been derived fi'om the
nucleotidic sequence in order to screen by PCR a library of T-DNA
l u s c r l [ o n a [ illUt~.tnts.
Il ] A
12l I.
13[ Y
141 S,

Quc~.ada et al. (1997) Pta.t Mot 13ioL. 3 4 265-274

Trtlclllall e/a/. (1996) P / a . l Ph.l.~tot Broth.. 5 621 627
I T~ay e t a / (1993) Cell. 72. 705-713
Idlctlr ,rod F. DanieI-Vedclc (19991 Planm. 207' 461 469

Methods for determining aflatoxins in red peppers

Ayse Erkahveci, Artemis Karaali

Toxigenic fungi may invade agricultural plants during growth, harvest or

storage stages The secondary metabolites of these include mycotoxins,
which may evoke pathological changes in man and animals Aflatoxins are
the most important of mycotoxins from the point of vie~,, of occurrence.
toxicity as well as of economy, therefore there is need for effective
methods of analyses for surveys and monitoring programs for individual
suscepuble commodities
This study aimed at selecting the most suitable analytical method for
quantitative determination of aflatoxins in dried red peppers For this
purpose, samples were spiked w;th Aflatoxin B I as the internal standard
The three methods used for extracting the toxin were adapted from BF,
CB[I] and TS methods[2]. Interfering substances were removed by
Steiner's TLC method[3] prior to the application of
classical
semiquanhtative TLC method for quantitative analysis of the toxin[I].
Reverse phase HPLC was also applied as the second quantitative
determination method using a C18 Bondapak column and acetonitrflewater (35 65) as the eluting solvent.
When accuracy of the extraction methods was evaluated based on solely
recovery rates, the TS method of extraction ranked the first, with 98%
recovery in TLC and 73 % recover3,, in HPLC method of detection The
highest repeatability, based on the smallest coefficient of variation was
attained also in samples extracted with TS method. These values were
2.04 in detectioff wlth TLC and 19.2 in detection with FIPLC.

[ 1]AOAC 1990 Official Methods of Analysis, Vol 2, 15t~ Edition, p. 1184.

[2]Trucksess et al, 1980. JAssoc Off..Anal. Chem,Vol 63, p.1052
[3] Steiner et al, 1988. L Agric Food. Chem., Vol 36, p 88

(Su/oth/238)

Risk factor for VtlL patients to develop a Renal Cell Carcinoma

C.Gallou ~, D.Joly t, A.M6jean :, F. Staroz t, N. Martin ~, G. Tarlet ~,
S. Richard 3, C. Junien ~, C B6roud ~.
t INSERM UR 383. : Service d'Ueologie. 3 Laboratoire de Neuro-Oncologie
E P H E : Hdpital Necker-Enfants Malades, PARIS, France.

Renal Cell Carcinoma (RCC) accounts for more than 90% of adult kidney
cancer and is the eleventh frequent cancers. RCC are mainly unilateral and
sporadic but can also be bilateral or associated with the yon HippeI-Lindau
disease (VHL).
The VHL gene, involved both in VHL disease and sporadic RCC, has
been cloned in 1993. It is a tumor suppressor gene localised on 3p25-26.
To investigate the nature of somatic VHL mutations, we analyzed 173
primary sporadic human renal cell carcinoma for this gene using single s~'and
conformational polymorphism analysis and sequencing. We detoct~ abnormal
SSCP patterns in 73 samples corresponding to microdeletinns in 58% of cases,
microinsertions in 17%, nonsense mutations in 8% and missense mutations in
17% of cases.
In addition to these results, we used data from the VHL database to
compare somatic and germline mutations. The study of mutational events
revealed a significant difference between somatic and germline mutations with
mutations leading to truncated proteins observed in 78% of somatic mutations vs
only 37% in germline mutations (p<0,001). We postulated that a specific pattern
of VHL mutations is associated with sporadic RCC. This pattern corresponds to
mutations leading mainly to Uxmcated proteins with few specific missense
mutations. We observed RCC in at least one member of the VHL families in
77% of cases with mutations leading to mmcated proteins vs 55% in cases with
missense mutations (p<0,05). Thus, mutations resulting in truncated proteins
may provide a higher risk to develop RCC for VHL patients.
Moreover, missense somatic mutations correspond to transversion in 68%
of cases and to transition in 32% vs 65% of transition and 35% of transversion in
germline mutations (p<0,001). Since transversions implicate a toxic factor, these
data support the hypothesis of the involvement of environmental factors in the
etiology of sporadic RCC in industrial countries.

Abstracts FEBS'99

(Su/oth/239)

s155

Variable number of antenna genes in

Prochlorococcus as a result of adaptation to different

(Su/oth/240)

light niches
L. Garczarek ~, W. Hess ~ and F. Partensky ~
IObservatoire Oc~anologlque de Roscoff, CNRS et UniversiM Paris
6, Station Biologique, BP 74, F-29682 Roscoff cedex, France
2Humboldt- Universitat Berhn, Institut .fur Biologie/Genetik,
Chausseestrasse l 17, D- 10115 Berlin, Germany
Prochlorococcus is a marine photosynthetic

prokaryote possessing
divinyl derivatives of chlorophylls (DV-Chls) a and b as the major light
harvesting pigments [1]. As the other two known prochloropbytes,
Prochloron and Prochlorothrix, this organism has been shown to possess
a new kind of antenna proteins (so-called Pcb's) [2]. These proteins seem
to have evolved by gene duplication and divergence of the core Chl abinding antenna family, also including CP43, CP47 and IsiA [3], Variations
in the apoprotein composition of this light-harvesting system could be
responsible for the remarkable capacity of adaptation of natural
populations of Prochlorococcus, which can grow from the light-saturated
surface waters down to poorly illuminated deep waters (150 m or more).
Using southern blots, we have shown that the SS120 strain, isolated from
the bottom of the euphoric zone, possesses 7 different pcb gene copies,
whereas only one pcb copy has been identified in the MED4 strain,
originating from the surface waters. Moreover, an RFLP analysis of two
other Prochlorococcus strains (one low- and one high-light adapted) also
indicated a large difference in the number of pcb gene copies. To our
knowledge, those results constitute the clearest example of the effect of
light on the evolution of light-harvesting systems within a single genus.
[1] Partensky F. et al., Microbiol. Mol, Biol. Rev., vol 63, (1999) p. 106.
[2] Laroche et al., Proc. Natl. Acad. Sci., vo193, (1996) p. 15244.
[3] van der Staay et al., Plant Mot. Biol., vol 36, (1998) p. 709.

(Su/oth/241)

ACUTE METABOUC ACTIVITY CHANGES IN RESPONSETO

ADMINISTERED CLENBUTEROL IN PIGS

T.Gojmeracl,B.Marldi62, N. Biland~i6",M. ~uri6~

Croatian VetednaryInstitute, Zagreb, Croatia; = Vuk Vrhovac Inst#ute
for Oiobetes,Endoerfno/ogyand Metabolic /~'soases,Zagreb, Croatia
Clenboterol is a synthetic selective 13~-adnmoceptor agonist widely used as a
bronehodilator in v e t ~
medicine.When administered in 5-to 10- fold
therapeutic dosages, clenlmteral has marked growth-l~omoting effects in slaughter
animals, reduces body lipids, end promotes muscle growth (l,2).Previous reports
have shown that the administration of elenbuterol influences the hormonal and
metabolic state of pigs (3).
The aim of this study was to assess whether acute exposure of female pigs to a
gowth-lrmmoting dose of clenbuterol could influence serum insulin, glucose and
non-esterified fatty acids concentrations. A group of six pigs (F1 generation of
Swedish Landrace x Large Yorkshire) were treated with 10 Ixg/kg body mass of
clenboterol intravenously 30 rain before the first feeding of the day. Blood
samples were collected by jugular veniptmcture at 30, 60, 90, 120, 180, 240 and
300 rain atter treatmem. Serum insulin was determined by the Pharmacia Insulin
RIA 100 kits (Pharmacia Diagnosfics,Ulrpsala,Swedan).Non-esterified fatty acids and
glucose were analyzed enzymatically(Randox, NEFA,Cmmlin, UK; Trace,enzymatic
glucose reagent).
The acute clenbuterol treatment influenced serum insulin, glucose and nonesterkfied fatty acids concentrations, since their mean levels were significantly
higher (!o<0.01;1o<0.01 and p< 0.001, re~tively).
The postprandial increase in serum glucose and insulin concentrations was
significantly higher (la<0.0t) at 120 mi~ after treatment with clenbuterol than in
the control animals, whereafltr the values of these biochemical parameters
decreased.
The results obtained suggested the acute treatment of pigs with a growthpromoting dose of clenbuterol showed a notable lipolytic and glycogenolytic
effects.
References: Peters, AR., Pig News and Inf. ll: (1990), 519
Zmmmrli,UV. and Blum, IW., LAnim.Physinl.AmmNutr: 63:(1990) 157
Baldi A., et al., J.Vet.Med. 41: (1994), 186

Effect of 6-bromo-6-deoxy-t~ascorbic
acid on
cisplatin-induced toxicity in mice
M Gavellah V. Sverko", M Rada&d". I Ljubenkov ~,
M Eckert-Maksic'. ~R 13o.~,'1,ovlchr./mite. Zagreh, I'll )'hovac
hl~tmae.Zagreb, Croatia. ' f l I O V I / l l ( Ka.Qe/-,S~tgttroc, ('/.oolla

The biological aspects and pharmacological significance of

combined treatment of 6-bromo-6-deoxy-ascorbic acid (6-BrAA)
and cis-dlam~nedichloroplatinum (cis-DDP) in mice model are
investigated The results indicate that effectiveness of 6-BrAA on
biological response(s) is sharply dependent on the dose of cisDDP. The treatment with lower dose of cis-DDP (10 mg/kg bw) and
pretreatment with 6-BrAA (480 mg/kg bw) 2 h before cis-DDP
contribute to the tissue protecting effect of modulating agent
reducing in some extent the nephro-, liver- and spleen- toxicity.
The blood n~trogen urea level and the lipid peroxidation process
detected as thJobarbituric reactive substances (TBARS) were
significantly lower (p<0.01 and p<0.05, respectively), while liver
lysosomal membrane permeability and total sialic acid content in
spleenocytes did not attained statistical significance compared to
cis-DDP treated mice. On the other hand, 6-BrAA in animals
treated with higher dose of cJs-DDP (15 mg/kg bw) leads to
exacerbation of the toxic cis-DDP effect and concurrent loss of
protective potenttal of the 6-BrAA toward hssue damage. Also, a
significant decrease in protein concentration (p<0.05) in
comparison to untreated as well as cis-DDP treated mice was
observed. The exact mechanism(s) of 6-BrAA protection and
exacerbation of the toxic cis-DDP effect is unclear, though
scavenging or generating of free radicals may play an important
role. The results obtained may be of importance in planing the
rational use of cis-DDP and 6-BrAA administration in the potential
treatment of cancer.

(SIi/oth]242)

Negative Temperature Effect on Drying Rate of Agadcus bisporus

Mushrooms under Low Temperature Drying Conditions
Ferda Gima~Seyhan.E. Ozgul E-~a'anuz
lstanbul Techmcal University.80626lstanbul. Turkey

Dehydrated mushrooms are valuable specialty ingredient in various

sauces and soups Since mushrooms are very sensitive to drying
temperature during hot air drying, drying at low temperature
conditions could b: .~ potential alternative in the prevention of the heat
damage to the product Cultivated mushrooms (AgwTcus blsporus)
slices 2 5 and 5 0 mm thick were dried at 20 , 30 and 40C with air
of 1 I%RIt or 32%RH and rehydration ability of dried mushrooms was
used as the criteria for the evaluation and determination of optimum
drying condition. Drying rate of mushrooms decreased as the
temperature was increased from 30 to 40C The negative effect of
temperature was more pronounced during drying with the air of
32%RH than I I%RH and for 5mm thick samples than for 2 5mm
thick samples. Rehydration studies showed that rate of water
absorption decreased for those samples dehydrated at 40C compared
to those dehydrated at 30C irrespective of the air relative humidity
and the sample thickness. It was concluded that the quality of
dehydrated mushrooms obtained under low temperature drying
conditions was depended on the rate of drying For 2 5ram thick
samples, good quality was obtained by drying at 30C with the air of
either } I%RH or ~z%RH For 5ram thick samples, good quality was
obtained by drying at 20",2 "vith the air of 1 I%RH and at 30C with
the air of 32%RH

s 156

Abstracts FEBS'99

(Su/oth/243) ~-Mannosidosis: Enzymatic activity and proteolytic

processing of ~-mannosidase mutants
GM Hansen~, HMF Riise I, 2, OK Tollersrud ~and ~ Nilssen2
1

"

:DepartmentofMedicalBtochemistry,
University of TromsO, Norway.

: Departmem ofMedwal Genetics,

c~-Mannosidosis (OMIM # 248500) is an autosomal recessive lysosomal

storagedisorder caused by deficient activity of lysosomal u-mannosidase
(LAMAN) (EC 3.2.1. 24). The disease is characterized by massive
intracellular accumulation of mannose-rich oligosaccharides and typical
clinical features are mental retardation, hearing loss, immune deficiency and
skeletal changes. ~-mannosidosishas also been described in cattle and cat.
We have previously identified 30 putative disease causing mutations in the
the LAMAN-gene, of which 3 were in animals and 27 in patients from
several countries.
In this study we have analyzed the effects of some of the disease-causing
mutations on the activities and proteolytic processing. By site-directed
mutagenesis of the human LAMAN-cDNA, we have reconstructed 9
missense mutations and two minor in-frame deletions. The mutant genes
were expressed in COS cells and the enzymatic activities were measured.
Preliminary results indicate that the activities of the mutant enzymes are
severely reduced (to < 2%). Pulse-chase experiments are in progress to
analyze how the mutations affect the proteolytic processing of the enzyme.

((Su/oth/244) In the yeast adaptive stress response to H202, cAMP differently

controls the Msn2/4p and the Yaplp transcriptional factors.
R. N. Hasana, M Tol~danob, J, Labarreb, M. Jacqueta,
and E. Boy-Marconea
aUniverstt4 Paris-Sud IGM, UMR 8621, B~timent 400, 91405 Orsav France
b CEA Saclay, SBGM, Bdtiment ]42, 91191 Gif-Sur.Yvette C~dex, France

In response to oxidative stress, the yeast transcriptional regulator

Yap lp induces the transcription of the Yap 1-dependent genes, most of
which code for antioxidant proteins. This response involves nuclear
localization of Yaplp and its binding to the YRE (Yapl Response
Element) present in the promoters of many such genes.
We have analysed the effect of cAMP on this stress response, at the
transcriptional level and through comparative two-dimentional gel
electropboresis of total cell proteins. Our results show that the
transcriptional induction in response to H 0,, of a YRE-IacZ construct
and of three Yaplp-dependent genes, is not affected by cAMP.
However, in the same conditions, the induction of three Msn2/4pdependent genes is repressed by cAMP, Msn2/4p being the
transcription factors activated by various stresses. Two-dimentional gel
analysis extends this observation to a larger sample of Yaplp and
Msn2/4p dependent genes.
These results seem paradoxal in view of those of Fernandes et al, [ 1]
showing that the Yaplp-dependent transcriptional induction is
abolished in the absence of Bcy lp, the regulatory subunit of the PKA
(cAMP-dependent protein kinase). This suggests that an excess of PKA
activity could lead to non-physiologic responses.
[11Mol. Cell. Biol. 1997, Vol 17:6982-6993

(Su/oth/245)

The Escherichla coil Xanthosine Transporter

M. H. Hauberg-Norholma, G. Dandanella
aDept, of Biological Chemistry, institute of z!:lolecular Biology. Umversi~' of.
Copenhagen, Solvgade 83[-I, DK-t307 Copenhagen K Denmark

(Su/oth/246)

Expression of matrix metalloproteinases during induction

of differentiation of human leukemic cells
P.Hollender~,L.Devyb,H.Nasrallah,A.No~lb,C.TrentesauxJ,J.
M.Foidart h, and P.Jeannesson ."Fac.Pharmacte, EA2063, Reims,
France ; blab. BioL Tumeurs et D~veloppement, Univ. Liege, Belgique.

The 52-rain region of the scherichia coli genome contains the xapABR operon
revolved m the metabolism of purine nucleosides. The xapR gene, which is
constitutwely transcribed, encodes a LysR family type regulatory protein.
W~en xanthosme is present XapR promotes transcription of xapA and xapB.
The xapA encoded product, xanthosine phosphorylase, can degrade all purine
nucleosides and the deoxy derivatives except (deoxy-) adenosine, but only
xanthosme can act as inducer. This salvage enzyme makes E. coli capable of
using xanthosine as a carbon and energy source.
Previously it has been suggested that together with xanthosme phosphorylase a
new uptake system was induced[l]. When the operon was sequenced in 1995,
it became clear that this property at least ha part could be assigned to the
presumed product of the xapB gene[2]. Supporting the role of xapB as a
nucleoside transporter, XapB was found to be enriched in the membrane
fraction in mmlcells. The hydropathic profile of the protein indicates that it is a
membrane protein with 12 transmembrane segments (12TM). This is m
contrast to identified nucleoside transporters from all other organisms,
Typically these are 9 or 14TM's. In this work we present strong evidence that
the protein indeed is a 12 TM nucleoside transporter. The ability of XapB to
transport nucleos~des is compared to that of the two well-characterised
transporters NupC and NupG. A yet tmcbaracterised ORF from the K coli
genome project is homologous XapB and NupG. Thus E. coli seems to have
three unusual 12 TM nucleoside transporters defining their own class in the
NHS superfamily.
i. K. Hammer-Jespersen et al., MoLGen.Genet. 179, (1980) 341-348
2. C. Seeger et al., ABactertol 177, (1995) 5506-5516

Leukemias are generally characterized by abnormal proliferation and

differentiation of immature white blood cells in the bone marrow. In acute
and chronic myelogenous leukemia, immature leukemic cells are able to
egress into peripheral blood to infiltrate extramedullary organs. We have
investigated the relationship between the invasive properties, the expression
of matrix metaUoproteinases (MMPs) and the differentiation state of human
promyelocytic HL60 cells, a model of acute leukemia. Boyden chambers
coated or not with the reconstituted basement membrane (Matrigel) were
used to quantify the migratory and the invasive activities of these cells.
After 6 h of incubation, a small fraction of ill60 (1 0.2%) were found to
migrate and to penetrate the Matrigel layer. Induction of granuloeytic
differentiation by all-trans-retinoic acid (ATRA) or by aclacinomycin
(ACLA), an anthracycline antitumor drug, strongly increased HL60 cell
invasiveness by 17-fold for ATRA and 42-fold for ACLA. As assessed by
gelatin zymography, HL60 cells constitutively released significant amounts
of proMMP-9 or progelatinase A (92kDa) and moderate amounts of
proMMP-2 or progelatinase B (72kDa). Granulocytie differentiation
induced by ACLA (71 + 2% NBT-positive cells) markedly enhanced
proMMP-9 production and this treatment is associated with an enhanced
migratory activity and invasiveness. Interestingly, ATRA- induced
differentiation (65 -+4% NBT-positive cells) decreased MMP-9 expression,
suggesting that other proteolytic enzymes could be involved in these in
vitro mechanisms. In conclusion, these results indicate that human leukemic
cells produce MMPs which may i) contribute to the degradation of
basement membrane barriers, ii) play a role in the progression of leukemias
and iii) be regulate by differentiating agents.

Abstracts F E B S ' 9 9

(Su/oth/247) Selective
expression
of
rat
7-glntamyl
transpeptidase promoters 4 and 5 in biliary and
immature hepatic ceils. N.Holic, T.Suzuki, I.Lamy,

s 157

(Stl/oth/248)

S. Huyghe, PE.Declercq, P.P. Van Veldhoven and M, Baes

MN.Chobert, A.Curlu, Y.Laperche. INSERM U-99. 94010 Cr~tetl

Laboratory of Climcal Chemistry, Katholieke Universiteit Leaven, 3000

Leaven, Belgium

~zndINSERM U.49, 35033 Rennes, France.

In adult liver, 7-glutamyl transpeptidase (GGT) activity is

associated to biliary cells [1]. However, GGT expression in hepatic foci
and periportal hepatocytes following xenobiotic treatments has
questionned the use of GGT expression as a biliary marker. This single
copy gene is transcribed from five alternate promoters into multiple
mRNAs (I to V) [2]. This diversity in the GGT gene expression is not
accessible at the protein level since all transcripts differ only in their 5'
non coding region and are translated into a unique polypeptidic chain.
GGT transcripts were analyzed by quantitative RT-PCR and in situ
hybridization during rat liver development and in vitro differentiation of
liver precursors cells (hepatoblasts).
In embryonic liver GGT mRNAs III, IV and V accumulation
was detected in hepatoblasts. Maturation of these cells into hepatocytes
leads to a sharp and early decrease of mRNAs IV and V, and a late
decrease in mRNA III accumulation initiated from birth. In contrast
differentiation of hepatoblasts into biliary cells induces a diseappearance
of GGT mRNAs III and V and an increase in GGT mRNA IV
accumulation.
In order to confirm these data, we induced in vitro
differentiation of hepatoblasts in biliary or hepatocytic lineage by Na+
butyrate or hydrocortisone, respectively. We observed that
differentiation in the biliary lineage is associated with a 175 fold
induction of mRNA IV and, conversely, differentiation of hepatoblasts
in the hepatocytic pathway leads to an extinction of GGT mRNA IV
expression.
All together these data indicate that GGT gene expression from
promoter 5 occurs only in hepatoblasts and expression from promoter 4
is a characteristic of cells differentiating in the biliary lineage. In
conclusion, the GGT gene is transcribed from multiple promoters in
liver cells depending upon their differentiation stage.
[1] Holic et al., Am J Pathol, 152, (1998) 1039.
[2] Nomura et al., Biochem. J., 326, (1997) 311.

Developmental studies of enzymes of the peroxisomal

13-oxidation by Northern hybridisation

Patients with peroxisome biogenesis disorders, including Zellweger syndrome, present with several organ defects (brain, kidney) that are already
present during embryonic development. In order to understand the pathogenesis of these disorders, it would be of interest to document the appearance of the different peroxisomal pathways during development (p-oxidation,
o~-oxidation, etherphospholipid synthesis and isoprenoid synthesis). However,
the current assays do not allow a reliable measurement of enzyme activities in
foetal rat or mouse tissues. Detection of the proteins is also hampered by the
lack of sensitive antisera. Therefore, the ontogeny of peroxisomal enzymes in
different mouse tissues was evaluated at the mRNA level by Northern analysis. Northern blots contained total RNA isolated from total embryos at day
9.5 post conception (E9.5), from liver and brain of Ell.5, E14.5 and E18.5
embryos, and from mice aged 1 day, 1 week, 3 weeks and 6 weeks.
In a first stage the enzymes of the different [3-oxidation pathways were examined. The mRNAs encoding the 3 peroxisomal oxidases were differentially
expressed in liver and brain, before birth palmitoyl-CoA oxidase was more
abundant in brain than in liver whereas postnatally liver was more enriched,
pristanoyl-CoA oxidase was ubiquitously expressed but at very low levels;
trihydroxycoprostanoyl-CoA-oxidase was selectively expressed in liver from
E 14.5 on. Transcripts for the L-specific multifunctional protein (MFP-1), believed to play a role in the oxidation of straight chain fatty acids [I], were
abundantly present in postnatal liver but only at very low levels in prenatal
liver, and were absent in brain at all stages. On the contrary, the transcripts
for the D-specific multifunctional protein (MFP-2), involved in the oxidation
of 2-methyl branched chain fatty acids and bile acid intermediates [1], were
present throughout development not only in liver but also in brain. This study
will be completed by analysing thiolase and SCP-X mRNA expression.
The early expression of mRNAs encoding palmitoyl-CoA oxidase and MFP-2
in liver and brain might indicate that peroxisomal !3-oxidation of certain substratcs is important for embryonic development.
[1] Dieuaidc-Noubhani M. ctal., Biochem J., 325, (1997) 367.

(Su/oth/249)

Use of biochemical parameters for the monitoring

of patients with diabete disorders
Dr J.C. Hyronimus,D.Canonge
UniversityHospitalof Fort-de-France97261 Fort-de-FranceCedexMartinique

Diabete disorders arc frequently associated with hyperlipidic deseases and

represent a significant percentage of the deseases diagnosed in Fort-deFrance University Hospital. This situation has led us to develop a system
that allow us to take into account selected biochemical parameters for the
follow up of this pathology.
This includes
glucosuria
blood glucose level (fasting)
glycemic 24 hour cycle
high provoked glycemia
hemoglobin Alc (HbAle) which is formed by the non-enzymatic
glycation of the hemoglobin A0, The level of HbAlc is proportional to the
level of glucose in the blood over a period of approximatively two months
(Usual value < 6% of total hemoglobin).
fructosamine measurement monitor to the glucose status over a
shorter time frame 1-3 weeks than glycohemoglobin (Usual value < 285
mmol/L),
and some lipidic parameters such as cholesterol, triglycerids,
apolipoprotcins A and B.
Patients diagnosed as having diabete has hyperglycemia that results in
glucosuria when the renal threshold for glucose is exceeded. With
glucosuria there is polyuria and thirst. With the need to metabolize protein
and then fats, ketone levels rise in the blood and urine, and with the
excretion of ketones and the accompanying base, metabolic acidosis ensues.
Measurement of blood pH (Usual value 7.38 - 7.44) , urea (Usual value :
2.49 - 7.47 mmol/L), and bicarbonate (Usual value : 23 - 29 Meq/L) allow
us to follow the acidosis level. A high level will be treated by intravenous
injection of sodium bicarbonate simultaneously with medical treatment of
diabete.

(Su/oth/250)

Fine specifieities of murine anti-Mg monoclonal antibodies.

E. Ja~kiewicz. D. Blanchard*, M. Rasamoelisolo*, M, J. Loirat*
J. J. Moulds and E. Lisowska
Institute of Immunology and Exp. Therapy, 53-114 Wrodaw. Poland
*Etablissement de Trans~ Sanquine, 44011 Names, France

The Mg antigen is a rare variant of glycophorin A (GPA), a major

sialoglycoprotein of human erythrocyte membranes, but anti-M g antibodies
are present in I-2% of human sera and their origin is not known. GPA
exists in two common genetic forms that give rise to the MN blood groups
[1).These two forms differ in amino acid residues at positions 1 and 5 of
the polypeptide chain (GPA-M: tSSTTGVAM8; GPA-N: ILSTTEVAM8).
GPA-M g has the sequence of GPA-N in which Thr4 is replaced by Asn
residue (LSTNEVAM). Due to this replacement the Ser2 and Thr4 residues,
which are glycosylated in normal GPA, are not or poorly glycosylated in
GPA-M g. Peptidic character of Mg antigenic site allows to examine the
specificity of antibodies by means of synthetic peptides.The specificities of
two murine anti-Mg monoclonal IgGl antibodies, 2D5 and 3B10, were
determined by pepscan analysis. Both antibodies bound strongly to the Nterminal Mg octapeptide LSTNEVAM. but they showed different
subspecificities. The essential fragment of the epitope 2D5 are amino acid
residues 2STNEVr. In contrast, the Leul and Asn4 residues were most
essential components of the epitope 3BI0, while Ser2, Thr3 and Glu5
seemed to be less important. Our present results and earlier ones on the
specificity of murine anti-M antibodies crossreacting with Mg [2], showed
that monoclonal antibodies reacting with Mg antigen recognize different
fragments and different amino acid residues of the amino-terminal
nonglycosylated domain of GPA-M g. Planned application of pepscan
analysis to human anti-M s antibodies may be helpful in elucidation of their
unknown origin.
[ 1] Lisowska, E. in The Molecular Immunology of Carbohydrates
(ed.Wu,A.M.), 1988, p.265,Plenum Press, New York.
[2] Ja~kiewicz, E., Czerwifiski. M., Syper, D. & Lisowska, E., Blood, 84,
1994, p.2340.

s 158

Abstracts FEBS'99

(Su/oth/251)

Endothelial nitric oxide synthase gene polymorphism and

hypertension.
M. J~ichymovd.K Hork~'. J Bultas, J. Pelegka. P Martasek*

((Su/oth/252)

2~aDept. oflnternal 3[edtctne aml *Dept of Paediatrtcs, Faculty of

Aledecme L Charles ( )Tzverstty. 128 O~Prague. Czech Republic.

Introduction: Mammalian tissues express three isoforms of nitric oxide

sznthases (NOS) which catalyze the formation of NO and L-citrulline from Larginine by two successive oxygenation reactions Endothelial NOS (eNOS)
produce nitric oxide (NO), which diffuses into vascular smooth muscle cells,
activating gnanylate cyctase and causing the down regulation of Ca2 and
vasodilatation
Objective: Hypertension, atherosclerosis, vasoconstriction and thrombosis
may exhibit endothelial dysfunction characterised by a reduction on binavailable endothelium-derived NO. The mechanism underlying impaired NO
production is not fully understood Studies of polymorphism of eNOS gene in
diseases characterised by endothelial dysfunction are in the first stage of
investigation. We studied G298A polymorphism in eNOS gene (localised in
exon 7) in distinct groups of hypertensive subjects and their offspring.
Design and Methods: Studies were performed in 4 groups The first group
(SN) consists of 102 normotensive men without family history of
hypertension The second group (SH) consists of 107 normotensive men
from families with hypertension at least in one of parents. The third group
(RH) of 74 hypertensive patients resistant to the conventional antihypertensive
therapy was compared with the group (C) of 58 age- and sex-matched
norrnotensive controls. DNA was extracted from white cells and genotyped
for G298A polymorphism
Results: In RH group the higher frequency of homozygous 298 Asp/TT
geootype was found as compared with group C (16% vs 8%, respectively).
In SH group with genotype GT and TT the significant higher levels of
systolic (SBP) and diastolic blood pressure (DBP) were found as compared to
corresponding genotypes in SN group
Conclusions: Our finding of association of T allele for eNOS gene with
higher BP in SH suggests that T allele might represent in these subjects
disadvantageous variant with respect to for the later manifestation of
hypertension
Supported by grant No.306 99 054 t)f GA('R. wld No. 74195-541101 o f
Howard Hughes Medtcal htstimte.

(Su/oth/253)

Characterizationof Nuclear
Selenium-containingProteins of the Rat
A. Kyriakopoulo~,D. R~thlein, S. Kappler, D. Behnc

Hahn-Mettner-ltTstlttlt, Dept "Trace Element~ m Health an Nutrltatton".

Ghemc'ker Sir IO0. 14109 B~rlm

The essentiality of selenium has been shown to be due to

the biological functions of several selenoproteins. After in
vivo labelling of rats with 75 Se, preparation of the kidney
homogenate and protein separation by SDS-PAGE, the
labelled selenium-containing proteins were determined by
autoradiographie (1). In the molecular mass range of 23 25 kDa. where the subunits of glutathionc peroxidase are
situated, two 75Se-containing bands were present. After
subcellular separation they were found to be enriched in
the nuclear fraction. For further characterization of these
proteins morphologically intact labelled nuclei were analyzed by 2D-electrophoresis. In this way the seleniumcontaining bands in the 23 - 25 kDa range were resolved
into (a) six double spots and (b) a single spot with isoelctric pl-values bet~,,een 5.7 and 6.9. The subunits of glutathione peroxidase were present among the double-spots.
After proteolytic degradation on the reelectrophoresis gel
and separation by SDS-PAGE the protein spot complex (a)
and the single spot (b) showed different peptidc patterns.
In the case of (a) six labelled peptides with molecular
masses of 20. 18. 16. 15. 13 an 12 kDa were lbund while
with (b) only tbur peptides "s,ith molecular masses of 21.
17. 14. and 12 kDa were visible. For more detailed intbrmarion the different peptidc fragments were analyzed by
means of RP-I-IPI.C.
(l) Behne. D. et. a[.; Biol. Trace Elem. Res. 55. (19961 99

Developing fish embryos fail to adjust membrane fluidity

to temperature
K. Kitajka~, E. Fodor a, I. Csengeri b, T. Farkasa
aBtologoeal ResearchCentre, Szeged, blast. Fish Res., Szarvas, Hungary

Adult fish has been shown to adapt membrane fluidity to reduced

temperatures. This adaptation process is accompanied by an accumulation
of phospholipid molecular species containing a monoenoic fatty acid in the
position sn-I and a polyenoic fatty acid in position sn-2 of the molecule
(like 18:1/22:6) [1] [2]. Carp (Cyprinus carpio) eggs were fertilized at 22C
and germinated at 18C and 26C. Development was complete in two days
at 26C and in three days in 18C. Plasma membranes, mitochondria and
endoplasmic retieula were prepared before fertilization, at 4-8 cell stage, at
the time of appearing of heart beating, and resorption of the yolk sac to
determine fluidity relationships by DPH fluorescence polarization technique
and the molecular species composition of phosphatidylcholines (PC) and
phosphatidylethanolamines (PE) by HPLC. There was an equal decrease in
fluidity of all membranes at the start of cell division irrespective of the
incubation temperature. Effect of the incubation temperature on membrane
physical state was evident only in free living larvae after the yolk sac had
been consumed. Similarly, accumulation of monoenoic/polyenoic PC and
PE at low incubation temperature was apparent only in free swimming
larvae. Levels of these species were considerably lower in embryos
throughout the whole development than in the unfertilized egg. It is
concluded that yolk supplies the membranes of embryos with the necessary
phospholipids while protein synthesis starts immediately after start of cell
division and these proteins may render the membranes more rigid. Embryos
at early developmental stages incorporate phospholipids offered by yolk
without
any
selection
or
modification.
Accumulation
of
monoenoic/polyenoic molecular species in free living larvae may be an
indication of expression of delta-9 desaturase induced sometimes before
resorption of yolk sac. Delta-9 desaturase was induced in two days in carp
liver in response to cold exposure [3].
[1]: Dey et al., PNAS, 90, (1993) 7498.
[2]: Fodor et al., Lipids, 30, (19951 1119.
[3]: Schfinke et al., BBA, 734, (1983) 70.

(Su/oth/254)

An illustrated database in Cell and Molecular Biologyt

H. Lorino*, P. Rouet**, M-P. Junier***, C Mariette **~*
INSE1LM*U492, "*U400and ***U421. ****CHUHenri Mondor
94010 Cr~tell. France (e-mail"iter,~a?im3msem&r)

In cortical collecting duct (CCD) of rat kidney, H,K-ATPase is

stimulated in response to salmon caleitonin (SCT) and isoproterenol (Iso) in oc
and ~ intercalated cells respectively. In these cells, SCT and Iso increase
cAMP production and activate protein kinase A (PKA). The aim of this study
was therefore to determine the role of PKA in SCT- and Iso-induced
stimulation of H,K-ATPase by evaluating the effect of protein kinase A
inhibitors on this stimulation in microdissected CCDs.
Inhibitors of PKA catalytic subunits, H89 and myristoylated peptide
14-22 amide, blocked Iso effect on H,K-ATPase, indicating that it is mediated
by the cAMP/PICA pathway. Conversely, these two inhibitors did not alter
SCT effect on H,K-ATPase, indicating its independence towards PKA.
Unexpectedly, SCT-induced stimulation of H,K-ATPase was blocked by
bisindolylmaleimide I (GF), a prot6ine kinase C (PKC) inhibitor. Because we
demonstrated that SCT did not activate PKC through the classical
phospholipase C pathway, we evaluated if this stimulation of PKC was
indiuced by cAMP.
Inhibition of AC by 2',5'-dideoxyadenosine abolished SCT-induced
stimulation of H,K-ATPase, demonstrating that activation of AC is necessary
for this effect. To further investigate the role of cAMP in SCT action, we
determined the effects of its permeant analogue 8-Br-cAMP and of forskolin
(FK) in the presence of H89 to abolish the 13intercalated cells response. Under
these conditions, 8-Br-cAMP and FK stimulated H,K-ATPase, and this
stimulation was fully inhibited by GF. Thus, 8-Br-cAMP and FK mimicked
the effect of SCT on H,K-ATPase in ~ intercalated cells.
In summary, these results demonstrate that in rat CCD o~ intercalated
cells SCT stimulates H,K-ATPase activity by an atypical AC/cAMP/PKC
pathway. They also suggest the existence of a cAMP-binding protein other
than PKA in these cells. Rp-cAMPS, a structural analogue of cAMP which
inhibits PKA regulatory subunits, nzimicked SCT effect on H,K-ATPase. This
demonstrates that, conversely to PKA, the cAMP-binding protein involved in
SCT action is equally stimulated by cAMP and by Rp-cAMPS.
Recently, Epae, a cAMP-activated protein has been cloned from a brain
library. Northen blot assays showed that Epac mRNAs are abundantly
expressed in kidney. By RT-PCR on microdissected nephron segments we
found that mRNA coding for Epac is expressed at a high level in rat CCD. This
makes Epac a possible canditate for the cAMP-binding protein involved in SCT
stimulation of H,K-ATPase in o~intercalated cells.

Abstracts FEBS'99

(Su/oth/255)

An illustrated database in Cell and Molecular Biology

s159

(Su/oth/256)

1] Lorino*, P Rouet**, M-P Junier***, C Mariette ****

INSERM *U492, **U400 and *~*U421, ****CLIOIlenri Mondor
94oln Crdlelt, France (e-mail citer@inl3.ulsermfr)

The interactive database BiOiB is an expanding encyclopedia which

aims at defining terms and abbreviations used in Cell and Molecular
Biology, in relation with cell structure, signaling, metabolic processes,
life and death. This database is more especially directed towards the
researchers, teachers and students in Biology and Medicine.
In 1999, BiOiB contains 2500 terms, 1200 figures, 16 movies and
20000 cross-references
Terms represent structures, biochemical compounds, mechanisms,
techniques .... Each entry contains a definition and an overview of the
main features, completed by coloured figures when useful Most terms
are grouped into general Topics. References from reference scientific
books are also included.
For each entry, a list of the BiOiB terms appearing in the definition
(eross-references) allows to surf through the database
Example. glucocorticoid receptor (GR) is cross-referenced with.
CREB protein (cross-referenced to. GRE, cAMP response element,
PKA, gene expression, ..)
cytokine (cross-referenced t o lymphocyte, T cell, antigen, ..)
Hsp90 (cross-referenced to' chaperone, steroid hormone, . )
GRE (cross-referenced to response element, gene, transcription. .. )
BiOiB is available on Macintosh- or PC-compatible CD-Rom.
A French version of the database is under development on Internet.

Molecular and genetic basis of neonatal diabetes mellitus,

implication of epigenetic factors.
E. Marquis*,J.J. Robert*, P. Bougn~res**,C Junien*,C Diatloff-Zito+. (+)
INSERM U383 (+)(*)H6pitalNecker-Enfants Malades Paris France,(**)
Hdpital Saint Vincentde Paul Paris France

Neonatal Diabetes Mellitus (NDM) is a rare entity characterized by

hyperglycemia, glucosuria frequently associated to intrauterine growth
retardation. Plasma insulin levels are undeteetable, patients require insulin
therapy to maintain blood glucose. In about half the neonates the diabetes is
transient (Transient Neonatal Diabetes Mellitus, TNDM) resolving within 6
months. In approximately 50% of the patients the diabetes is permanent
(Permanent Neonatal Diabetes Mellitus, PNDM). TNDM predisposes to
diabetes type 2. A failure of insulin-releasing mechanism or a delayed
maturation of pancreatic 13 cells may explain this phenotype. Paternal Uni
Parental Disomy of chromosome 6 (UPD6) has been reported in some TNDM
suggesting the ~mplication of an imprinted gene. A candidate region has been
narrowed to the 6@3-@4.
In order to contribute to the characterization of NDM, we have studied
14 patients with NDM collected m France. We analyzed 23 microsatellite
markers spread along the chromosome 6. UPD6 (isodisomy) was found in two
out of six patients with TNDM. It is found that the patients have inherited one
paternal allele all along the chromosome 6 with an absence of maternal
contribution. The different pre and post-natal growth patterns of these two
patients points out the complex inter-relationships of the genotype/phenotype.
No alteration of the parental specific methylation marks of the [GF2R gene
proximal to the TNDM candidate region could be found in the patients
examined. In a consanguineous family with PNDM the haplotypes for the
chromosome 6 markers lead to the exclusion of IDDM5, 8, 15 loci, and of the
TNDM candidate region. The results are m favor of the involvement of an
imprinted gene in some TNDM. It can be suggested that at least one other
locus, which needs to be determined, should be implicated in NDM.

BiOiB will be presented on a computer at the poster site.

(Su/oth/257)

nm23-HS, a testis specific nm23/NDP kinase homologue

A. Mtmiera, C. Feral b, L, Milon a, J. Capeau a, G. Guellaenb,
M.-L. Lacombe n. aINSERM U402.CHUSt-Antome,75012 Parts,
blNSERM U99 H@ital H. Mondor, 94010 CrOteil. France

Nucleoside diphosphate (NDP) kinases are ubiquitous enzymes

responsible for synthesis of most cellular nucleoside triphosphates. Four
human NDP kinases have been identified. NDP kinase A, the product of the
nm23-H1 gene, is involved in tumor progression and metastasis (reviewed in
[1]) while NDP kinase B, the product of nm23-H2, is the transcription factor
PuF for the proto-oncogene c-myc. DR-nm23, is involved in differentiation
and apoptosis of myeloid cell lines while Nm23-H4, identified in our
laboratory, is a mitochondrial NDP kinase [2].
We report here the identification of another member of the human nm23
gene family, nm23-H5 [3]. The cDNA was obtained by sequencing RT-PCR
fragments generated using primers designed from "expressed sequence tags"
(ESTs) homologous to nm23-H4. The sequence predicts a 212 amino acid
protein possessing a region about 30% identical to the other human NDP
kinases and a COOH-terminal extension of 51 residues which exhibits no
significant homology to known proteins. Despite the presence of most of the
conserved residues shown to be crucial for catalysis, Nm23-H5 expressed in
E. coli does not exhibit NDP kinase activity. This suggests that additional
factor(s) could be required to reveal the activity or that the protein is endowed
with other, yet undefined, function(s). The nm23-H5 gene is located on
chromosome 5q23-31. Its distribution is very tissue specific, with one main
transcript of 1.1 kb being highly expressed in testis. In situ hybridization
experiments show that the expression is selective for germinal cells,
spermatogonia and early spermatocytes, suggesting a role in early stages of
spermatogenesis.
[1]
[2]
[3]

De La Rosa A, et al., BioEssays, 17, (1995) 53.

Milon L. et al., Hum. Genet., 99, (1997) 550.
Munier A. et al., FEBS Let., 434, (1998) 289.

(Su/oth/258)

Sulfation of thyroglobulin and its relation with

thyroid hormonosynthesis.
MC. Nlend, D. Cauvi, N. Venot, O. Chabaud
INSERM U38. Factdtd de mddecine, 13385Marseille. France

We investigated the possible role of sulfate group bound to thymglobulin

(Tg), the precursor of thyroid hormones, in the hormonogenic process
which consist of iodination of tyrosines followed by the coupling of
certain iodotyrosines. The binding of sulfate to tyrosine and to complex
oligosaecharides is modulated by thyrotropin (TSH) ll 1. We hypothesized
that sulfated tyrosine (Tyr-S) could be involved in this process since the
consensus sequence required for tyrosine sulfation was found in the Tg
hormonogenic sites. In a first study Tg was secreted by porcine thyrocytes
cultured on collagen-coated filters with TSH and without iodide in the
presence of 35S-sulfate. In vitro hormonosynthesis was performed on this
35S-Tg with increasing concentrations of iodide (KI). From 10 atoms of
iodide incorporated into Tg the amount of Tyr-S was decreased by about
twofold. In these conditions the rate of hnrmonosynthesis reflecting
essentially the coupling of iodotyrosines was about 40%. An approach
was carried out to decrease Tg sulfation: cells were incubated with or
without sodium chlorate (3raM) known to be a good inhibitor of sulfation.
In chlorate-treated cells, 35S-sulfate incorporation into Tg was decreased by
90%, and within this inhibition only 50% of the amount Tyr-S was
recovered. In vitro hormonosynthesis was then performed with ~25I-iodide,
and the rate of hormonosynthesis was found decreased by about 40% in
Tg secreted by chlorate-treated cells. Comparatively when cells were
incubated with ~2~I-iodide and with or without chlorate, the rate of
hormonosynthesis was also decreased by the same range in L25 I-Tg
secreted by chlorate-treated cells.
In conclusion these results lead us to think that there could be a relation
between the sulfated tyrosine of Tg and thyroid hormonosynthesis.
I I / . Nlend et aL, J. Endocrinol. Lnvest. 19(6): 38(abs 76)

s160

Abstracts FEBS'99

(Su/oth/259) Reactive Oxygen Species assessment, in live adherent cells

using Microplate CytofluorimetD - Application to Tacrine

((Su/oth/260)

R. Osseni a, C. Debbasch a, M.O Christen b p. Rat a J.-),l. V~'arneta

a I/ran:de Plutrma~, To~t~oh~et~" CelluhI~re CH,~O 1~/20 7~012 P~ol~
h ~1~ al Ptuo sll~lBP22 ~92151 S'ule,~m ,~ (~,dcl F~aln e

. The mechanisms leading to tacrine (THA) hepatotoxic effects arc not ye!
fully understood. Reactive oxygen species (ROS) overproduction and
intracellular reduced glutathione /GSH) depletion are common mechanisms
involved in drug toxicity.
, The aim of this study was to investigate, on the human liver cell line
HepG2, whether THA at human blood concentrations mduces ROS
production stimulation and/or GSH depletion. A possible effect of a free
radical scavenger, anethole dithiolethione (ADT), was also assessed.
, ROS production was measured with a fluorogen probe 2',7'dichlorofluorescin diacetate (DCFH-DA). Reduced GSH and cell viability
were measured with, respectively, monochlorobimane (mBCI) and neutral
red probes. Assays were performed directly on living adherent cells in 96well microplates,(MiFALC test-Micrototration Fluorimetric Assays on Ltve
cells)[1] and sensitive fluorescent detection used microplate cytofluorimetry
with cold light fluorimetry technology.
,The results showed that THA induced a concentration-dependent increase
in ROS production and a decrease in GSH [2]. Furthermore, for THA
concentratruns between 10 and 100 lxM~ ADT protected cells from ROS
production stimulation and GSH depletion induced by THA.
, In conclusion, These MiFALC tests is adapted to reactive oxygen
species and glutathione assessment, directly in live adherent cells in
microplates. So our in vitro study demonstrates that oxidative stress,
evidenced by enhanced ROS production and GSH depletion, is a mechanism
involved in THA cytotoxicity. Moreover, ADT is effective in preventing
THA-induced mnjury,

Phosphorylation of photolyzed rhodopsin is calciuminsensitive in retina permeabilized by s-toxin

A. Otto-Bruc ~, R.N. Fariss-', J.P. Van Hooser2, K. Palczewsk? 34
JCNRS-1PMc, 660 route dess Lucioles, 06560 Valbonne, France.
Departments o f 2Ophthalmology, Pharmacology anti'Chemistry, University
o f Washington, Seanle, WA 98 [95-6485, USA.

Light triggers the phototransduction cascade by activating the visual

pigment rhodopsin (Rho--)Rho*). Phosphorylation of Rho* by rhodopsin
kinase (RK) is necessary for the fast recovery of sensitivity after intense
illumination. Ca2 ions, acting through Ca2*-binding proteins, have been
implicated in the desensitization of phototransduction. One such protein,
recoverin, has been proposed to regulate RK activity contributing to
adaptation to background illumination in retinal photoreceptor cells. Here we
describe an in vitro assay system using isolated retinas which is well suited
for a variety of biochemical assays, including assessing Ca2+ effects on Rho*
phosphorylation. Pieces of bovine retina, with intact ROS, were treated with
pore-forming c~-toxin, including an or-toxin mutant which forms pores whose
permeability is modulated by Zn z. The pores formed through the plasma
membranes of rod cells permit the diffusion of small molecules <2 kDa, but
prevent the loss of proteins, including recoverin (25 kDa). The selective
permeability of these pores was confirmed using the small intracellular tracer
neurobiotin. Application of [7-32P]ATP to e~-toxin-treated, isolated retina
allowed us to monitor and quantify phosphorylation of Rho*. Under various
experimental conditions, including low and high [Ca2+]free, the same level of
Rho* phosphorylation was measured. No differences were observed between
low and high [Ca2+]freeconditions, even when rods were loaded with ATP and
the pores were closed by Znz+. These results suggest that trader physiological
conditions, Rho* phosphorylation is insensitive to regulation by Caz+ and
Ca2+-binding proteins, including recoverin.

I l l - R a t P. et a l , Animal Alternatives, Welfare & Ethics. Elsewer, DAVS 27,(I 997) 813
[ 2 ] - R Ossenl et al.. Toxicok in Vitro, 1_33,(1999), in press

(Su/oth/261)

IN'rF_JtYACL~L
PROPERTIESOF
H A Z E L N U T OIL AND EMULSIFIER BLENDS

Beraat Oz~elik, Ozgiar Koq, Artemis Karaali

L 12 U Chemical andAIetallurgwal Faculty, A4aslak, lstanb ul, Turkey

Hydrogenated vegetable oils are being used for improving the

polymorphic properties of food emulsions because of their 13'
stability and creaming ability. The plasticizing and stabilizing
effect is assumed to be due the increased chain length diversity
of the fatty acids in new oil blend of emulsion.
In this research study , the effects of two commercial
emulsifiers (Palsgaard 6111 and 4125) on polymorphic
properties of hazelnut puree, a major Turkish export commodity,
were investigated.
To determine the optimum concentration for Palsgnard 6111, it
was added into puree in the concentration range 0.5-2 g / 100 g.
Changes in polymorphic properties of puree were observed by
measuring its rheologieal properties and % oil separation. It was
seen that although 1.5 % (g/g) Palsgaard 6111 was the most
effective concentration preventing oil-phase separation, it
resulted in an undesired increase in product viscosity.
In the second part of the study, Palsgaard 4125 was used in
combination with the Palsgaard 4125 to optimize the viscosity.
Using Response Surface Methodology, the optimal proportions
and concentrations of the two ingredients were determined for
obtaining the ideal plasticity in hazelnut puree.

(Su/oth/262)

eADPR-indueed Ca 2. release from SR: involvement of SH.

I.Panfoli ~, B. Burlando and A.Viarengo
~lst. Chimica Biologica, Untv. Genova, Genova. Italy
bDtp. ScL e Tecno Avanzate, Univ. Piemonte Oriental,Alessandxta. Italy.

Cyclic ADP-Ribose (cADPR) a natural metabolite of nicotinamide adenin,

dinucleotide (NAD+),elicits calcium-induced calcium release (CICR) in ;
variety of cell types [1]. In mammalian skeletal muscle, membran~
depolarization activates the dihydropyridine receptor (DHPR)-ryanodinq
channel (RyRl) complex to give the initial calcium burst from SR lumen
which in turn, through CICR, recruits those RyRls which are not directl:
coupled to DHPR [2].We studied the effect of cADPR on Caz+ release il
muscle cells by incubating SR vescicles from scallop (Pecten jacobaeus
adductor muscle in the presence of the calcium tracer fluo-3. Exposure of SR t~
eADPR (20 I~1) produced Ca2+release, which was a function of free [Ca2+]it
a range between 150 and 3000 nM, over which Ca2was inhibitory, indicatinl
RyR involvement. Ca2+ release was not significantly enhanced by calmoduli~
(7 p.g/ml), but it was enhanced by equimolar addition of ADPR. The Ca2
release elicited by ADPR/cADPR was a function of free [Ca2] too. cADPR
self inactivation was observed at low free [Ca2+] (about 150 nM), but it tende~
to disappear upon [Ca2+] elevation. An ADP-ribosyl cyclase activity wa:
detected in our preparations. Caffeine or ryanodine induced a Ca 2+ releas~
which was ruthenium-red (RR, 2.5 laM) sensitive as far as the free Caz+ leve
was low. Such hindering effect on the RR inhibition, would suggest that Ca2
can drive the channel to an activated state insensitive to the inhibitor. S1;
incubation with Thiolyte, a fluorescent tracer for sulfhydryls showed
reduction of SH availability upon cADPR addition, suggesting that the actim
of cADPR needs SH involvement. This result is in accordance with tht
presence of functional SH groups regulating the receptor gating [3], Based ot
our data, a model is proposed for Ca2+ signalling in muscle cells, where ,'
steady-state cADPR cytosolic level would trigger a [Ca2+]-dependent Ca2
release when free [Ca2+] reaches a threshold (slightly above the resting level)
hence producing cascade RyR recruitment along SR cisternae from initia
calcium burst.
[1] Lee, H. C., Physiol. Rev., 77, (1997) 1133.
[2] Klein, M. G., et al, Nature, 379, (1996) 455.
[3] Xu, L. et at., Science, 279, (1998) 234,

Abstracts FEBS'99

(Su/oth/263)

Prostaglandin F2~z modulation of oxytocin-induced

oscillations in cultured human uterine myocytes.

s161

calcium

C'P-PLESTED~, R.BOBE2, S.WATSON2 and A.L(~PEZ-BERNAL~.

University of Oxford Departmentsof 1Obstetricsand Gynaecoiogy,
John Radcliffe Hospital, Headington, Oxford OX3 9DU and
2Pharmacology,Mansfield Rd, Oxford OX1 3QT.
Oxytocin is a potent inducer of uterine contraction, but the
modulation of this contraction during pregnancy and parturition
remain to be fully understood. Clinically, the failure to understand
the mechanisms of labour impede effective treatment for conditions
such as pre-term labour.
Here, we have investigated the actions of oxytocin on
intracellular calcium using single cell methodology. Isolated
human uterine myocytes were loaded with Fura-2 and the rise of
intracellu~ar calcium ([Ca~],) in response to oxytocin was
investigated. As in a previous investigation, some cells exhibited a
delay of up to 1 minute before a rise in [Ca2+], was observed
(Phaneuf et al., 1993). Therefore, this current investigation asked
whether other agonists, such as prostaglandin F2ct (PGF2~t)
modulate oxytocin-induced [Ca~+],.
Using sub-confluent myocytes, addition of PGF2c~ (llaM)
had little effect on [Ca2*], whilst oxytocin (lrtM) alone induced a
single peak of [Ca2*],. However, if PGF2o~ was applied five minutes
before oxytocin, the cells exhibited a cycling of [Ca2*], peaks which
occurred every 2-4 minutes. This [Ca2 +], cycling was not observed
if PGF2~ was applied after oxytocin. Thus, it appears that PGF2~
may have some modulatory effect on the mechanism by which
oxytocin-raises [Ca2+], and consequently uterine contraction.

(Slffoth/264) Phylogenetie variation in the peptide composition of

lysosomal ct-mannosidase: Two conserved cleavage sites

HMF R.iise, GM Hansen, G Evjen, K-E Eilertsen, OK Tollersrud
Dept Medical Biochemistry, University of Tromso, Tromso, NORWAY

Lysosomal ~-mannosidases(LAMANs) are complexes that consist of two to

ten different peptides depending on the species. The peptides originate from
the proteolytic processing of a single-chain precursor of about 1000 amino
acids. To characterize these cleavages, LAMANs from eleven species
representing the mammalian, bird, fish, amphibium and insect classes were
partially purified and characterized. All enzymes had pH optima of 4.2-4.4,
were heat stable and had native molecular masses of about 250-300 kDa. The
peptide patterns were characterized by western blot analyses and compared
to previous reports on LAMANs from slime mold, cattle, man and cat. Nterminal sequencing was carried out for some peptides to locate the exact
cleavage sites. Two cleavage sites were phylogenetically conserved and could
thus be of physiological importance. Cleavage at the first site separated
LAMAN peptides '%" and "d", and was conserved in all species investigated.
The second site separated peptides "d" and "e" and was found only in
mammals, chicken and frog. Apparently, this site was introduced close to the
separation of the fish and amphibium lineages about 450 million years ago.
Five additional cleavage sites were observed. Cleavage at these sites was
mostly partial and not restricted to any phylogenetic classes, and probably
resulted from slow proteolysis in the lysosomes.

Reference

Phaneuf et al., (1993) J Endocrinoll. 136, 497-509

(Su/oth/265)

Expression and induction of YP1A1 in Black

Seabream (Spondyliosoma cantharus) hepatocyte
cultures : Effects of heavy metals
C. Risso de Faverney 1'~, G. de Sousa 2, M. Lafauxie 2,
R. R a h m a n i ~
ILaboratoire de Pharmaco-toxicologie cenulaire et
raolEculaire - Centre de Recherches 1NRA - 41, Bd du Cap, BP
2078, 06606 Antibes eedex (France)
ZLaboratoire de Toxicologic environnementale, Facult[ des
Sciences de Nice, Parc Valrose 06000, Nice (France)

We developed a new method, which allowed black seabream hepatocytes to

be maintained in primo-culture for several days. This method was shown to
be suitable for studying sea fish CYPs expression and regulation following
xenobiotic treatment. After isolation, hepatocytes were directly mixed with
a type I collagen gel and culture medium, seeded in 6-well tissue culture
plates (2.106 cells/35 mm well) and incubated under atmospheric air at
20C. After 24 hrs, the cells were exposed for 1, 2 and 3 days to 3methylcholanthrene (3MC), heavy metals (Cd (II), Cu (II), Pb (II), Zn (II)),
or 3MC with increasing concentrations of these metals. Cytotoxicity was
assessed by the neutral red test : the sequence was Cd (II) > Cu (II) > Pb (II)
> Zn (II) (ECs0 : 57,234, 544 and 722 }aM respectively) and appeared to be
correlated with the metal solubility. CYP1AI expression was monitored by
EROD activity as well as by Western and Northern blots. As expected, 3MC
induced the CYP1Al-related EROD activity in a time- and dose-dependent
manner. These data were confirmed by Western blot (intense band of 55-60
KDa) and Northern blot analysis. Maximal induction (17-fold over control)
was obtained at 1 pM after 72 hrs of exposure. This induction was inhibited
by simultaneous exposure to Cd (lI), Cu (II), Pb (II) or Zn (II). This effect
was dose-dependent and EROD activity was decreased to less than 50% of
the control (1 pM 3MC), with ECso ranging from 1.3 taM for Cd (II) to 16
}aM for Zn (II). CYP1AI protein and mRNA levels were also reduced.
Taken together, since CYP 1A induction response in fish liver is increasingly
being used in ecotoxicological studies, these results lead to the conclusion
that heavy metals significantly affect CYPs expression and therefore have to
be taken into consideration in biomarker monitoring

(Su/oth/266)

Thyroid hormone receptors and glucose transporter

genes expression in rat brain after gestational hypoxia
C. Royer, G. Crouzoulon, JC. Roux, Y. Dalmaz, J. Lachuer
[23/IR ('NRS-U('BI~','onl, 43Bd du llnovembre 1918,
69622 I'dleHrbam~ecedex, I,)'a~ce

Hypoxta modifies the embryonic development and could lead to brain

dysfunctions in adult rat. The mechanisms remain unknown but could be
due to an impairment of neural divisions, migrations and differentiation
processes. Modification of specific genes expression involved in brain
development or energetic supply regulation may explain such
dysfunctions. Thus, we studied hypoxia effects (10% oxygen) on cerebral
ontogenesis of 1) thyroid hormones receptors (TR) genes expression,
involved m cellular division and differentiation, and 2) glucose
transporters (GLUT) genes expression which regulate glucose utilisation.
Semi-quantitative RT-PCR shows that gestational hypoxia involves : 1) a
drastic reduction of body mass, an increase of cerebral mass and a oneday parturition delay 2) a increase of the TRot2 isoform at day 14 of
embryonic stage (El4) 3) an gene over-expression of GLUT-3 and
GLUT-4 at specific ages. The neurone-specific transporter GLUT-3, is
significatively more expressed at El4 and at birth (P0) in hypoxia group.
The insulin-dependant transporter GLUT-4 has been observed for the first
time in this study in brain during embryogenesis. This transporter is
strongly expressed at El4. And rts expression is increased by gestatlonal
hypoxia at El4 and 7 days after birth (P7).
In conclusion, a gestational hypoxia induces an adaptative response that
led to compensate the energetic deficit. Some post-partum disorders
however persist and led to think that this response is insufficient.

s 162

Abstracts FEBS'99

(Su/oth/267) Dehydroaseorbate reduction is glutathione-independent

in human keratinoeytes cell line (HaCaT).
I. Savini and L. Avigliano

((Su/oth/268)

Department o f Experimental Medicine and Biochemical Sciences,

University T o t Vergata, Rome, Italy

Vitamin C is present at high concentrations in human skin where plays a key

role in protecting cutaneous tissue from injury induced by oxidative stress.
Because humans cannot synthesize vitamin C, recycling of ascorbic acid (AA)
from the oxidized forms, dehydroascorbic acid (DHA) and ascorbic acid free
radical (AFR), is required to maintain intracellular stores of the vitamin. Another
mechanism that contributes to intracellular concentrations of vitamin C is the
uptake from the surrounding interstitial space. Transport studies have revealed
that mammalian cells possess at least two different systems involved in the
cellular uptake of vitamin C, a Na+-dependent cotransporter for AA and a
facilitative glucose transporter for DHA [l]. Upon intracellular entry, DHA is
immediately reduced to AA. The systems involved in this reaction are both
glutathione-dependent and glutathione-independent [2].
In order to clarify the contribute of g/utathione-dependent and indipendent
systems on DHA reduction we investgated the effect of glutathione depletion and
the effect ofquercetin, a potent inhibitor o f a glutathione-independent (NADPHdependent) DHA reductase [3], on the ability of HaCaT cells to accumulate AA
when incubated with DHA. Glutathione depletion was achieved by treatment of
the cells with different compounds. Diethyl maleate (DEM) and ethacrynic acid
(EA) bound covalently and irreversibly to free cysteines or protein thiol groups;
in particular EA depleted both cytosolic and mitochondrial glutathione.
Buthionine sulfoximine (BSO) acted as an irreversible inhibitor of glutathione
synthesis.
HaCaT cells incubated in the presence of 0.1 mM DHA accumulated high
intracellular concentrations of AA (7 raM), the maximum level was reached after
30 min and thereatter rapidly decreased. HaCaT cells treated with I mM BSO for
40 hours had a 92% decreased level of intracellular glutahione but they did not
have alteration in their ability to reduce DHA. Also glutathione depletion (95%)
obtained by treatment of HaCaT cells with lmM DEM or 0.2 mM EA for I hour
did not alter the ability of the cells to reduce intracellular DHA despite these two
compounds impaired the DHA transport (50%). Incubation for 2 hours of
HaCaT cells with 0.1 mM quercetin before DHA uptake resulted in an altered
intracellular DHA reduction (40%).
These findings show that HaCaT cells posses efficient systems for DHA
reduction that are glutathione-independent but quercetin sensible.
[1] Welch RW et al. J. Biol. Chem, 270 (1995) 12585
[2] Washburn MP etal. Biochem. 38 (1999) 268
[3] Del Bello Bet al. Biochem. J.340 (1994) 385
(Su/oth/269)

Affinity labeling and purification of NLS-binding

proteins of the nuclear pore complex
Sabine Schmalz, Markns Kircher and Hugo Fasold
lnst)tut~ur Biochem~e, J W Goethe-~'mversttat b)'ankfurt 31,,
~lIarte Curte ,';tr 9, 60439 Frankfurt 3[, Germany

The transport of nuclear proteins from the cytoplasm into the nucleus is
denpending on a nuclear location sequence (NLS) The protein import
without the help of cytoplasmic factors is a two-step process involving NLSdependent docking of the substrate at the nuclear envelope followed by
translocation through the membrane. In order to mimick the attachement to
the nuclear pore complex we synthesized nuclear location sequence
combined with Ferritin and a carboxyterminal His-tag Import of NLScontaining proteins was inhibited by these compounds The NLS was
derivated with an azido-group The transport inhibitor (Ferritin) may be
removed by reducing agents The irradiation of the sensitized membrane
permitted the specific labeling of two proteins. The covatently marked NLSbinding proteins of the nuclear pore complex can be extracted from nuclear
envelopes and isolated by metal affinity chromatography The apparent
molecular weight of the most dominant protein band was determined to 65
kD.

Microscopic studies on
crystals of insulin mutated at crystal contact surfaces.
U. Schell & R. Hilgenfeld
Institute of Molecular Btotechnolog3; Department of Structural Btology &
Crystallography, Beutenbergstr 1 I. 07745 Jena, Germany.

Insulins mutated at crystal contact surfaces are excellent objects to study

the effects of the alterations on crystallisation conditions, crystal
contacts, crystal quality and stability. The mutant DesPheBl of porcine
insulin was crystallised in the presence of Zn 2+ and phenol, which is
often added as preservative in pharmaceutical insulin preparations. It has
been shown previously that phenolic compounds bind to specific sites on
the insulin hexamer and act as allosteric effectors, inducing a
transformation of the T~, hexamer (in rhombohedral spacegroup R3) to
the R 6 hexamer (mostly in monoclinic spacegroup P21) [1]. However,
mutant DesPheB 1, in the presence of phenol, exclusively crystallised in
the rhombohedral spacegroup R3. This indicates that residue BI is
obviously essential for interhexameric contacts in monoelinic crystal
forms. The structure of this mutant is presently under investigation. We
will present preliminary results on the visualization of the crystal faces
by atomic force microscopy which has been used only recently to detect
packing defects at near molecular resolution [2]. A novel highmagnification stereo-microscope constructed in our department is used to
follow crystal growth.
[1] U. Derewenda et al., Nature, 338. (1989) 594.
[2] C. M. Yip et al.. Biophys. J., 74. (1998) 2199.

(Su/oth/270)

Characterization of zinc transport in MDCK cells

M Tauc, O.Ducoudret, M.Fuchs and P.Poujeol
UA[R-CNRS 6548, Universltd de N~ce-Sophta Anttpolis, parc
Vah'ose 06108 Nwe cedex 2. France

The aim of this study was to characterize the mechanisms of Zn 2*

transport in MDCK cells. The use of the potential sensitive probe oxonol
used on isolated cells have shown that zinc induced a cell depolarization
in a dose dependent manner (1C~ = 10~aM) as well as cadmium (IC~o=
501aM) or copper (ICs0 = 90~tM). This effect was inhibited by La 3- or
Gd 3. but was not modified in the presence of NPPB or 10 t.tM amiloride.
Zinc did not induce depolarization in the absence of extracellular Na"
This depolarization was also observed by using the whole-cell clamp
technique on partly trypsinized cells. Fluorescent video-microscopy
experiments performed with BCECF showed that the application of zinc
induced a reversible cell acidification, the amplitude of which depended
on the extracellular pH To determine the implication ofNa" in the
depolarization process, we performed 22Na" uptake experiments in the
absence or the presence ofZn 2+. In these conditions, Zn 2- stimulated a
Na + influx depending on the extraceflular Zn 2" concentration. Finally, we
performed ~Zn 2. uptake experiments The results indicate that zinc
enters MDCK cells via a carrier mediated process depending on
extracellular Na + Uptake experiments led in the presence of cadmium
indicated no competition with Zn 2transport
Overall, this study suggests that Zn z* could enter in MDCK cells
in combination with H ~ions via a transporter inhibited by La 3and Gd 3*.
The increase of 22Na+ stimulated by Zinc could be in part due to the
activation of the Na+/l-l- exchanger resulting of the Zn induced cell
acidification However, the role of sodium in zinc transport itself
remains to be clarified.
Although the molecular identity of this transporter is not yet
known, its physiological properties differs from those of the recently
cloned proton-coupled metal ion transporter (Nramp2/DCT1) and from
the cadmium inhibitable ZnT-1 transporter expressed in the kidney.

Abstracts FEBS'99

(Su/oth/271)

s163

Effect of starvation on glutamine metabolism in the rat

renal cortex: A ~3C NMR study
B. Vercout~re a, G. Martin a, J. Mispelterb, G. Baverei a
acentre d'Etudes M~ta~ollques par SRM, INSERM U499, HJpttal E.
Herriot. Lyon, France, INSERM U350, Orsay

Studies performed in vivo both m man and experimental animals

suggest that starvation stimulates the production of glucose by the
kidney. This is why, in the present study, we have tested if starvation
alters the fate of glutamine, a physiological renal substrate, in isolated rat
renal proximal tubules. For this, we have incubated rat proximal tubules
prepared from fed and 48hr-fasted rats in the presence of variously ~3Cand ~4C-labelled glutamines.
The results obtained by c.zymauc,

radioactive and

13C-NMR

measurements m combination with a new mathematical model of

glutamine reveal that starvation induces a stimulation of glutamine
removal associated with an increased synthesis of glucose, an increase in
the pyruvate carboxylase/pyruvate dehydrogenase flux ratio but no
change of glutamine complete oxidation.
It is concluded that, during starvation glutamine may contribute to the
increase in glucose production by the kidney.

(Su/oth/273)

Identification of five new PMM2 mutations in

carbohydrate-deficient glycoprotein syndrome type IA
S. Vuillaumier-Barrota, G. Hetet b, A. Baruier", M. Cuer a,
G. Durand a, B. Grandchamp b, N. Seta a. ~Bioctumte A. h6pttal btchat
PARIS, France. t, INSERM U409. PARIS, France

Carbohydrate-deficient glycoprotein (CDG) syndrome type 1 A is an

autosomal recessive disorder, characterized by a defective N-glycosylation
associated with central nervous system dysfonction. Patients have a
phosphomannomutase (PMM) deficiency and mutations in the PMM2 gene.
A total of 25 different missence mutations and one single base pair deletion
have already been described.
Nineteen unrelated French patients with CDG type 1A were screened for
PMM activity and for mutations. All patients had less than 15% residual
activity of phosphomannomutase in either leucocytes or fibroblasts. Thirty
two missense mutations on 38 disease chromosomes were identified (84%).
In two patients, no mutation could be found in the entire eDNA and in
intron/exon junctions. In two other patients, only one mutated allele was
identified. No mutation was observed at the homozygous state. The most
frequent mutation RI41H in exon 5 was found in 15 out of 38 chromosomes
CDG type 1A (39.5%). The mutation 1132T in exon 5 and V231M in exon 8
were found three times each (8%). Five new mutations were identified: C9Y
(G26A) in exon 1, L32R (TA95GC) in exon 2, EI39K (G415A) in exon 5
(found in two unrelated patients), T226S (C677G) and C241S (G722C) in
exon 8.
Our data confirms the absence of homozygoty for the most frequent
mutation RI41H and the existence of hot spots for mutations in exon 5 and
8. The C9Y mutation, which is the first one described in this exon, was
found in two brothers of 36 and 33 years old. Furthermore, leucocytes and
fibroblasts from the few patients with compound heterozygoty for either
C9Y or E139K and RI41H mutations showed a higher residual PMM
activity than cells from other patients suggesting that these two new
mutations are mild ones. Expression of these five new mutant proteins in
vitro are underway to confirm the role of these mutations in the enzymatic
activity of the protein.

(Su/oth/272)

Free radicals metabolism in patients with RA and OA

D. Vipartien/:, A. Kmpovrtiekien, K. Valiukiene
The Institute of Experimental and Clinical Medtcine, ~ygtmantt~ 9, 2600
Vilnius, Lithuania

During metabolic stress in patients with rheumatoid arthritis (RA) and

osteoartrosis (OA) free radical production increases, however, antioxidant
activity might be important in protection against inflammatory and
degenerative diseases including the enzymatic defense system [I].
Malondialdehyde (MDA) is one of the most frequently used indicators of
lipid peroxidation [2]. Also the activity of catalase (CAT) can reflect the
treatment efficiency of RA and OA [3]. We investigated changes of the end
product of lipid peroxidation - MDA, antioxidant enzyme - CAT activity,
total antioxidant activity (AOA). The purpose of the present study was to
compare MDA, CAT, AOA in patients with RA and OA blood serum with
control group. The main finding of our study was that patients with RA and
OA have an increased production of MDA and decreased activities of AOA,
CAT. Considering the examined biochemical parameters, the changes of
lipid peroxidation MDA, AOA and CAT in patients with RA and OA
significantly differ from the control group. The mean level of serum MDA
was 5.120.25grnol/1 (P<0.05) among RA and 5.33:~0.26~mol/1 (P<0.02)
among OA. It is 30% and 35% higher than in control group accordingly. The
AOA in RA was 44.591.69% (P<0.05) and in OA it was 43.912.15%
(P<0.02), while in control group it was 57.532.65% CAT activity was
extremely decreased in RA and OA groups. The level of CAT in RA was
slightly higher than in OA patients, 181.8233.96U/p and 165.0931.91U/p
accordingly. In healthy patients there was found correlation between MDA
and AOA (r=-0.448), also of MDA and CAT (r=-0.679), AOA and CAT
(r=0.475). However, the correlation between such indexes have disappeared,
decreased or changed correlation coefficient sign in both RA and OA. The
data of MDA and AOA in our study show that free radicals metabolism
plays the role in RA and OA development, because these indicies differ
from healthy donors control group. We suggest that CAT activity is the main
free radicals scavenger indicating the RA development.
[1] Heli6vaara M e t al, Ann Rheum Dis, 53, (1994) 51.
[2] Chapple I, J Clin Periodontol, 24, (1997) 287.
[3] Bartosiewicz G e t al, Therapia Hungarica, 41, (1993) 67.

(Su/oth/274) "Suppressors of pyruvate decarboxylase in Saccharomyces

cerevisiae

"

Sanjeev K. Waghmare* and Zita Lobo*

*Department of Biological Sciences, Tara Institute of Fundamental
Research, Mumbai - 400003 lndla.

In yeast Saccharomyces cerevisiae,two structural genes have been

characterised viz. PDC1 and PDC5 for pyruvate decarboxylase enzyme.
In the wild type, PDC1 is active however, PDC5 becomes active when
PDC1 is deleted. Their common regulatory gene PDC2 has been
characterised. PDC2 is known to be a transcriptional activator for the
synthesis of this enzyme, pdc2 mutant fails to grow on glucose. Two
distinct classes of extragenic suppressors - XSP9 and xsp17 have been
isolated from a pdc2 deletion mutant strain. Both these suppressors
support the growth on glucose and also regulate the synthesis of pyruvate
decarboxylase. The suppressors XSP9 and xspl 7 restore PDC1 transcript
on both alcohol -and glucose-grown cultures. The suppressor XSP9 is
dominant and has flocculent property which is recessive. It restores about
70-80% of pyruvate decarboxylase activity. In addition, most of the
glycolytic enzymes levels are higher in both glucose -and alcohol-grown
cultures. The rate of glucose utilisation and alcohol production are high in
XSP9 suppressor bearing strain as compared to the wild type. A putative
clone complementing the flocculent phenotype has been obtained. This
clone encodes zinc-finger protein and adenylate kinase. However, further
characterisation will unravel the mechanism of suppression. The
suppressor x s p l 7 is recessive and restores 30-40% pyruvate
decarboxylase activity but fails to support anaerobic growth. This
suppressor bearing strain, showed loss of 80-90% aldolase activity as
compared to wild type. xsp17 is shown to be a mutated allele of the locus
f o a l which is the structural gene for aldolase. The xspl 7 suppressor strain
showed approximately 10 - to 20 - fold accumulation of fructose 1,6bisphosphate. The double mutant strain pdc2A fba] shows induction of
pyruvate decarboxylase.
References:
[1] Velmurugan et al., Genetics, 145, 1997

s164

(Su/oth/275)

Abstracts FEBS'99

A contribution to the ab-lnitio globular protein folding.

Denis Znamenski & Jean-Paul Mornon

Syst~mesMoleculalres et Biologic Structurale,LMCP, UP6, UP7, CNRS
UMR 7590, case 115, 4 place Jusieu 75252 Pans CEDEX 05, France

Different approaches can be applied to the problem of ab-initio

prediction of protein structures. Here we present an approach
which consists in assembling the secondary structures as rigid
blocks. First we use an algorithm which predicts secondary
structures from a sequence. Loops are not considered in the
procedure. Then we construct alpha hehces wlth radius of 2.3~
and with 3.6 residues per turn. Beta sheets are modeled by
hehcoid surface w~th a variable degree of twist. Side chains are
represented as spheres with the centre at Ccc position and with
radius varying from 0 to 0.8 of the mean length of rotameres
for each residue. Using hydrophobic residues as attractants
belonging to secondary structures we generate a set of tightly
assembled tertiary structures by successive adjustments
produced by translations and rotations of the secondary
structures considered as rigid blocks. The halting criteria for
this dynamic phase of the algorithm is attaining the minimum
of the greatest distance between centres of hydrophob~c areas
of secondary structures and the centre of gravity of the protein.
Then we select the most compactly assembled structures
among all generated ones. An application of this algorithm for
four helix bundles (ferritin, myohemerythrin, granulocytemacrophage stimulating factor) resulted in RMS difference
between the model and the native structures of 3,~, 1.SA and
2.4,~,, respectively. At present we continue developping the
algorithm to better adapt it for proteins with a greater number
of secondary structures.

Abstracts FEBS'99

s165

1.1 New genomes

(Mo/1.1/O01)

Insertional Inactivation of Treponema denticola cfpA

Gene Results In Altered Cell Growth and Division.
J. Izard and R. J. Limberger
NI'S Dept. of Heoltlt P.O. Box 22002, Albany, NY 12201, USA

(Mo/1.1/002) Cloning of the eDNA encoding tachykinin related

peptides of echluroid worm
T, Kawada, H. Satake, H. Minakata, M. Yojiro and
N. Kyosuke
Suntory Institute for Bioorganic Research, Osaka, Japan

Treponema denticola is an anaerobic spirochete associated with

periodontal disease. Treponema cells possess a helical ribbon of filaments
in the cytoplasm. By electron microscopy it was determined that these
filaments run the length of the cell but the point of termination of this
structure at the ends of the cell is unclear. The filaments are composed of
one major protein, CfpA. The gene was first sequenced in Treponema
pallidum. There is no known homologous structure or function associated
with these filaments.
To determine the function and potential domain conservation of
these filaments, we looked at the inter-species sequence conservation of
CfpA. The sequencing of the cf-pA gene from T. phagedenis, T. vmcenm
and T. denticola revealed short insertions and deletions as compared to T.
pallidum cfpA. However these changes do not drastically affect the amino
acid sequence, and more than 80% of identity was observed between the
sequences.
Insertional inactivation was used to explore the role of CfpA. A
segment of cfpA was amplified from T. denticola chromosomal DNA and
cloned into a suicide plasmid. The erythromycin resistance cassette
ermF/AM was inserted into the DraI site in the beginning of the gene.
Electroporation-mediated allelic exchange incorporated the interrupted
cfpA gene into the T. denticola chromosome creating erythromycin
resistant cfpA mutants.
The mutant did not spread as well as the wild type strain on agar
plates. The cells are longer than the wild type; under a single outer
membrane few cytoplasmic cylinders can be observed by electron
microscopy. Fluorescent dye binding to DNA shows dense chromosome
area in the cytoplasmic cylinder and some anucleated cells. The insertional
inactivation of cfpA affects cell shape, motility and cell division.

(134o/1.1/003)

Direct eDNA selection within a 1.2 Mb

of human chromosome 5q31.1
E. Levsherlkova, M. Markelov, A. Bashirova, E. Frolova
Sherayakin-Ovchinmkov Institute of Bioorgamc Cheraistry,
Mtklukho-Makl~a St. 16 10, 1l 7871, GSP-7, Moscaw, t'-437. Russia

Human chromosome 5q31.1 contains an evolutionary conserved cluster of

genes encoding factors of proliferation and differentiation for hematopoietic
cells. This region is frequently deleted in the malignant cells of patients
with mielodisplastic syndrom (MDS) and acute nonlymphocyte leukemia
(ANNL). The recent data showed locus 5q31.1 was also deleted in renal
carcinoma, esophageal cancer and differentiated gastric adenocarcinomas
[1]. observed in human tumors strongly suggests the existence of(a) tumorsuppressor gene(s) at the concerned locus. Furthermore, it has been shown
that the interferon regulatory factor-1 (IRF-I) locus on chromosome 5q31 1
is one of the common minimal regions of LOH in these cancers. Previously
we have constructed the YAC and cosmid contigs overlapping the IRF-1
locus and we have generated a physical map of this contig. The next step
was to search for novel genes in this region. There was some probability to
find a novel member of the cytokine gene cluster or a gene whose function
is associated with the tumor suppression. In order to find the novel genes,
we used direct eDNA selection technique[2] from cosmids and successfully
isolated and mapped to human chromosome 5 the eDNA clone
corresponding to RIL gene [3]. The second step was an applying eDNA
selection for YAC clones. As a result we selected a number of eDNA
clones corresponding to already known unique genes, pseudogenes and
structural genes. Among unique genes there were RIL (a positive control of
selection), pI20E4F transcription factor and EDFI (endothelial cell
differentiation factor) Genomic location of these genes was previously
unknown, we located them within the locus 5q31.1. Moreover, we selected
putative pseudogene of ASGR1. The two of selected eDNA clones revealed
homology only with dbEST GenBank Database and three did non revealed
homology with any GenBank Databases. One of these eDNA probes was
undertaken to the most detailed analysis, including Northern and Southern
hybridization, RT-PCR and the isolation of full-length eDNA.
[1]. Nozawa, H., et al., nt. J. Cancer, vol 77(4), 1998, p.522
[2]. Parimoo, S., et al., Meth. In Mol. Genet., vol 1, 1993, p 23
[3]. Bashirova, A., et al., Gene, vol 210, 1998, p. 239

Tachykinin family is known one of the most famous mammalian

neuropeptides which are involved the regulation of pains and emotions
in the nervous system. StructuaUy and functionally related peptides
have also been isolated from various invertebrate species. Urn-TK I
(Leu-Arg-Gln-Ser-Gln-Phe-Val-Gly-Ser-Arg-NH2) and I1 (Ala-AlaGly-Met-Gly-Phe-Phe-Gly-Ala-Arg-NH2) were isolated from the
ventral nerve codes of echiuroid worm, Urechis unicinctus. These
peptides have been shown to exhibit a tachykinin-like excitatory effect
on the circular body-wall muscle of echiuroid worm and the hindgut of
cockroach. Furthermore, the replacement of Arg-amide by Met-amide at
the C terminus of Uru-TK I and II resulted in stimulation of the ginia
pig ileum muscle contraction, although neither Uru-TK ! nor II itself
made any effect on mammalian tissues. However, a cDNA encoding an
invertebrate tachykinin-related peptides has not been characterized. In
this presentation, we show the novel eDNA sequence encoding the
urechistachykinin precursor. An amino acid sequence analysis of the
deduced polypeplide revealed that the urechistachyk~nin precursor
included seven structurally related peptides, unlike vertebrate tachykinin
precursors. In addition, Northern blot analysis demo:~.strated that
urechistachykinin gene was transcribed only the mRNA. It suggests
that no splicing variants are produced from urcchistachykinin gene in
nervous tissue unlike mammalian tachykinin PPTA gene. This is the
first identification of invertabrate tachykinin-related peptide eDNA
sequence.

(Mo/1.1/004)

Cloning of the cytosolic branched-chain amino acid

aminotransferase from sheep brain
1. Papet, J. Bonfils, F. Glomot, M. Faure
Unitd d'Etude du Mdtabolisme Azotd, INRA Theix. 63122 Ceyrat, France

Branched-chain amino acid aminotransferase (BCAT, EC 2.6.1.42) catalyses

the reversible transamination of brancbed-chain amino acids (BCAA: leueine.
isoleueine and valine). Two isoenzymes (mitochondrial and cytosolic (BCATc))
have been identified in mammals. In rats and humans BCATc expression is
restricted to only few tissues. By contrast, in sheep BCATc is expressed in
several tissues included skeletal muscle which is the main in vivo site of
BCAA transaminatiom In order to study mechanisms involved in BCATc
expression, we first have isolated the sheep BCATc eDNA.
A 1073 bp fragment of the sheep BCATc eDNA has been obtained by RT-PCR
using a sense and a reverse primers corresponding to nucleotides 14 to 47 and
1121 to 1153 of the human BCATc eDNA sequence (accession number
U21551), respectively. This partial eDNA is very similar to human, murine and
mt BCATc sequences (76 to 79 % of identity), This fragment missed both start
and stop codons. The 3' end has been obtained using the Marathon eDNA
Amplification kit (CIontech). A first PCR has been performed with API and a
specific sense primer corresponding to nucleotides 907 to 931 of the 1073 bp
fragment. A second PCR has been realized with a nested primer to get a
specific product of about 1500 bp. Its 5' end has been sequenced over 390 pb. It
includes the stop codon. Experiments are in progress to obtain the 5' end.
The amino acid sequence deduced from the almost full-length sheep BCATc
eDNA (1108 bp) is 368 residue long. This partial protein exhibits 60 % of
identity with the ovine BCATm protein (accession number AF050173) and 90,
84 and 78 % of identity with human, routine (accession number p24288) and
rat (accession number U35774) BCATc proteins, respectively. The few amino
acids specific to the ovine BCATc are located within the 160 first N-terminal
residues, at the position 324 and at the C-terminal end. The lysine residue (204)
which binds the cofactor PLP and amino acids involved in the active site are
conserved in the present sequence. So, sheep BCATc likely belongs to the Fold
IV class of PLP-dependent enzymes.
Despite high sequence identities among mammalian BCATc, the tissue-specific
expression of sheep BCATe is peculiar and deserves more investigations.

s 166

(Moil.I/005)

Abstracts FEB S' 99

Evidence for conservation of the

vassopressin/oxytocin superfamily in Annelida.
H. Satake, K. Takuwa, H. Minakata
SuntoryInstitutefor BioorganicResearch,Osaka618-8503, Japan

Annetocin is a structurally and functionally oxytocin-related peptide

isolated from the earthworm Eisenia foetida. We present the
characterization of the annetocin cDNA. Sequence analyses of the
deduced precttrsor polypeptide revealed that the armetocin precursor is
composed of three segments: a signal peptide, an annetocin sequence
flanked by a C-terminal amidation signal Gly and the dibasic processing
site Lys-Arg, and a neurophysin domain, similar to other oxytocin-family
precursors. The proannetocin showed 37.4%-45.8% amino acid
homology with other prohormones. In the neurophysin domain, 14
cysteins and amino acid residues essential for association of a
neurophysin with a vassopressin/oxyocin-superfamily peptide were
conserved, suggesting that the Eisenia neurophysin can bind to
annetocin. Furthermore, in situ hybridization experiments demonstrated
that the annetocin gene is expressed exclusively in neurons of the
central nervous system predicted to be involved in regulation of
reproductive behavior. These findings confirm that armetocin is a
member of the vasopressin/oxytocin-superfamily. This is the first
identification of the cDNA encoding the precursor of an invertebrate
oxytocin-related peptide and also the first report of identification of an
Annelid vasopressin/oxytocin-related precursor [ 1]. We will also show
the structure of the armetocin genomic DNA.
[1] H. Satake et al, d. Biol. Chem., in press.

Abstracts FEBS'99

s167

7.2 Translational control and molecular mimicry

(Mo/7.2/006) Programmed translational frameshifting in insertion sequence

(Mo/7.2/007)

IS911 : influence of nucleotides following the slippery sequence

C. Bertrand ~, M-F. Pr~re 1, J. ALldns~, O. Fayetl

J. Caillet a, M. Graffe a, P. Romby b, B. Ehresmannb,

C. Ehresmann b , D. Moras c & M. Springera

LMGM, CNRS UPR9007. I18 route de Narbonne, 310.~4Toulouse, France, 2

Department of Human Genetics, Universityof Utah, USA

Insertion sequence IS911 is a transposable element of the iS3 family

found in the genome of all Shigella species. It exhibits two consecutive and
partially overlapping open reading frames, orfA and orfB, that are in the 0 and
-1 reading phases respectively. Translation of these two ORF leads to the
production of three proteins : two are the expected products of orfA and or~B,
but the third results from a programmed -1 translational frameshifting on an
A6G heptanucleotide located in the overlap region [1]. The A6G slippery
sequence is surrounded by two frameshift enhancers: a Shine-Dalgarno
sequence, 11 nucleotides upstream, and a stem-loop structure 6 nucleotides
downstream [2].
This 100 bp long region was cloned in a reporter plasmid, between
gene 10 of bacteriophage T7 (0 phase) and LacZ (-1 phase). The three
products of translation can easily be quantitated after in vivo labelling,
allowing precise calculation of frameshifting efficiency.
The six nucleotides localized between the slippery motif and the stemloop (called spacer 2 region) were randomly modified in order to determine
context effect on frameshifting. Analysis of 150 mutants shows, that the first
nucleotide of the spacer 2, and thus the first codon in -1 phase after the shift,
determines the quantity of the fusion protein : 30% fusion protein is produced
with a pyrimidine, 2 0 0 with an adenine and 15% with a guanine. These
results suggest that the first postshift codon has to be in the A site of the
ribosome to exert such an effect on stability of the translation complex right
after rephasing of the two tRNA-Iys. Our results therefore lend support to the
post-translocational shift mode[ proposed by Horsfield et al [3].

[1] P.Potard et al., .1. Moi. Biol., 222 ,(1991) 465

[2] Rettberg el al., J. Mol. Biol., 286, (1999) 1365
[3] Horsfield et al., N. A. R., 23, (1995) ]487

(Mo]'7.2/008) Elongation Factor-2 kinase may mediate inhibition of

protein synthesis by acidic intracdlular pH.

Multicopy dominant lethal mutants of E.coli

threonyl-tRNA synthetase
a IBPC, Paris.b IBMC, Strasbourg,ClGBMC, Illkirch, France

The recent determination of the threonyl-tRNA synthetase (ThrRS) structure

complexed with tRNAT M indicates the presence of a zinc ion at the cata/ytic
centre. The co-ordinated ligands include H385 from motif 2, H511 from motif
3 and C334 from a helix that precedes motif 2. A water molecule completes the
tetrahedral co-ordination of the zinc. The co-ordinated water molecule and the
localisation of the zinc within the active site, strongly suggest a catalytic
function.
To assess the role of the catalytic zinc, the three residues interacting with the
metal (C334, H385, H51 I) were mutated independently. The changes strongly
affect the aminoacylation activity as judged by the incapacity of the mutant
genes, even when overexpressed, to complement a strain carrying an
inactivated chromosomal copy of fllrS, the gene encoding ThrRS.
Interestingly, the mutated genes, if overexpressed from a multicopy plasm~d,
cause a dominant lethal phenotype in a wild-type strain, i.e., they inhibit
bacterial growth even in the presence of a wild-type copy of the thrS gene. To
understand this phenotype, we first introduced the multicopy plasmids
harbouring the mutated copies of thrS in a strain carrying a mutation in the
operator of the chromosomal copy of thrS that abolishes its negative feedback
regulation. In the presence of this mutation, no lethality is observed. This
permitted us to study the thrS regulation in vivo under steady state conditions
using a thrS-lacZ fusion cloned in bacteriophage ~. and integrated at art ~. in the
E. coil chromosome. With these tools, we were able to show that the
repression efficiency of the mutated ThrRSs was only decreased by a factor of
two, indicating that control is only marginally affected by the mutations.
Second, we constructed a strain were both a thrS-lacZ fusion (integrated in the
chromosome) and mutated thrS (on a multicopy plasmid) are under p/ac
control. Upon induction, we were able to follow the expression of the fusion
for a sufficient time before growth arrest to show that regulation is only
marginally affected by the mutations.
These experiments indicate that the mutant ThrRSs are inactive for
aminoacylation but active for feedback regulation. Although we cannot, at the
present time, completely exclude other explanations, we believe that the
multicopy dominant lethal phenotype is mainly related to the repression, by the
mutant synthetases, of the expression of the active chromosomal copy of the
thrS gene. This repression leaves the cell without active synthetase and causes
growth arrest.

(Mo/'/.2/009)

Interaction between the regulatory region of the

human transferrin receptor messenger RNA and IRP-I
V Gegout. J. Schlegl, M Hentze*. E Westhof. C Ehresmaral.B.
Ehresmann& P. Romby
1BMC, UPR 9002, 15 rue Ren~Descartes 67084 $trasbourg Cedex
*EMBL, Meyerhofstrasse1, D-691t7 Heidelberg. Gemlany

M~ V. Dorovkov, K. S. Pavur, A. N. Petrov, M. A. Brostrom. C. O.

Brostrom,M. M. Sanders, and A. G. Ryazanov.
Dept. of Pharmacology, UMDNJ, 675 Hoes Lane, Ptscataway, NJ 08854

Cellular ion homeostasis is regulated

in part through a post-

transcriptional mechanism. A portion of the 3' untranslated region of the

Changes in intracellular pH (pHi) accompany many physiological

and pathologieal conditions. During hypoxia and ischemia, for example,
phi drops by 0.5-1 pH unit. This decrease in pHi is accompanied by a
significant inhibition of protein synthesis, the mechanism of which is
unknown. We found that eEF-2 kinase, a ubiquitous protein kinase that
phosphorylates and inactivates eEF-2, is dramatically activated by a
similar decrease in pH. eEF-2 kinase displays low levels of activity at the
normal pH of 7.4, but becomes 10 to 20-fold more active when the pH
drops to 6.8. Analysis of protein phosphorylation as a function o f p H in
rat heart extracts reveals that eEF-2 is the only protein whose
phosphorylation increases in response to a decrease in pH. Thus, in
heart, activation of eEF-2 kinase correlates with increased eEF-2
phosphorylation and inhibition of protein synthesis.
To extend this study to other cells types, we have manipulated
pHi in GH3 cells and NIH3T3 fibroblasts by incubation in media buffered
at pH's between 6.0 and 8.0. We observed that inhibition of protein
synthesis at acidic pH, correlated with activation of eEF-2 kinase. Our
results suggest that eEF-2 phosphorylation may be responsible for
protein synthesis inhibition during acidosis, and may play a significant
role in hypoxia and ischemia. Since inhibition of protein synthesis is
known to block ceU death during ischemia and hypoxia, pH-dependent
activation of eEF-2 kinase can be a major protective mechanism against
cell death caused by isehemia

human transferrin receptor mRNA determines the cytoplasmic stability of the

mRNA and ultimately affects iron uptake The minimal RNA regulatory
region contains three conserved stem-loop structures, the iron-responsive
elements (IRE) which are specifically recognized by the iron regulatory
protein-I (IRP-I) under iron starvation conditions. Iron accumulation
regulates IRP-l function by promoting the assembly of an iron-sulfur cluster,
conferring aconitase activity and hinderring RNA binding
In this study, the structure of the regulatory region and the effect of
IRP binding were studied using a combination of enzymatic and chemical
probes Based on these data, a three-dimensional model of an IRE motif was
built by computer modeling. The binding of IRP results in a protection of

conserved residues in each of the three IRE elements but also induces
reactivity changes outside of the IRE structures, restricted to a U-rich hairpin
loop located in close vicinity to the endonucleolytic cleavage site
Footprinting experiments using proteases with different specificities
were used to map the surface of the IRE binding site and to study iron-sulfur
reconstitution. The IRE induces protection in two define areas located in a
putative cleft of IRP-I. Interestingly, the reconstitution of the iron sulfurcluster induces strong protection in two regions which overlap the R N A
binding site

These data suggest that IRP-I

undergoes a structural

rearrangement upon cluster formation that affect the accessibility of residues

constituting the IRE binding site

s168

(Mo/7.2/010)

Abstracts FEBS'99

EXPRESSION OF POLIOVIRUS 2A PROTEASE

RESULTS IN THE INDUCTION OF APOPTOSIS
D. Goldstaub, Z. Bercovitch, C. Kahana
Department of Molecular Genetics, the Weizmann Institute of Science,
Rehovot 76100, Israel.

A cell line was constructed expressing the poliovirus 2A protease in an

inducible manner. Tightly controlled expression was achieved by utilizing
the novel muristeron regulated expression system. Upon induction of the
2A protease expression, a rapid and efficient cleavage of the translation
initiation factor elF4GI is observed which is accompanied by a severe
inhibition of protein synthesis activity. Cells in which the poliovirus 2A
protease was induced displayed fragmented nuclei, chromatin
condensation and an oligonucleosome-size DNA ladder, hence, their death
can be characterized as apoptosis, Although it is tempting to speculate
that the apoptotic death of these cells is a direct cause of the observed
inhibition of protein synthesis, this does not seem to be the case as the
2A protease was more efficient then cycloheximide in provoking
apoptotic cell death.

(Mo/7.2/011) Mapping of the binding site of the initiator IMet-tRNA~ ~t to

Bacillus Stearotherraophilus IF2
M. Guenneugues a, R. Spuriob, E. Caserta ~', S. Meunier ~, C.O.
Gualerzi~', R. Boelensa
"Bovoet Center for Biomolecular Researeh. Utrecht University, Padualaan 8.
3584 CH Utrecht, The Netherlands
~Dipartimenm di Biologica M.C.A.. Univer.sita degh Studi di Camerino,
Via Camerlm 2, 62032 Camermo (MC), Italy

Initiation of the protein translation in prokaryotes is promoted by

three initiation factors. IF2 is the largest one and interacts with GTP,
fMet-tRNA~ met and the ribosome. The binding site for the initiator
tRNA is located in the 24.5 kDa C-terminal part of the protein [1] which
was shown to be composed of two domains of approximately 100 residues each [2]. The C-terminal domain, referred as IF2-C2 (residues
632-741 in Bacillus Stearothermophilus) binds the initiator tRNA as
the intact 1F2. It adopts a ~-barrel fold in solution, its 3D structure being
very close to that of EF-Tu domain 2 (Meunier S. et al., manuscript in
preparation). In the latter protein, domains 1 and 2 form a cleft where
the acceptor arm of the tRNA interacts to form the EF-Tu/tRNA complex [3].
The observed structural relationship suggests that IF2-C2 may
play a role similar to EF-Tu domain 2 upon binding to the aminoacylated tRNA. To assess this assumption, we investigated the possible
binding of formylat~d methioniue (flYlet) to IF2-C2. Using 2D heteronuclear NMR ~pectrn~enpy. we monitored the 1HN and Isl~ ehe~mieal
shifts upon addition of increasing amounts of fMet to a ~5N labeled
IF2-C2 sample. When compared to methionine, specific variations
were observed enabling to delineate a region at the surface of the protein composed of residues matching those of EF-Tu domain 2 that
interact with phenyl-alanine in the complex with Phe-tRNA P~:. An excellent agreement is found with site-directed mutagenesis experiments.
Since IF2 also binds NacPhe-tRNA r'he, we also titrated IF2-C2 with
Nac-Phe or the CACCA 3' end of tRNA phe and found very similar
results.

(Mo/7.2/012)

Effect of a 5' monophosphate end

expression o f t h e p n p gene.
A-C. Jarrige, C. Pottier

on

the

UPR 9073 du CNRS, Institut de Btologie Physico-chiraique , 13

rue Pterre etMarie Curie, 75005 Paris, France.

Polynueleotide phosphorylase is an exoribonuclease which degrades

processively polyribonucleotides from 3' to 5'. It has been shown
previously that the synthesis of polynueleotide phosphorylase is
autoregulated at the translational level by an endonuclease dependent
mechanism. Cleavage of the p u p mRNA by RNase III eighty
nueleotides upstream of the initiation codon is necessary for
autoregulation of polynucleotide phosphorylase synthesis [1].
Although this autoregulation induces a drastic change in the p n p
message stability, no clear mechanism has been described accounting
for a recognition of this new 5' end of the message by polynucleotide
phosphorylase. Translational fusions between p u p and lacZ
demonstrated that the autoregulation site is located at the 5' end of the
p n p messenger.
To have a further insight into the mechanism involved and to identify
if the new 5' monophosphate end created by the RNase 11I cleavage
corresponds to a determinant specifically recognized by
polynucleotide phosphorylase, a messenger identical to the processed
message but carrying a 5' triphosphate end group, has been
constructed. The stability of this message and its translational
properties have been analysed by comparison to a processed
transcript.
[1] Robert-LeMeur, M., Portier, C. Nucleic Acids Res. 22, (1994) 397.

(Mo/7.2/013)

[1 [ Gualerzi C.O. et al., J. Biol. Chem., 266, (1991), 16356

[2] Misselwitz R. etal., Biochemistry. 36. (1997l, 3170
[3] Nissen P. etal., Science, 270, (1995), 1464
The influence of mutation in the 3'UTR
of the MSXi gene on selective tooth agenesis
A. Kobielak ~, K Kobielak~, W H. Trzeciak~
~l)el~artment ~?fPhy~lologwal ('hemtstry UtttverMly Sch~gd of MedwaL St't.
6 Swlectcklego St.. 60- 781 Poz,a,. Polamt

It has been reported that a missense mutation in human ,~s'xl gone,

resulting in substitution of Arg--*Pro within the homeodomain protein
MSX1, causes selective tooth agenesis We have investigated the
structure of ,~/s:rl gone in patients with familial and sporadic tooth
agenesis and found no mutation m the entire amino acid coding sequence
of.~'xl gene by both single strand conformation polymorphism analysis
and direct sequencing However, in 7 out of 12 paUents a C-->T transition
was evidenced, 6 bp downstream beyond the amino acid coding
sequence. Analysis of MSX1 mRNA with the use of DNASIS program,
demonstrated that this mutation, prevented formation of a loop structure
which constitutes a potential binding site for regulator)' proteins, m the 3'
untranslated region of MSXI mRNA We have decided verify the
stabilhty of MSX1 transcript m culture of limfocytes derived from
patients with mutation m th~s gone. We postulate that this mutation might
change stability of mRNA coding for the homeodomain protein MSXI
and thus constitutes a major causative factor in selective tooth agenesis
Suported by grant No 4 P05A 059 15 from the State Committee for
Scientific Research

Abstracts FEBS'99

(Mo/7.2/014)

Antisense regulation of plasmid R1 replication: analysis of the

antisease - messenger RNA interaction
F. Kolb, H. M. Engdald (*), C. Malmgren (*), B. Ehrcsmann, C.
Ehresmann,E. Westhof,E.G.H. Wagner(*) and P. Romby

s169

(Moll.2/015)

UPR 9002 du CNt~. I.BJd.C, 15 rue Descartes, gtrasbourg (F) and (*) Dpt of
Microbiology. Genett Center. SLU . Box 7025, Genet~kvaegen 5, S-75007 Uppsala, Sweden.

Replication frequency of plamid R1 is controlled by an antisense RNA

(CopA) that binds its target (CopT) in the 5' untranslated region of repA
mRNA, thereby inhibiting synthesis of the replication protein RepA.
Previous studies have shown that both RNAs contain a major stem-loop
structure important for the interaction. We have shown that the formation of
a full duplex between the two RNAs is a very slow process in vitro and have
identified a stable binding intermediate. This complex was further studied
using enzymatic and chemical probing and graphic modeling. We also have
identified nucleotides in the stems required for fast binding and optimal
control. Our data show that the recognition pathway involved a multistep
process : the first loop-loop interaction is rapidly followed by a partial
unfolding of the upper stem-loop II in CopA and in its complementary
region in CopT. This extension is facilitated by bulged-out residues in the
stem of each molecule and results in the formation of two intermolecular
helices in the upper stem regions. The resulting four-way junction adopts an
asymetrical X-shape structure wich imposes a side-by-side alignment of two
long helical domains. This unusual structure facilitates the formation of a
third intermolecular helix between the thirty 5' nucleotides in CopA and the
complementary sequence in CopT. Finally, we also bring evidences that this
unique structure, never identified so far, is responsible for an efficient
repression of RepA synthesis.

(Mo/7.2/016)

Colicin ES, a novel cytotoxic tRNase targeting a specific

group of tRNAs

-1".Ogawa ~, K. Tomita-" ~, T. Ueda-', K. Watanabe-', T. Uozumi ~

and 1t. Masaki t
Department of Biotechnology. The UmversiO~ of Ibk),o, Yayoi I-1-I, Bunkyo-ku, Tokyo
113-8657, Japan: "- Department of Chemtstry and Btotechnolobo', The Umverst O, t?JTokyo,
Hongo 7-3-1, Bunkyo-ku, Tokyo I13-8654, Japan. : Present address: Institut de Bu)logte
Moldculaire des Plantes do CNRS, 12 rue du Gdn&al Zimmer, 1-67084, Strasbourg
Cedex, France.

E-group coticins are ColE-plasmid-encoded toxins which kill E.

coli cells after binding to a common cell surface receptor, BtuB. Among
them colicin E3 is known to cleave 16S-rRNA to inactivate ribosomes by its
C-terminal RNase domain (CRD). Colicin E5 was also believed to stop
protein synthesis possibly in a similar fashion to E3 [1]. But their Cterminal domains show no sequence homology [2], suggesting different
cytoplasmic targets of E3 and E5.
We constructed a plasmid which produces the complex of E5-CRD ot"115
amino acids and its inhibitor, ImmE5. ImmE5 consists of 108 amino acids,
including the N-terminal 26 amino acid extension from that previously
speculated [2]. E5-CRD and lmmE5 were separated from the complex and
purified individually. E5-CRD in lhct inhibited protein synthesis of the E.
coli S-30 fraction but apparent inactivation of ribosomes was not found.
Instead, tRNAs for Tyr, ttis, Ash and Asp were specifically cleaved at the 3'
side of the wobble position of the anticodons leaving a 2", 3"-cyclic
phosphate. This was also the case for the cytoplasmic tRNA pool after the
challenge of colicin E5 to the growing E. coli cells. Moreover, a tight
correlation of the cell viability and the cleavage of the tRNAs concerned
showed that the primary target ofcolicin E5 is this group of tRNAs.
[ 1] M. Mock and A. P. Pugsley, ~ Bacteriol., 150, (1982) 1069.
[2] P. C. K. Lau and J. A. Condie, Mol Gen. Genet., 217, (1989) 269.
[3] H. Masaki, et. al., NucL Acids Syrup. Set., 37. (1998) 287.

The role of the universal core m R N P protein, p50, in

regulation of m R N A activity in translation
E Kovrigina, D. Nashchekin, V. Evdokimova, L Ovchinnikov
]nsntute o f Protein Research o f RAS; Pushchino, 142292, Russia

p50, the major core protein of messenger ribonucleoprotein particles

(mRNPs) found mainly in association with different mRNA species in the
cytoplasm of somatic mammalian cells, has been identified as a member of
the Y-box binding transcription factor family. Proteins of this family interacte
with both DNA and liaNA and control the transcription and translation [1]. A
detailed study of the role of p50 in protein synthesis revealed that the amount
of p50 attached to mRNA varies as dependent on the mRNA functional
state, inactive free mRNPs contain a two times higher amount of p50 than
active polysomal mRNPs, p50 stimulates mRNA translation in the cell-free
protein synthesis system at a low p50/mRNA ratio, while p50 inhibits mRNA
translation when added in amounts corresponding to those of inactive free
mRNPs [2]. This intriguing result made us elucidate how a variety of
functions could be accomplished by the same protein and propose the
mechanism of both the positive and negative effects of p50 in protein
synthesis
We have shown that the reticulocyte lysates depleted of p50 by
monospecific antibodies are inactive in initiation of translation and their
activity can be restored by addition of p50 In the p50 depleted system
mRNA is accumulated within 48S preinitiation complexes. We speculate that
at low concentrations, p50 might facilitate either attachment of the 60S
ribosomal subunit or scanning of the 5' UTR of mRNA by the 43S
preinitiation complex [3]. The inhibition of translation at a high p50/mRNA
ratio also occurs at the initiation stage, but in this case mRNA is accumulated
within free mRNPs, p50 was shown to destabilize the mRNA secondary
structure and to form large multimeric complexes; therefore, at a low
p50/mRNA ratio, p50 is supposed to provide mRNA activity by promoting
the general mRNA conformation optimal for translational initiation, while at
a high pS0/mRNA ratio, the inhibition of translation initiation can be a result
of mRNP condensation due to p50-p50 interactions.
[1] V.M Evdokimova et al., J. Biol. Chem., 270, (1995) 3186.
[2] W B Minich et at., Biochimie, 74, (1992) 477.
[3] V.M. Evdokimova et at., J Biol. Chem., 273, (1998) 3574.
Supported by RFBR grant 96-15-98043 and INTAS grant 97-0501.

(Moll.2/017)

Effects of mutations into the RNA binding sites of

polynucleotide phosphorylase
C. Portier, D. Brechemier, N. Mathy, A-C. Jarrige
UPR 9073 du CNRS, lnstitul de Btologie Phystco-chtratque , 13

rue Pierre et Marie Curie, 75005Parts, France.

Polynucleotide phosphorylase, an exoribonuclease phosphorolysing

processively polyribonucleotides from 3' to 5', is involved in the
control of its own synthesis at the messenger level. The first step of
this mechanism is the cleavage of the pnp messenger near its 5' end
by RNase III [1]. This processing is a primary event in polynucleotide
pbosphorylase autoregulation. This cleavage permits polynucleotide
phosphorylase to act at a subsequent step which results in decreased
expression and mRNA instability [2]. However, no specific direct
interaction between polynucleotide phosphorylase and its processed
messenger has been shown till now. The presence in this enzyme of a
KH domain and a S1 like domain, both corresponding to RNA binding
sites, suggests that a specific interaction with the messenger is
possible.
By using a translational fusion between pnp and lacZ, the effects of
mutations inside these two regions were analysed in vivo. The
mutations do considerably reduce autoregulation but do not abolish
polynucleotide phosphorylase catalytic and degradative activities
suggesting that a direct and specific interaction can occur between
polynucleotide phosphorylase and its messenger, which is necessary
for autocontrol and is independent of its catalytic activities.
[1] Robert-LeMeur, M., Portier, C. EMBO J., 11, (1992) 2633.
[2] Robert-LeMeur, M, Portier, C. Nucleic Acids Res. 22, (1994) 397.

s170

Abstracts FEBS'99

(Mo/7.2/018)

Translation of a short ORF is a prerequisite for

ribosome shunt on a plant pararetrovirus RNA leader
M. M. Pooggin a"b, j. Fiitterer c, K. G. Skryabin b and T. Hohn a

(Mo/7.2/019)

aFriedrich Miescherlnstttute, Basel, ~InstUutefor Plant Sciences, Zurwh,

bCentre "Btoengmeermg", RusmanAcademyofSciences, Moscow. Russta

The 5'-leader of pregenomic RNA of plant pararetroviruses contains

several short ORFs and has a potential to form a large stem-loop
structure. In cauliflower mosaic (CaMV) and rice tungro bacilliform
(RTBV) viruses representing two major groups of plant pararetroviruses,
initiation of translation downstream of the leader occurs via ribosome
shunt that makes ribosomes bypass the inhibitory short ORFs and the
structure. A computer-aided comparison revealed that the Y-proximal
short ORF terminating in front of the stem-loop structure is the
conserved feature of the leader in most plant pararetroviruses [1]. By an
extensive, point mutagenesis we investigated the role of this short ORF
in (i) translation downstream of the CaMV and RTBV leaders in
transiently transfected protoplasts and (ii) infectivity of CaMV in
mechanically inoculated plants. The analysis of stable and revertant
progenies of the mutant viruses [partially documented in ref. 2] and the
effects of mutations and ensuing reversions on shunt-mediated
translation strongly suggested that the 5'-proximal short ORF should be
translated in order to allow efficient ribosome shunt and viability of the
virus. Proper termination of this translational event, both positionally
and mechanistically, appeared to be the most critical step, whereas
initiation and elongation steps and nature of the encoded product turned
out to be of minor importance. Based on the obtained results we propose
a mechanistic model of ribosome shunt.
1. Pooggin et al., J. Gen. Virol., submitted, (1999).
2. Pooggin et al., J. Virol., 72, (1998) 4157.

(Mo/7.2/020)

Endonuclease RNase E, which degrades most E. coli

mRNAs, can be titrated by excess substrate in vivo
S. Sousa, I. Marchand and M. Dreyfus
Laboratoire de G~ndttque Moldculaire (UMR CNRS 8541), Paris, France

RNase E is an essential E. coli endonuclease which controls the decay of most

mRNAs together with the maturation of 5S rRNA. In particular, it efficiently
cleaves the 5' UTR of its own message ( m e mRNA), thereby autoregulating
its synthesis [1]. Recently, we pointed out that due to this autoregulatory
loop, RNase E presumably never exist in excess with respect to its substrates
in vivo: indeed, any excess must lead to destabilisation of the rne m R N A ,
reducing RNase E production [2]. This view yields the simple prediction that,
if the synthesis of substrates were suddently boosted, RNase E would be
titrated, yielding stabilization of most mRNAs including its own. However,
stabilization should only be transient, because following rue mRNA
stabilization RNase E production will increase until it matches again the
concentration of its substrates. To test this prediction, we transformed E. coli
with a plasmid on which a non-functional rRNA operon retaining the 5S
sequence and the bracketting RNase E sites is fused to a strong inducible
promoter. Following induction of this promoter, the cellular concentration of
the rne mRNA increases transiently, reaching a maximum after 10-20 min.
The same result was obtained with rne-lacZ fusions retaining the intact rne 5'
UTR, but not with a similar fusion lacking most of it. Moreover, we have also
shown that this accumulation, which causes an increased synthesis of RNase
E (or RNase E-B-galactosidase)protein, reflects mRNA stabilization. In
addition some typical cellular mRNAs were also transiently stabilized
together with the rne mRNA. Altogether, these observations, which support
the main features of the above model, document an unexpected coupling
between rRNA synthesis and mRNA stability.
[1] Jain, C. etal. Genes & Dev., 9 (1995) 84.
[2]. Lopez, P. J. et aL Proc. NatL Acad. Sci. USA., 95 (I 998) 6067.

Direct identification of translationally controlled

mRNAs
B. Pradet-Balade, W. Mikulits, H. Beudt, J.A. Garcia-Sanz,
E. Mtillner
Departement d'lmmunologia et d'Oncologia, CNB,
Univ. Autonomia, 28049, Madrid (SP)

Translational control appears to be one of the most widespread

post-transcriptional mechanism controlling gene expression. Indeed,
we have shown that it affects 13% of the total mRNA species (7.9%
translationally activated and 4.7% translationally repressed) in T
lymphocytes upon activation (Garcia-Sanz et al., 1998. Faseb
Journal 12, 299-306). T lymphocyte activation is one of the major
stages in an immune response, involving important changes in gene
expression. Thus, it is an example of a physiological transition where
translational control has a substantial contribution.
We then used a differential screening technique to identify the
cDNAs coding for translationally regulated mRNAs. First, we
fractionated ribosome-free and polysome-bound mRNAs, from either
quiescent or activated cells, trough sucrose gradients. These four
mRNA pools were used to label probes, to hybridise either four
replicas of a cDNA microarray or a cDNA library. For each clone,
the distribution of the signal between the free-mRNP's and the
polysome associated fractions was assayed in resting and activated "lcells. A change in this distribution would indicate a translational
control of the corresponding mRNA upon activation. To validate the
results, redistribution of the corresponding mRNAs in polysome
gradients was demonstrated.
In conclusion, this technique allowed us to identify mRNAs
translationally regulated upon T cell activation. It may also be
successfully applied to a variety of other cell transitions.

(Mo/7.2/021)

Mice protein synthesis during experimental

intoxication with cadmium salt
D. Viezeliene, I. Sadauskiene, G. Cherkashin,
R. Stapulionis, L. Ivanov

Institute for Biomedical Research at the Kaunas University of

Medicine, Eiveniu 4, LT-3007 Kaunas, Lithuania

Cadmium is known to be one of the most severely damaging heavy

metals, acting on a variety of biological systems of the living organism.
However, the molecular mechanism of its damaging effect is rather
purely understood so far. One of the likely targets of cadmium action is
the protein synthesis system.
We studied effects of CdCL on the protein synthesis in various organs of
white mice in vivo. 24 hours after the i.p. injection of CdC12 (80 ~g/kg
body weight which equals to the LDI~) there is a tendency for
insignificant increase in total protein synthesis in heart, kidney and
muscle tissue and a decrease in liver. LD2s causes suppression of protein
synthesis in all these organs which is more pronounced in kidney and
liver. Day after the LD~ injection a significant inhibition of amino acid
incorporation into proteins was observed in all tested organs. Heart and
skeletal muscle protein synthesis was the most heavily affected, and
amounted to only around 60 % of the control level. Follow up of the
protein synthesis level within 24 h after the LDzs intoxication revealed a
dramatic inhibition of translation within the first two hours, which
progressed into stimulation, reaching its maximum at 8 hours, and
subsequent decrease at 16 and 24 h. This pattern was displayed by all
four organs, however, amplitude of variations was much wider in the case
of liver and kidney protein synthesis.
Taken together, these results indicate that protein synthesis in vivo can
resist low concentration cadmium poisoning, and there are attempts to
rescue it even at sublethal CdCI 2 concentration. Studies intended to figure
out individual components of translation system, targeted by the
cadmium ions, are required in order to provide details on these processes.

Abstracts FEB S' 99

(Mo/7.2/022)

Translational regulation of cytochromefin Chlamydomonas

chloroplast
K. Wostrlkoff,J. Girard-Bascou,Y. Choquet,D. Stem* and F.-A. Wollman
Inst. Blol. Phys -Chlm. UPR/CNRS 1261, 13 i". P. & M. Curw, 75005 Paris.
France (*) goyce Thompson Inst.. Cornell Un . Ithaca. USA

Studies of the biogenesis of the photosynthetic protein complexes in the

unicellular green algae Chlarnydomonas reinhardtii, have pointed to the
importance of the concerted expression of nuclear and chloroplast genomes.
The accumulation of chloroplast and nuclear encoded subunits is coordinated;
the post-transcriptional expression of chloroplast genes requires gene-specific
nuclear factors. Furthermore, the synthesis of some chloroplast-encoded
subunits, designed as CES proteins (Controlled by Epistasy of Synthesis), is
regulated by the availability of their assembly partners from the same complex.
Both aspects of chloroplast gene expression have been studied for the
petA gene encoding the cytochrome f a major subunit of the cytoehrome b6f
complex. This is a model protein for the study of the CES process. In the
absence of the subunit IV, another subunit of the cyt. b6f complex, its synthesis
is decreased by 90%. This results from a negative autoregulation of cyt.J
translation initiation, mediated by the carboxy-terminal domain of the
unassembled protein [1]. A putative translational activator, capable of
competitive binding to the C-terminal domain of cytf or the 5' untranslated
region o f p e t A mRNA, may be involved in this regulation.
Several nuclear mutants specifically affected in the translation of cyt f
have been isolated; they define a single nuclear locus TCA1. Using chimeric
genes, we have shown that the TCA1 factor and the CES process have the
same target: the 5' tmtranslated region of the petA gene. Several suppressors of
tcal restoring partially the translation of cytf were isolated, the genetic
analysis of which is in progress. Besides numerous nuclear suppressors, some
putative chloroplast suppressors will allow the molecular characterization of
the TCA1 target. In these suppressed strains, the CES process still occurs, but
less efficiently than in wild-type context. These arguments suggest that TCA1
could indeed mediate the CES process.
This leads us to propose a model for the translational regulation of cyt.f,
reflecting the cooperation between nuclear and chloroplast compartments, that
may be general in the biosynthesis of organelle protein complexes.
[1] Y. Choquet, D.B. Stem, K. Wostrikoff, R. Kuras, J. Girard-Bascou and F.-A.
Wollman. Proc. Natl. Acad. Sci. USA (1998) 95, 4380-4385: Translationof cytochromef is
autoregulated through the 5' untranslated region of petA mRNA in Chlamydomonas
chloroplast.

s 171

s172

Abstracts FEBS'99

11.2 Lipids in signal transduction

(Mo/11.2/023) Arachidonic acid is a possible trigger of cardiomyoeyte
hypertrophy
A. Amadou,, C. Pavoine and F Pecker

(Mo/11.2/02A) Nuclear phospholipases and phosphatidyleholine

metabolism in LA-N-1 neuroblastoma cells
P. Antony, J.N. Kanfer and L. Freysz
LNMIC, 67085 Strasbourg Cedex, France and University of Manitoba,
Faculty of Medicine, Winnipeg, Canada R3E OW3

1NSERM Unit~ 99, H6pttal Henri A.[ondor, 94010 Crdted, France

We have recently shown that arachidonic acid (AA) mediates the

contractile response of embryonic chick ventricular cardiomyocytes to
positive inotropic factors (1-2). This action of AA is potentiated by
cyclic AMP (2) Herein, using Fura 2-loaded myocytes, we show that
the addition to the cellular medium of low concentrations of AA (3-10
I.tM) leads, after 10 to 30 min, to a decrease in systolic [Ca]i and the
cessation of electrically stimulated Ca transients. This action of AA is
potentiated by indomethacin, an inhibitor of cycloxygenase, and
mimicked by NDGA, an inhibitor of lipoxygenase, suggesting that a
desequilibfium of AA metabolism in the cardiomyoeyte, which results in
AA accumulation, can rapidly lead to a dramatic perturbation of cell
contraction Agents which evoke elevation of cellular cyclic AMP
protects the cell against AA effect Mathematical modeling designs the
SERCA pump as a possible target of AA action
Hypertrophic hormones, TNF~ (50 n g / m l ) and angiotensin 17
(0 3 ~tM), which evoke AA release, cause a similar cessation of
electrically stimulated Ca transients
These observations pose the question of AA as a possible trigger
of cardiommyocyte hypertrophy
[l]
[2]

Sauvadet A et al, J Biol Chem, 272, (1997) 12437

Pavoine C et al, J Biol Chem, 274, (1999) 628

(Mo/11.2/025)

Nuclear Phosphoinositide 3-kinase y

in pig aortic smooth muscle cells.
D. Bacquevillea, LC. Thiersb, B. Pen'eta, H. Chapa, M.T. Pierraggib,
M. BretonS,lNSERMaU326, CHU Purpan, bU466, CHU Rangueil,
31000 Toulouse, France.

Last years, the existence of an autonomous nuclear polyphosphoinositide

(PI) metabolism which is related to both mitogen-stimulated cell growth and
differentiation, has been etablished. More recently, immunocytochemical
and biochemical evidence suggested the presence of a phosphoinositide 3kinase (PI3K) in the nuclei of rat and human cells. PI3K phosphorylates the
D-3 position of the inositol ring of PI to generate the putative second
messengers PI(3,4,5)P3, PI(3,4)P2 and PI(3)P. However, information
concerning PI3K isoforms and regulation in the nucleus are by now very
limited. Also, we investigated the PI3K distribution and activity in nuclei of
vascular smooth muscle cells (VSMC).
Highly purified nuclei, assessed by biochemical and electron microscopy
analysis, were isolated after a hypotonic shock combined with non-ionic
detergents and sedimentation through a sucrose discontinuous density
gradient (0.3M/2M). In contrast with the previous studies, Western blotting
analysis failed to detect the p85~x regulatory subunit of the PI3K~[3
isotypes in membrane-depleted nuclei. However, a 117 kDa specific nuclear
protein was detected with an antibody directed against the catalytic subunit
of the PI3K7 isotype suggesting the presence of a GTP-regulated PI3K in
VSMC nuclei. PI3K activity was assayed in vitro without exogenous
substrates. Nuclei were unable to produce inositol lipid phosphorylation.
Addition of 100 I.tM GTPyS stimulated only a PI(3,4,5)P3 synthesis whereas
GDPI3S was ineffective. Nuclei preincubation with 20 nM Wortmarmin and
10 ~M LY294002, two PI3K inhibitors, inhibited 60-80% of the GTPySinduced PI(3,4,5)P3 synthesis. Finally, accumulation of PI(3,4,5)P3 was
reduced about 60% by 5 ng/ml pertussis toxin pretreatment.
Taken together, our data suggest that a Gi/Go-dependent PI3K could
phosphorylate an intranuclear pool of PI(4,5)P2 and that PI(3,4,5)P3 may
play a specific role in the nucleus to regulate VSMC proliferation involved
in atherosclerosis and restenosis.

In recent years, it has become evident that agonist stimulation of a variety of

cells results in the induction of specific lipid metabolism in the nuclear
membrane, supporting the hypothesis that the lipids play an important role
in the nuclear signal transduction. While several studies indicate the
existence of a PI cycle in cellular nuclei, little attention has been given to
other nuclear lipids like phosphatidylcholine, known to be a reservoir for the
production of lipid second messengers. To address this question the
metabolism of phosphatidylcholine in the nuclei of neuroblastoma cells LAN-I has been investigated. The cells or nuclei prelabelled with [all]choline
or [3H] 16:0 have been stimulated with phorbol ester (TPA). In presence of
ethanol the stimulation of [3H]16:0 prelabelled cells produced an increase of
DAG, PtdET and PtdOH in the nuclei. Similarly the stimulation of isolated
nuclei with TPA led to the formation of DAG, PtdEt and PtdOH. These
results indicate the presence of PLD and PLC activity in the nuclei of LAN-I which can be activated by TPA, The activation of PLD by TPA is
enhanced in presence of GTP'fS suggesting that the LA-N-1 nuclear
enzymes is regulated by G proteins. Moreover, the stimulation of
[3H]choline prelabelled nuclei led to the release of free choline suggesting
the involvement of PtdCho in the production of lipid second messengers in
the nuclei. This hypothesis has been confirmed by the effect of TPA on the
synthesis of PtdCho in the nuclei. The results showed in LA-N-1 nuclei the
presence of CTP phosphocholine: cytidylyltransferase and CDP choline
diacylglycerol phosphocholine transferase activities but no choline kinase
activity. The treatment of the cells with TPA enhance the incorporation of
P-choline hut not of CDP choline into the nuclear PtdCho. Moreover, the
CTP-phosphocholine cytidylyltransferase activity but not the CDP choline:
diacylglycerol phosphocholine transferase is activated in nuclei of
stimulated cells suggesting the translocation of the P-choline
cytidylyltransferase from cytosol to nuclei. The correlation in LA-N-1
nuclei of the disappearance of PC, the enrichment in PtdOH, DAG and
choline with the stimulation of PtdCho synthesis in TPA treated cells
suggest the existence in LA-N-I nuclei of a PtdCho cycle involved in the
regulation of nuclear function.
(Mo/11.2/026)

SRC-mediated activation of c~diacylglyeerol kinase

is required for HGF signalling
G. Baldanzi, S. Cutrupi, D. Gramaglia*, A. Maffe*,
P.M. Comoblio*, F. Bussolino*, A. Graziani
Dept of Genetics, Biology and Biochemistry, University of Torino,
*Institute for Cancer Research (IRCC), Torino,
** Division of Cellular Biochemistry,
The Netherlands Cancer Institute, Amsterdam

Diacylglycerol kinases, which con,,ert diacylglycero[ into phosphatidic

acid, have been suggested to be involved in cell signaling, either as
regulator of diacylglycerol levels or as intracellular signal generating
enzymes However neither their role in s~gnal transduction nor their
biochemical regulation have been elucidated Hepatocyte Growth Factor
(HGF), upon binding to its tyrosine kinase receptor, activates multiple
signaling pathways stimulating cell motility, scattering and proliferation,
which lead to branching morphogenesis in epithelial cells and
angiogenesis in endothelium. Herein we demonstrate that (i) the
enzymatic activity of ~xDiacylglycerol kinase ((xDgk) is stimulated by
HGF in epithelial, endothelial cells and in ctDgk-transfected cells; (ii) an
otDgk-specific inhibitor
(R59949),
prevents
HGF-induced
morphogenesis, cell movement and DNA synthesis in epithelial cells and
endothelial cells, (iii) ctDgk associates in a complex with Src in HGFstimulated cells, and inhibition of Src by either Src dominant negative
mutant or by PP1, a Src-specific inhibitor, inhibit HGF-induced ~Dgk
activation The data reported herein provide the first evidence for
involvement of Dgk in tyrosine kinase receptor signaling and suggest
that Src mediates its activation

Abstracts F E B S ' 9 9

(lVlo/11.2/027)

Sphingosine and signal transduetion in the heart.

V. E. Benediktsd6ttir, B. H. Skfilad6ttir, A. Grynberg and S.
Gudbjamason

s 173

(Mo/11.2/028) Do sphingosine and sphingosine derivatives stimulate

phospholipase D in C6 glioma cells?
M Bobeszko, A Dygas, J Baraflska
Nenck~ Institute of Expemmental Biology, Laborato O" of Signals
Transductton, Pasteura 3, 02-093 Warsaw, Poland

Science Institute, University of Iceland, Vatnsm~jrarveg 16, IS-I01

The purpose of this study was to examine the effects of sphingosine and
sphingosine-l-phosphate on signal transmission in the B-adrenergic pathway
in cultured neonathal rat cardiomyocytes. The effects of these agents on
cAMP levels in stimulated and unstimulated myocytes were determined as
well as the effect on the rate of contraction of the cardiomyocytes. The cells
were incubated with 10~dvl sphingolipids for different periods of time after
which the cAMP levels or beating rates were measured without or after
stimulation with l~tM isoproterenol for 10 rain.
The results demonstrated that sphingosine and sphingosine-l-phosphate
may be involved in signal transmission in the B-adrenergic pathway.
Incubation with sphingosine or sphingosine-l-phosphate for 30 rain
decreased the cAMP levels after isoproterenol stimulation by 50% and 30%,
respectively, compared to control. Both these agents also lowered basal
cAMP levels after 30 min incubation and this effect of sphingosine was
observed already after 3 min. Furthermore sphingosine had a long-term (24
h incubation) negative effect on basal cAMP levels. The negative effect of
sphingosine on signal transmission in the B-adrenergic pathway was also
demonstrated by a significant reduction in the rate of contraction of the

In the present study we investigate an effect of exogenous sphingosine,

sphingosine l-phosphate and sphingosylphosphorylcholine on
phospholipase D activity in glioma C6 cells. The cells were prelabeled
with [1)4C]palmitic acid and phospholipase D-mediated synthesis of
[]4C]phosphatidylethanol was measured. We have found that
sphingosine i-phosphate and sphingosylphosphorylcholine do not
stimulate ['4C]phosphatidylethanol formation both at low (0.1-10) and
high (25-I00) [aM concentrations. On the other hand, sphingosine at
concentrations of 100-250 ~tM exerts strong stimulatory effect on
phospholipase D as compared to the effect of phorbol ester, 12-Otetradecanoylphorbol 13-acetate, known as a phospholipase D activator.
The effect of 12-O-tetradecanoylphorbol 13-acetate on phospholipase D
is linked to the activation of protein kinase C. The present study also
shows that sphingosine additively enhances TPA-mediated
phospholipase D activity. This is in contrast to the postulated role of
sphingosine as a protein kinase C inhibitor. These results demonstrate
that in glioma C6 cells sphingosine not only affects phospholipase D
independently of protein kinase C, but is also unable to block TPAmediated phospholipase D activity.

cardiomyocytes when exposed to sphingosine.

Conclusion: Sphingosine and sphingosine-l-phosphate modulate the signal
transduction of the 13-adrenoceptor pathway in rat cardiomyocytes.

(Mo/11.2/029)

Phytosphingostne and sphingoslne are synthettzed by different

pathways in flsb leukecytes. Incorporation into sphingolipids,
J. Bodermeca,b, G. Zwingelstemb , G. Brichon b, O. Koul c, J. Portoukalian a
aLaboratory of Tumor Glycobiology, Faculty of Medicine Lyon-Sod, 69921

Oallins, France. blnstimte Michel Pacha, 83500 La Seyne Sur Met, France.
cDepartment of Neurology, Harvard Medical School, MA 02114, USA

To better understand the de novo biosynthesis of sphingolipids,

assumed to begin by condensation of palmitoyl-CoA with serine, we have
been studying the biosynthesis of sphingoid bases from their precursors. We
have studied the incorporation of three different radioprecursors {(3H)serine,
(3H-methyl)methionine and (14C-methyl)methionine} into free ceramides and
sphingolipids. Peripheral blood mononuclear leukocytes isolated from the
fish Dicentrarchus labrax, were incubated for up to 5 hr with each of the
precursors. Lipids were extracted and purified according to the procedure of
Folch. Free ceramides, neutral glycosphingolipids (n-gly) and sphingomyelin
(sph) were separated by a solid phase extraction procedure modified from
Bodennec et al [I]. The extracted lipids were sequentially hydrolysed to
obtain the com]~onent moieties to localize and determine the extent of
incorporation. (3H)serine was preferentially incorporated into sphingosine,
while (3H-methyl)methionine incorporated into phytosphingosine backbone
of free ceramides (respectively 87 and 77% of total radioactivity in total free
ceramides), confirming our earlier observations [2}. In order to avoid
contribution of tritium exchange reactions, latter experiments were done with
(14C-methyl)methionine instead of the tritiated precursor. The extent and
preferential localization of the (14C) precursor into phytosphingosine was
similar to that obtained with the (3H) precursor, indicating the en bloc
incorporation of the methyl group from methionine into this sphingoid base.
Further, a follow up of radioactivity into sph indicated a preferential
incorporation of (3H)-serine into sphingosine and sphinganine backbone
(respectively 72 and 9% of total radioactivity in sph). [n contrast, the
radioactivity incorporated into n-gly came from (3H-methyl)methionine, and
was preferentially localized in phytosphingosine bases (76% of total n-gly
bases). These results indicate that in fish leukocytes phytosphingosin and
sphingosine bases are synthesized from different precursors. Moreover, the
newly synthesized phytosphingosine is incorporated into n-gly. We conclude
that, during biosynthesis, certain precursors are preferentially incorporated
into specific sphingoid bases through probably different biosynthetic
pathways. The observed differential incorporation of the two precursors into
complex sphingolipids is consistent with a selective orientation
(compartmentation?) of the newly formed bases within the cell.
[ 1] Bodennec, J. et al. J. Lipid Res. 3 8, (1997), 1702.
[2] Bodennec, J. et al. Biochem. Biophys. Res. Comm. 250, (1998), 88.

(M0/11.2/030)

ILII~ induces type-ll-sPLA2 gene in vascular smooth

muscle cells by a NF ~B and PPARs-mediated process.
A. Brouillet, C. Couturier, G. B~r~ziat. M. Andr~ani
UPRES-A 7079, 27 rue de Chahgny 75012 Paris, France

Type-II-secreted phospholipase A2 (type-II-sPLA2) is expressed in

smooth muscle cells during atherosclerosis or in response to Imerleukin113. The present study shows that the induction of type-II-sPLA2 rat gene
by lnterleukin-113 requires activation of the NFrd3 pathway and cytosolicPLA2/PPAR~/ pathway, which are both necessary to achieve the
transcriptional process. Interleukin-l{3-induced type-II-sPLA2 gene doseand time-dependently and increased the binding of NFr,B to a specific site
of type-II-sPLA2 rat promoter (-194, -174). This effect was abolished by
proteinase inhibitors which block the proteasome machinery and NFrd3
nuclear translocation. Type-II-sPLA2 induction was also obtained by free
arachidonic acid and blocked by either AACOCF3, a specific cytosolicPLA2 inhibitor, PD98059, a MAPkinase-kinase inhibitor which prevents
cytosolic-PLA2 activation, or NDGA, a lipoxygenase inhibitor, but not by
the cyclooxygenase inibitor indomethacin, suggesting a role for a
lipoxygenase product. Type-II-sPLA2 induction was obtained after
treatment of the ceils by 15-deoxy-Al2']4dehydroPGJ2, carba-prostacyclin
and 9-hydroxyoctadecadienoic acid, which are ligands of PPAR% whereas
PPAR<x ligands were ineffective. Interleukin-113 as well as PPARy-ligands
stimulate the activity of a reporter gene construct containing two PPRE
binding sites (PPREx2-TK-Luc). We also identify a weak PPAR binding
site in type-lI-sPLA2 promoter (-160, -133) presenting extensive
homology with DRI element of a consensus PPRE, which binding is
nevertheless induced by Interleukin-II3 stimulation. In the present study,
we demonstrated that the binding of both NTr,B and PPAR3' is required to
stimulate the transcriptional process because inhibitors of each
transduction pathway block Interleukin-113-induced type-II-sPLA2 gene
activation. We therefore suggest that NFr,B and PPAR7 cooperate at the
enhanceosome-coactivator level to turn on transcription of the
proinfiammatory type-II-sPLA2 gene.

s 174

Abstracts F E B S ' 9 9

(M0/11.2/031)

Role of glycosphingolipid-rich domains in

CD77.mediated apoptosis of Burkitt's ceils.

Sphingosine and sphingosine 1-phosphate

modulate calcium signals in glioma C6 cells

K. Carlier,C. Tetaud, B. Clausse, P. Busson, J. Wiels

R. Czajkowski, P Sabata and J Baraflska

Interactions moleculaires et caneer.CNRS UMR1598, IGR, 39 rue

C. Desmoulins,94805 Villejuif Cedex

Nencki Institute of Experimental Biology, Laboratory of Signals

Transduction, 3 Pasteura St.. 02-093 Warsaw. Poland

In the hsematopoietic system, CD77 is restricted to Buddtt's

lymphoma (BL) cell lines and a subset of germinal center B lymphocytes.
Previously, we have reported that CD77" B lymphocytes undergo rapid and
spontaneous apoptosis when isolated and cultured in vitro and that specific
binding of CD77 induce apoptosis of CD77 BL cell lines.
Recently, glycosphingolipid (GSL)-rich domains (also called "lipid
rafts" or "DIGS: detergent-insoluble glycolipid-enriched structures") have
been described. In these microdomains of the plasma membrane, GSL and
sphingomyelin are self assembled .to form clus~rs which also ~clude
cholesterol but exclude gjyceropnospnotipias, beverm proteins are specmcaity
concentrated in these lipid rafts: GPI-anchored proteins, some transmembrane
proteins and, in the inner leaflet of the membrane, tyrosine kinases of the Src
family and the Ga subunit of the heterotrimeric G proteins. The GSL-rich
domains are involved in membrane trafficking and in signal transduction.
Since CD77 is a GSL surface antigen, we have analysed its subcellular
localization. BL cells have been fractionated using a method based on Triton
X-100 extraction and flotation through a sucrose step gradient. This method
allowed us to recover three subcellular fractions: HSP which contains the
cytosqueletal elements, F40 which contains most of the cellular proteins and
the GSL-rich fraction. The distribution of CD77 in these fractions was then
determined by thin layer chromatography and orcinol staining. As expected,
more than 80% of CD77 was found in the GSL-rich domain fraction. We
have also shown, by immunoprecipitation experiments that Lyn, a Src-family
tyrosine kinase, is mostly located in this fraction as well as most of the
tyrosine phosphorylated proteins.
In order to elucidate the signal transduction mechanism which leads to
apoptosis of the ceils after triggering of CD77, we then investigated the
relationship between Lyn and CD77 and whether or not modifications of
tyrosine-phosphorylation occured after CD77 ligation.
By coimmanoprecipitation experiments, we have been able to demonstrate that,
within GSL-rich domains, CD77 and Lyn are physically associated. We have
also shown, by Western-blot analysis, that cross-linking of CD77 is
followed, very rapidly (2 min.), by an increase in tyrosine-phosphorylation of
various proteins. These proteins, with molecular weights in between 65 and
80kDa, are also associated with CD77 since they are co-precipitated by an
anti-CD77 mAb. We are currently trying to identify these proteins.

(M0/11.2/033)

(Mo/11.2/032)

Isolation of a PI-PLC-associated protein from soybean

Sphingosine (SPH) and its derivative, sphingosine-l-phosphate

(SPP), natural constituents of animal cells, attend considerable
attraction as bioactive lipids that regulate cell physiology. They act as
first and second messengers and may modulate the calcium signalling
pathways. This study was aimed to investigate the effect of SPH and
SPP on changes in intracellular Ca ~+ concentration in glioma C6 cells
and was studied with Fura-2 video imaging technique.
SPH, at high concentration of 100 gM caused a rise in intracellular
calcium level, ranging from 50 to 200 nM. It also diminished the
response for thapsigargin (TG - an inhibitor of calcium pump in
endoplasmic reticulum) and ATP (acting via IP3) when applied 5 rain
before. Preincubation with neomycin, which inhibits PLC partially
eliminates this effect. This suggests the involvement of PLC in
sphingosine-mediated calcium mobilisation.
However, SPH added 1 rain. before ATP increases the calcium
response for this agonist. This is probably due to inhibition of PKC.
TPA has an opposite effect. It diminishes calcium response for ATP
by activating PKC. SPH and TPA added simultaneously also cause a
decrease in IP3-mediated response for ATP.
SPP added extracellularly in nM concentrations caused an
immediate and high rise in cytosolic Ca z+ level, in PTX-sensitive
manner. It is therefore suggested that when SPH acts inside the cell
and modulates Ca 2~ signals in a pleiothropic manner, SPP has a
property of the first messenger, acting on glioma C6 plasma
membrane receptor.

(Mo/11.2/034)

SHIPI and SHIP2 in human blood platelets

C. Dammann and M.K. Bhattacharyya

S. Giuriato a, X. Pesseseb , B. Payrastre ~, C. Emeux b, H. Chap a

Samuel Roberts Noble Foundation, Plant Biology Division, P.O Box

2180. Ardmore OK 73402. USA

INSERM U. 326, CHU Purpan, 31059 Toulouse, France

blRIBHN, ULB, 808 route de LenntL 1070 Brussels, Belgique

Using the yeast two hybrid system we have identified a protein that
binds to phosphoinositide-specific phospholipase C1 (PI-PLCI) from
soybean. A cDNA clone of 2.7 kb has been isolated that encodes a
protein of 800 amino acids. No significant homology to known genes
could be found. However, a search in the "prints" database revealed
footprints of the rhodopsin superfamily of G-protein-coupled
receptors. The footprints map in the predicted transmembrane
domains of this new protein.
Southernblot analysis indicates that the gene is present in not more
than two copies in soybean. The gene is constitutively expressed in all
organs of the plant with very little variation in the expression level.
Moreover, almost identical amounts of mRNA were detected in
developing and mature tissues from leaves, flowers and fruits.
Homologous genes are present in Arabidopsis and rice.

The SH2 domain-containing inositol 5-phosphatases, SHIP1 and SHIP2,

are important regulators of the phosphoinositide metabolism. Their primary
structures present several functional protein interaction domains such as
SH2 domain, proline rich sequences and NPXY motives able to bind PTB
domains. Their catalytic activity is specifically directed against
Ptdlns(3,4,5)P~ and Ins(l,3,4,5)P~ from which they remove the 5 phosphate
of the inositol ring to produce PtdIns(3,4)P, and Ins(1,3,4)P, respectively.
These t,~,~, proteins are expressed in human blood pl~:~lets and may
participate in the production of PtdIns(3,4)P2 observed upon stimulation
downstream of integrin engagement and platelet aggregation. Indeed,
platelet activation induces a rapid and transient production of PtdIns(3,4,5)P3
followed by a late phase of PtdIns(3,4)P2 accumulation. We have studied the
activities of SHIP 1 and SHIP2 immunoprecipitated from resting or thrombin
stimulated platelets. It appears that SHIP1 presents a basal PtdIns(3,4,5)P~
and Ins(1,3,4,5)P, 5-phosphatase activity which is not upregulated during
the activation. In contrast, SHIP2 is activated in a stimulation dependent
manner as a Ptdlns(3,4,5)P~ 5-phosphatase but its Ins(1,3,4,5)P, 5phosphatase activity is undetectable. Therefore, it appears that these
isoenzymes have distinct behaviour. This is probably due to very precise
regulatory mechanisms such as phosphorylation, subcellular relocation or
specific protein-protein interactions. Concerning SHIP1, we have shown
that its tyrosine phosphorylation and cytoskeleton translocation increased
upon thrombin stimulation in an integrin and aggregation-dependent
manner. Moreover, our results suggest that the tyrosine phosphorylation of
SHIPI could be due to the kinase c-Src since: 1) SHIP1 and c-Src are
constitutively associated. 2) Src kinase activity is recovered in SHIP1
immunoprecipitates. 3) Src kinase inhibition by PP1 abolished the tyrosine
phosphorylation of SHIPI without affecting the aggregation response of
platelets. Nevertheless, this tyrosine phosphorylation did not change the 5phosphatase activity of SHIP1 suggesting that the relocation of this enzyme
is very important for its regulation.

Abstracts FEBS'99

s175

(Mo11.2/035) Diaeylglycerol kinase 0, a novel effeetor of RhoA

B. Houssa, J. de Widt, O. Kranenburg W. Moolenaar and W.
van Blitterswijk. Division of Cellular Biochemistry, The Netherlands

(Mo/11.2/036)

T. Kanematsu, H. Takeuchi and M. Hirata

Department of Biochemistry, Faculty of Demistry, Kyushu University.
Fukuoka 812-8582, JAPAN

Cancer Institute, Plesmanlaan121, 1066 CX Amsterdam, The Netherlands.

Diacylglycerol kinase (DGK) phosphorylates the second messenger

diacylglycerol to yield phosphatidic acid. To date, nine mammalian DGK
isotypes have been cloned, but little is known about the regulation of DGK
activity. We have previously identified the DGK0 isotype, which is
predominantly expressed in brain [ 1]. DGK0 binds specifically to activated
RboA in transfected COS cells as well as in non-transfected neuronal N1E115 cells [2]. Binding is abolished by a point mutation (Y34N) in the
effector loop of RhoA. DGK0 does not bind to inactive RhoA, nor to the
other Rho-family GTPases, Rac or Cdc42. Like active RhoA, DGK0
localizes to the plasma membrane. Strikingly, the binding of activated
RhoA to DGK0 completely inhibits DGK catalytic activity. Our results
suggest that DGK0 is a downstream effector of RhoA and that its activity is
negatively regulated by RhoA. Through accumulation of newly pmduced
diacylglycerol, RhoA-mediated inhibition of DGK0 may lead to enhanced
PKC activity in response to external stimuli.
[ 1] Houssa etal., J Biol. Chem. 272, (I 997) 10422.
[2] Houssa etal., J Biol. Chem. In press

(Mo/11.2/037)

Specific inhibition of collagen-stimulated platelet

aggregation by a novel, fatty acid-binding protein
R. J. Keogh and R. W. Famdale
Deparmwnt of Biochemistry, Universio'of Cambridge.
Tennis Court Road, Cambridge. CB2 I QW. Umted K~ngdom

Injury to the wall of a blood vessel results in the exposure of subendothelial

collagens to which platelets adhere and then aggregate, resulting in formation of
a thrombus. Anti-platelet factors act to inhibit the aggregation of platelets and
are commonly found in the saliva of blood-sucking arthropods. One of these
materials is pallidipin, a novel, 19 kDa protein isolated from the saliva of the
assassin bug Triatoma pallidipennis [I]. The aim of this work was to
characterise the effects of pallidipin on platelet activation and to determine the
mechanism of its anti-platelet action. Experiments were performed on washed
platelets prepared from human blood (for aggregation and adhesion) or platelet
concentrates. Aggregation was measured turbidimetrically, adhesion was
measured colorimetrically by a microtitre plate assay, thromboxane A2 (TxA2)
was measured as its stable metabolite TxB2 by ELISA and fatty acid binding
was measured using a modified Lipidex method. Aggregation of platelets
induced by collagen f~bres was specifically inhibited by pallidipin with no effect
on that induced by a collagen related peptide (CRP-XL), thrombin or the TxA2
mimetic U46619. Pallidipin was without effect on platelet adhesion to
monomeric collagen (via integrin ot2~]1), demonstrating that the inhibition of
aggregation observed does not result from pallidipin binding to collagen
receptors. Adhesion studies also showed that pallidipin had no effect on
adhesion to CRP-XL, a GPVI agonist [2], or to OKM5, an anti-CD36
monoclonal antibody. Pallidipin treatment of platelets was without effect on
several signalling pathways activated by collagen including the activation of
protein kinase C, phospholipase Cy2, p125FAK and p72syk. Pallidipin was
found to inhibit TxA2 synthesis induced by collagen, CRP-XL and thrombin
suggesting an effect on arachidonic acid (AA) metabolism, the substrate for
TxA2 production. Aggregation induced by AA was inhibited by pallidipin and
the activity of the cyclooxygenase which catalyses the first step in TxA2
synthesis was inhibited by pallidipin treatment. Based on amino acid sequence
analysis of pallidipin and related proteins it was proposed that pallidipin can
bind AA, which was confirmed by demonstrating that pallidipin binds AA in a
concentration-dependent manner. In conclusion, we propose that pallidipin
abrogates collagen-induced platelet aggregation by binding AA and inhibiting
TxA2 synthesis, Collagen-induced aggregation is dependent on TxA2
production unlike aggregation induced by other ligands [3]. This makes
pallidipin a novel and collagen-specific anti-platelet factor.
[1] Noeske-Jungblut, C. etal., J Biol Chem, 269, (1994) 5050.
[2] Kehrel, B. etal., Blood, 91 (1998) 491.
[3] Takahara, K. etal., l Biol Chore, 265 (1990) 6836.

Possible function of a new inositol 1,4,5trisphosphate binding protein (p130)

We isolated a novel inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) binding protein

with a molecular mass of 130kDa (p130), which was later found to be a similar
protein to 61-isozyme of phospholipase C (PLC-bl), but had no catalytic
activity as PLC. In-the present study, we established a cell-line which was
stably over-expressing p130 (COS-pl30) in order to assess the physiological
function of p130. Fura-2-1oaded COS-pl30 were stimulated with either
bradykinin (BK) or epidermal growth factor (EGF) and the changes in free
Ca 2. concentration was monitored. Compared to the control cells, the
responses of free Ca x+ change were diminished, and thus dose-response curve
shifted to the right in both cases of stimulation. Since p130 bound to
Ins(1,4,5)P 3 through its pleckstrin homology (PH) domain, green fluorescence
protein (GFP) fused p130, PH domain deleted p130 (p130APH) or GFP alone
was transiently expressed in COS-I and stimulated with ATP in order to clarify
whether Ins(1,4,5)P 3 binding was necessary for the effeCt of p130 on COS-I
ceil mentioned above. While decreased Ca2+ response to ATP was observed by
GFP-pl30 expression in COS-t, GFP alone or GFP-p130APH expression in
COS-1 had no effect on Ca 2 response to ATP. Two possibilities could be
assumed for the reason of the diminution of Ca"``+response; one is the blockade
of phosphatidylinositol 4,5-bisphosphate (PIP,_.) hydrolysis by binding of p130
to PIP2, or another is the binding to cellular lns(1,4,5)P3, not PIP2, thus
reducing Ins(1,4,5)P3 available to the receptor molecules. To discriminate
these possibilities, Ins(1,4,5)P3 productions in response to BK and EGF were
measured. The production of Ins(1,4,5)P3 was not inhibited by the overexpression of p130, rather it seemed a little bit enhanced. In fact, we observed
the reduction of the metabolizing activity to Ins(1,4,5)P3 when lns(1,4,5)P3 5phosphatase or lns('t,4,5)P3 3-kinase was measured in the presence of p130.
These results indicate that the diminution of Ca 2 response by p130 could be a
result of binding of cellular lns(1,4,5)P3 produced by PLC activation,
indicating that p130 might function as a negative regulator for Ca"-* signaling
pathways in cells.

(Mo/11.2/038)

Engagement of T cell receptor triggers

its recruitment into raft domains
C.Langlet, A-M. Bernard, P. Drevot, P, T H e
Centre d'immunologie INSERM-CNRS de Marseille
Lumlny. caseg06, 13288Marsetlle Codex 9,France

T cell receptors (TCRs) upon binding to peptide-MHC ligands

transduce
signals
in
T
lymphocytes.
Tyrosine
phosphorylations in the cytoplasmic domains of the CD3 (yge)
and ~ subunits of the TCR complex by src famaly kinases are
known to initiate the signaling cascades via the docking and
activation of ZAP-70 kinase and other signaling components.
Here, we examined the role of detergent-insoluble membrane
rafts (DIMs) in TCR signaling. Using mouse thymocytes as a
model, we first characterized in detail the structural
organization of DIMs. We then demonstrated that TCR
engagement triggered an immediate increase in the amount of
TCR/CD3 molecules present in DIMs. The TCR/CD3
recruitment is accompanied by the accumulation of a series of
prominent tyrosine phosphorylated substrates and by an
increase of Lck activity in DIMs. Upon TCR stimulation, the
DIM-associated receptor complexes are highly enriched in the
hyper-phosphorylated p23 ~ chains, contain most of
TCR/CD3-associated,
phosphorylation-activated ZAP-70
kinases and seem to integrate into higher-order, multl-tyrosine
phosphorylated substrate-containing protein complexes, In
addition, the TCR/CD3 recruitment was found to depend on
the activity of src family kinases. We thus provide the first
demonstration of a recruitment of TCR/CD3 to DIMs upon
receptor stimulation and propose it as a mechanism by which
TCR engagement is coupled to downstream signaling
cascades.

s 176

(Mo/11.2/039)

Abstracts FEBS'99

N-ethylmaleimide-stimulated arachidonic acid release in

human platelets
G. Leoncini, M.G. Signorello

(Mo/11.2/040)

DIMES Sezione Biochimica, V.le Benedetto XV, 1 16132 Genova -haly

Treatment of human platelets with the alkylating agent N-ethylmaleimide

(NEM) induces amchidonic acid release. The effect is time and dosedependent. NEM-stimulated arachidonic acid mobilisation can be prevented
by pretreating platelets with the cytosolic phospholipase A_, (cPLA2) specific
inhibitor arachidonyltrifluoromethylketone (AACOCF~). Moreover the
tyrosine kinase inhibitor genistein is able to significantly inhibit arachidonic
acid mobilisation. NEM-stimulated release of arachidonic acid appears to be a
Ca-'* dependent mechanism as shown by the observation that arachidonic
mobilisation is significantly reduced by platelet treatment with EGTA and is
abolished by preloading platelets with the intracellular chelator BAPTA/AM.
In FURA 2-loaded platelets, NEM is able to significantly increase intracellular
Ca2+ level. The Ca 2 elevation is suppressed by cell treatment with
BAPTA/AM. Arachidonic acid released by cPLA2 activation produces a
significant elevation of reactive oxygen species (ROS) intracellular levels. The
accumulation of these reactive compounds is almost completely inhibited by
5,8,11,14-eicosatetrayonic acid (ETYA), which blocks arachidonic acid
metabolism, suggesting that in NEM-treated platelets ROS are in large part
generated during arachidonic acid metabolisation. In addition ROS
accumulation is partially inhibited by diphenyliodonium (DPI), indicating that
other mechanisms such as the direct or indirect activation of NADP(H)oxidase
by arachidonic acid are involved.

INSERM Unit~ 99, HOpital Henri Mondor, 94010 Cr~teil, France

We have recently demonstrated [1] that the contractile effect

elicited by 5z-adrenergic receptor stimulation in embryonic chick
ventricular cardiomyocytes is mediated by arachidonic acid via a
pathway sensitive to AACOCF3, the cytosolic phospholipase A2
(cPLA2) inhibitor. In the present study, we investigated the role of the
Mitogen-Activated Protein Kinase (MAPK) pathway in the ~ adrenergic receptor stimulation. By using Fura 2-loaded myocytes, we
show that stimulation of the amplitude of Ca 2+ transients by the specific
~-adrenergic agonist, zinterol, was abolished after treatment of the
myocytes with 10 ~tM SB203580, a specific p38 MAPK inhibitor.
Western blot analyses indicated that zinterol triggered a rapid and dosedependent phosphorylation of both p38 and p42-44 MAPK and
suggested that zinterol may induce MAPK activation. Preparation of cell
lysates from zinterol treated myocytes and subsequent dosage of cPLA2
demonstrated that zinterol enhanced cPLA2 activity, inducing the
translocation of the enzyme from the 100,000g supernatant fraction to
the 100,000g pellet fraction. Treatment of the myocytes with the p42-44
MAPK inhibitor, PD98059, or with SB203580 affected zinterol
activation of the cPLA2.
We conclude that MAPK activation is required for cPLA2mediated AA release by B2-adrenergic receptors. These results highlight
the key role of the MAPK pathway in ~-adrenergic receptor induced
contractility of cardiomyocytes
[1]

(Mo/11.2/041) Non-covalent opsinlall-trans-retinal complex activates

transducin (Gt) depending on receptor palmitoylation
D. Maretzki, K. Sachs, K.P. Hofmann
Instttut far Medlzmtsche Physzk & Btoph_vsik, A,ledtzintsche Fakultdt
(Charltb), Humboldt-Umversltdt, D-10098 Berhn, Germany

Opsin with the chromophore site empty has a very low catalytic rate of
transducin (Gt) activation m vitro. Binding ofaU-trans-retinal (atr) to opsin
enhances its activity towards Gtup to a maximum of 5% of the light-induced
activity (Meta II photoproduct). The activity is also seen with reductively
methylated opsin, in which the original binding site is blocked [1].
Moreover, air does not compete with 1 l-c/s-retinal incorporation into the
light-sensitive binding site during regeneration of opsin to rhodopsin, even
in an amount that saturates the activity of opsin/atr complex. This suggests
that the atr/opsin-complcx catalyzes Gt activation by different mechanisms
then Meta IL We have now studied the influence of phosphorylation and
palmitoylation on the atr promoted activity of opsin. Rhodopsin is palmitoylated at cysteines (Cyss22 and Cys323),thus forming the putative fourth
cytoplasmic loop of the receptor Atr recombines equally well with opsin
or phosphorylated opsin, leading to the same level of activity in the Gtct
intrinsic fluorescence assay. Reductive chemical depalmitoylation of opsin
reduces the activity to 25 % of the palmitoylated control. Repalmitoylation
of opsin by autoacylation with palmitoyl-Coenzyme A partially restores the
original rates of Gt activation.
The non-covalent opsin/atr complex represents an ,,agonist-like" second
signaling state (R**) which differs from photoactivated Meta II (R*).
It is not deactivated by phosphorylation The observation that its catalytic
efficiency is enhanced by palmitoylation suggests a functional role for this
modification.
[1] J~ger, S. et al., Biochemistry, 35 (1996) 2901

1$2-adrenergic agonists activate the cPLA2 via the

MAPkinase pathway in ventricular cardiomyocytes.
S Magne, F. Pecker, C. Pavoine

Pavoine C. et al., J Biol Chem, 274, (1999) 628

(Mo/11.2/042)
Molecular and biochemical characterisation of type 2 phosphatidic
acid phosphatases from guinea pig airway smooth muscle
A. E. McKie, P. L Darroch and S. Pyne
Department of Physio/og)' and Pharmacology, Untverstty of Strathclyde,
Glasgow, Scotland

Type 2 phosphatidic acid phosphatases (PAP2) catalyse the

dephosphorylation of phosphatidic acid (PA), lysophosphatidate and
sphingosine l-phosphate. Two PAP2 cDNAs have been isolated and
characterised from guinea pig airway smooth muscle cells (gpPAP2AI and
gpPAP2A2). The gpPAP2AI and gpPAP2A2 genes are 923bp and 926bp in
length, respectively and are predicted to encode proteins of 32.1kDa.
Analysis of the predicted amino acid sequences shows 91% identity between
gpPAP2A~ and gpPAP2A2 with 94% and 84% identity, respectively, for
human PAP2A.
We have cloned the two guinea pig PAP2A genes into a protein expression
vector for use in a bacterial system. Recombinant gpPAP2At and gpPAP2A2
possess a 6-Histidine tag at their N-terminus, which allows purification of
the expressed protein using metal chelate affinity chromatography. Results
of the biochemical characterisation of the recombinant PAP2As will be
presented here.
We are also using a degenerate oligonucleotide/PCR approach to determine
whether there are other isoforms of PAP2 present in the airway smooth
muscle of guinea pig. Data from this molecular characterisation will be
shown.

Abstracts FEBS'99

(Mo111.21043)

s177

Lipid products of phosphoinositide 3-kinase interact in vitro

with Racl GTPase and stimulate GDP dissociation
K. Missy. V Van Poucke. P. Raynal, C. Vlala, G. Mauco,
M. Plantavid, H. Chap, B. Payrastre
INSERM Umtc;326. H3pttalPmpaJt. 31059 Toutous-e c~ctet 03. France

Phosphoinostide 3-kinase (PI 3-kinase) is a key player in several cellular

functions, including cell proliferation, cell differentiation, cytoskeleton
reorganisation or intracellular trafficking [1]. PI(3,4)Pz and PI(3,4,5)P3, two
PI 3-kinase products, accumulate after cell stimulation by various agonists,
and are now considered as second messengers. Recent studies have reported
many signalling proteins that interact with PI 3-kinase products, resulting in
their membrane relocalisation and/or in the modulation of their activity [2].
Small G-proteins of the Rho family are known to regulate the reorganisation
of actin cytoskeleton [3], and a number of reports suggest that under different
conditions leading to this reorganisation, the GTPases Racl and possibly
RhoA are downstream targets of PI 3-kinase.
In order to bring more information on the role of PI 3-kinase in the regulation
of Rho GTPases, we have investigated the hypothesis of a direct interaction
between D3-phosphoinositides and the members of Rho family Using
recombinant proteins, we have observed that Racl, and to a lesser extend
RhoA, but not Cdc42, were capable to selectively bind to PI(3,4,5)P3 in a
mixture of crude brain phosphoinositides. In contrast to the interactions
described for Pleckstrin Homology domains, we have demonstrated that this
binding with Racl and RhoA require both specific positions of phosphate
groups on the inositol ring and the two fatty-acyl chains. The analysis of Racl
primary sequence allowed us to identify two potential binding sites. Their
deletion led to the complete loss of binding to D3-phosphoinositides. Finally,
we have demonstrated that PI(3,4,5)P3 strongly stimulate, in vitro, the GDP
dissociation from Racl in a dose dependent manner. In agreement, we have
observed in Vero cells, that a pool of activated Racl was targeted to the
plasma membrane upon stimulation with epidermal growth factor, in a PI 3kinase dependent manner To conclude, we can hypothesise that PI(3,4,5)P3
might help the translocation of Racl to specific membrane clusters containing
not only small G-proteins, but other partners like exchange factors, required
for the Racl activation.

(Mo/11.2/044)

Inhibition of LPA-induced DNA synthesis by

KN-62 and Tyrphostin AG 1478 in human SMCs
U.K. Nilsson and S.P.S. Svensson
Dtv. of Pharmacology, Dept. of Medicine and Care, Faculty of llealth
Sciences. Linkrpings Universitet. SE-581 85 Linkrping, Sweden

Lysophosphatidic acid (LPA) is an intercellular lipid messenger which is

produced and released from membrane microvesicles from activated ceils.
LPA signals ceils through G-protein-coupled receptors and can exert such
diverse effects as smooth muscle contraction, platelet aggregation and
fibroblast proliferation. We have previously reported that LPA has the
ability to increase DNA synthesis and to rapid and transient rise the
intracellular concentration of calcium in smooth muscle cells (SMCs) from
the human myometrium. The exact role of calcium in the regulation of
cellular growth is unclear and seems to be cell specific. Here we have
investigated the eventual role of calcium/calmodulin-dependent protein
kinase and epidermal growth factor (EGF) receptor kinase in regard to the
proliferative activity of LPA in human myometrial SMCs. The growthpromoting effect of LPA was quantified by measuring [3H]thymidine
incorporation into DNA for 24 hours. First we used KN-62, which is an
inhibitor of calcium/calmodulin-dependent protein kinase I1, together with
LPA. KN-62 decreased the LPA-stimulated [3H]thymidine incorporation
dose-dependently. Secondly we used Tyrphostin AG 1478 which is an
inhibitor of the EGF receptor tyrosine kinase. It has been suggested that
the LPA receptor indirectly activates the EGF receptor and that this ligandindependent activation can be stimulated through a rapid transient calcium
signal. Tyrphostin AG 1478 inhibited LPA-stimulated DNA synthesis in
the SMCs. Whereas the negative control, Tyrphostin A1, was without
effect. The EGF receptor has been suggested to be important for the
proliferative response of LPA. Our results confirm this but also suggest
that calcium/calmodulin-dependent protein kinase II may play a crucial role
on the growth stimulatory effect of LPA in SMCs.

[1] Vanhaesebroeck. B. et aL, Trends Btochem. Sci. 2, (1997), 26Z

[2] Toker, A., and Cantlev L.C., Nature, 387, (1997), 673.
[3] Nobe6; C. andHalLA., Cell, 81, (1995), 53.

(Mo/11.2/045)

Macrophages Inhibit
Lymphocyte Proliferation byTransfering Phosphatidylcholine
Nishiyama-Naruke, A.; Curl, R. University ofSao Paulo, Physwlogy
and Btophysics Department, S~o Paulo,BrazlL

Previous studies have shown that cholesterol and fatty acids can be
transfered from macrophages to lymphocytes. In this work, we examined
whether phosphatidylcholine (PC) could be transfered between these cells
and also if this process could alter eoncanavalin A-stimulated lymphocyte
proliferation. To carry out the incorporation and tranference experiments,
we
used
PC- 1-stearoyl-2-[14C]-arachidonoy1 (14C-AA-PC) and
phosphatidyl.[n.methylJ4C]-eholine- 1,2 dipalmitoyl (t4C-choline-PC).
Macrophages incorporated t4C-AA-PC and exported the radioactivity to the
medium as fatty acids and PC. On the other hand, v~C-choline-PC
incorporated-macrophages exported the radioactivity basically as PC.
Compared to lymphocytes, macrophages exhibited a much higher rate of
~4C-AA-PC uptake, being mainly incorporated into phospholipid classes.
When co-cultured, V~C-AA-PC and ~4C.choline-PC incorporated by
macrophages were transfered to lymphocytes indicating that
phosphatidylcholine molecules were, in fact, transfered to lymphocytes.
Non-radiolabeled compounds: arachidonic acid-rich PC (AA-PC) and
arachidonic acid-poor PC (FA-PC) were used to study the transference
effects on lymphocyte proliferation. AA-PC and FA-PC added directly to
the medium of eoncanavalin A-stimulated lymphocytes or AA-PC- and FAPC-loaded macrophages co-cultured with lymphocytes were both able to
inhibit [~4C]-thymidine incorporation into DNA. In both cases, the inhibitory
effect of AA-PC was significantly more pronounced than that of FA-PC. In
conclusion: 1) PC is incorporated into macrophages and lymphoeytes, 2)
PC is transfered from macrophages to lymphocytes, 3) PC alone or PCloaded macrophages inhibit lymphocyte proliferation, 4) the PC inhibitory
effect on lymphocyte proliferation is more pronounced when AA-PC is
used.
Financial Support: FAPESP (95/9015-0; 93/3498-4), CNPq and PRONEX.

(Mo/11.2/046) INTERACTION
BETWEEN
GM1-GANGLIOSIDE AND
MEMBRANE-ASSOCIATED TUBULIN.
PALESTINI P.,
PITTO M., TEDESCHI G., FERRARETTO A., * BRUNNER
J.and MASSERINI M. Dept. of Med. Chem. and Biochemistry,
Milan, Italy and * E.T.H., Zurich, Switzerland
Gangliosides interact with other membrane components, including proteins,
affecting their functions and suggesting their involvement in events
occuring at the call surface. The aim of this work is to study the interaction
of gangliosides with specific proteins in neurons. The experimental model
system was constituted by rat carebellar granule calls in culture and by a
photoreactive, radiolabeled
GMl-ganglioside
derivative
TID-GM1,
s~nthesized in our laboratories. TID-GM1 is a 3-(trifiuoromethyl)-3-(m[ 5I]iodophenyl)-diazirin GM1, carrying both the photoreactive and
radioactive group in the caramide portion. This probe, after insertion into
the call membrane and illumination, covalently crosslinks neighbouring
proteins, that become labeled. TID-GM1 was dissolved in the medium for
call culture ( 10"e M final concentration). After 2 h at 37C. a number of
radiolabeled proteins was detected by 2D-EF and autoradiography. When
the experiment was repeated at 40C, a temperature at which no
endocytosis is reported to occur, only a few proteins were still radiolabeled,
suggesting their interaction with TID-GM1 at the plasma membrane level, in
particular, a major radioactive spot (MW of about 55 kDa and apparent pl
of 5 ). Immunoprecipitation experiments carried out with anti-s, anti-~ and
anti-acatyl-tubulin, showed that this is the main protein crosslinked by TIDGM1, suggesting the existence of a ganglioside-tubulin interaction within
the hydrophobic core of the plasmamembrane. The preparation of low
density detergent insoluble membrane fractions showed that tubulin
interacting with TID-GM1 was enriched in this fraction 10 fold with respect
to homogenate. The ganglioside-tubulin crosslinking product is susceptible
to KOH, but not to hydroxylamine treatment, suggesting that the interaction
is likely occurring between the caramide portion of the former and an
esterically-linked fatty acid anchor of the protein. The information that
membrane-associated tubulin and G proteins can form complexes makes
membrane-associated tubulin a candidate for endogenous modulator of G
protein-mediated signal transduction. Therefore, our results suggest that
also GM1 may participate to this process

s 178

(Mo/11.2/047)

Abstracts FEB S' 99

Evidence that macrophages transfer cholesterol and

arachidonic acid to several tissues in vivo.
C. M. Peres, P. I. Homem de Bittencourt Jr., R. Curt
Department of Ph~'iologv and B,>php~ic,~ - ICB-I, ( nlver.~l(v of S~o Paulo,
1524, Av Pro/? Lmeu Preste.~; 05508-900 SOo Paulo, Braztl.

Our previous results have shown that [14C]-Iabelled cholesterol

(CHOL) [1] and arachidonic acid (AA) [2] were transferred from
macrophages (M~) to lymphocytes when these cells were co-cultured To
investigate if this process can occur m vivo, resident and inflammatory
(thioglyeollate-stimulated) rat peritoneal Mq were cultured in the presence of
[3H]-CHOL or [14C]-AA After 18h, ~lxl0 ~ labelled cells (with ~llaCi) were
injected in a canule connected to the jugular vein of untreated animals. Rats
were killed after 24h Tissues were collected, lipids were extracted with ether
and the radioactivity measured in a beta-counter Plasma radioactivity was
low indicating that few Md~ lost their integrity [t4C]-AA was incorporated in
great amounts in the adrenal, liver, spleen, and aorta, and [3H]-CHOL in the
adrenal, spleen, aorta, lung and liver The radioactivity incorporation was low
in the brain, possibly due to the encephalic- barrier, which does not allow Md~
to reach the tissue. The thioglyeollate stimulation of MO labelled with [~4C]AA induced a reduction in the incorporation of radioactivity in the brain,
mesenteric lymph nodes, heart and epididimal tissue compared with nonsimulated M~ In [3H]-CHOL labelled M~, thioglycollate induced a reduction
in the aorta radioactivity. These results suggest that the intercellular transfer
of CHOL and AA from Mdp can occur in vivo and that this process depends
on the tissue and the activation state of the donor cells. The mechanism and
meaning of this process is now been studied in our laboratory. [1] P. 1.
Homem de Bitteneourt Jr. et al. Biochem. Mol. Biol. Int. 44(1998)'347, and
[2] C M. Peres et al. Biochem. 3/lol. Biol. Int. 43(1997):1137
Financial support FAPESP, FAPERGS, CNPq, PRONEX (168/97)

(Mo/11.21049)

Sphingomyelinase treatment of hepatoeytes inhibits

glycogen synthesis by decreasing cell volume
D.A. van Sluijters, G.M. van Woerkom, A.J. Meijer
Department of Bzochemlstr),, Academic Medtcal Centre, Amsterdam,
The Netherlands

Insulin reststance plays a critical role in obesity and non-insulin dependent

diabetes mellitus. Recently, TNF-ot has been implicated in insulin
resistance. Many of the effects of TNF-a can be mimicked by
sphingomyelinase (SMase) treatment of cells. In adipocytes, ceramide
produced by TNF-c~ stimulated SMase activity interferes with insulin
signaling [ 1]. SMase also inhibits insulin-stimulated glucose transport and
glycogen synthesis in skeletal muscle cells [2].
We showed previously that hepatic glycogen synthesis from glucose is
stimulated by amino acid-induced cell swelling [3]. This stimulation does
not reqmre the presence of insulin. In order to establish whether SMase also
interferes with glycogen synthesis in the absence of insulin-dependent
signal transduction, we investigated the effect of SMase (50 mU/ml)
treatment on amino acid-stimulated glycogen synthesis from glucose in
fasted rat hepatocytes, m the presence or in the absence of amino acads at
physiological concentrations.
SMase decreased glycogen synthesis by about 40% either under basal
conditions or in presence of amino acids. Gluconeogenesis from either
lactate or dihydroxyacetone was not affected. Inhibition by SMase of
glycogen synthesis was accompanied by cell shrinkage. There was a unique
linear relationship between cell volume and either the rate of glycogen
production or the activity of glycogen synthase a. Cell-permeable short
chain ceramides had no effect on glycogen metabolism, indicating that the
inhibition of glycogen synthesis by SMase may not be medmted by signal
transduction events. Indeed, amino acid-dependent signal transduction, as
measured by p70S6 kinase phosphorylation, was not affected by SMase.
It is concluded that in hepatocytes SMase inhibits glycogen production
independent of signal transduction. Rather, glycogen synthesis in
hepatocytes is controlled by changes in cell volume.
1. Hotamisligil GS et al., Science, 271, (1996) 665
2. Begum N et al., Endocrinology,137, (1996) 2441

(Mo/11.2/050)

Differential Localisation of PITP~ and PITP~

in RBL-2H3 mast cdls
C Wiedemann, A Ball, G Way and S Cockcroft
( )~lver~ity College London. Lomlon WCIE ~lJ. UK

Phosphatidylinositol transfer protein (PITP) was originally identified as a

protein capable of transferring phosphatidylinositol
(PI) and
phosphatidylcholine (PC) During the last six years, PITP was recognized
as a key player in numerous signalling events relying on PI metabolites,
such as pbospholipase C signaling, PI 3-kinase activity, biogenesis of
secretory vesicles from the trans Golgi Network (TGN), and regulated
exocytosis In our studies, we investigated the amount, membrane
association and localisation of the two mammalian isoforms PITPa and
PITP[3 in RBL-2H3 mast cells The relative amount of PITPs in RBL-2H3
cells was investigated by Western blot analysis It was found that PITP[3
expression is about 5-fold higher than PITP(* expression. In quiescent
cells, PITPt, is found exclusively in the cytosol, while PITP[~ is partly
membrane associated Consistant with these data, cells, which are
permeabilised with streptolysin O are completely depleted for P1TP<~ after
5 minutes, while a substantial amount of PITPb is still associated with the
cells even after extensive permeabilisatlon Stimulation of RBL-2H3 cells
leads to membrane association ofPITPa, while the membrane associated
amount of PITP[3 decreases and more PITP[5 is found in the soluble
fraction. This antiparallel behaviour of the two mammalian PITP isoforms
detected by Western blot analysis is further supported by
immunolocalisation experiments. In quiescent cells, PITPa is seen
throughout the cell, not localized at any particular subcellutar structure.
Upon stimulation, PITP(x is detected in the periphery" of the cell Current
investigations aim to identify, the cellular structures PITPc~ is associated
with upon stimulation In contrast, PITP(3 is tocalised in the periphery of
the cell and colocalises with TGN38 at the Golgi in resting cells Upon
stimulation, it appears that PITP[') is removed from the periphery while
localisation at the TGN is retained We are currently investigating what
determines the antiparallel movement of PITPot and PITP[3 following
stimulation

Abstracts F E B S ' 99

s 179

Mo/12.1 Mitochondrial biogenesis function and pathologies

(Mo/12.1/051)

Two ATP synthase 13-subunit mRNA binding proteins interact

with 3'-untranslated region in an interdependent manner
H. Antonickfia, U. Anderssonb, B. Cannon b, J. Hougt~k a
"Dep. of Bioenergetics, Institute of Physiology ASCR, CZ-142 20 Prague
~Dep. of Physiology, The Wenner-Gren Institute, S-106 91 Stockholm

There are several reports suggesting that the catalytic [3-subunit of

mitochondrial ATP synthase ([3-ATPase) is post-transcriptionaly regulated
[1,2].
We present an evidence for the existence of two mammalian cytosolic
proteins that selectively interact with the T-untranslated region of the
mRNA coding for [3-ATPase. One of the proteins BARBI (Beta-ATPase
mRNA Binding protein), is a novel poly(A) binding protein that specifically
binds the poly(A) tail of the !3-AYPase transcript. BARB1 achieves its 13ATPase poly(A) selectivity through its interadtion with a second protein BARB2, that specifically binds to a 22 bp element with a uridylic core
located 75 bp upstream of the poly(A) tail. In the absence of BARB1,
however, BARB2 is not able to bind the [3-ATPase mRNA with high
affinity. Thus the interaction between BARBI and BARB2 and [3-ATPase
mRNA involves the formation of a complex between the two BARB
proteins.
Considering that the poly(A) tail in general plays a role in regulation of
mRNA half-life and translatability, it is likely that these proteins are
involved in tissue-specific post-transcriptional mechanisms regulating [3ATPase gene expression.

[1] Tvrdik, P. et al., FEBS Lett. 313, (1992) 23

[2] Izquierdo, J.M. et al., Mol. Cell Biol. 17, (1997) 5255

(Mo/12.1/053)

Inhibition of pyrimidine biosynthesis

by nitric oxide at the mitochondrial step
C Beuneu, B. Roy, G. Lemaire, M. Lepoivre
CNRS UAIR 8619, Univeestt# Parts.Sud, 91405 Orsav. France

Dihydoorotate dehydrogenase (DHODH) catalyzes the oxidation of

dihydroorotate to orotate, Le. the fourth step of de n o v o pyrimidine
biosynthesis. In eukaryotic cells, the enzyme is located in the inner
mitochondrial membrane and is a component of the respiratory chain.
Electrons resulting from dihydroorotate reduction are accepted by
ubiquinone and finally by cytochrome c oxidase It is now well known
that nitric oxide (NO) inhibits mitochondrial respiration. In particular
cytocbrome c oxidase (complex IV) is reversibly inhibited by NO.
Irreversible inhibition of complexes I and II by NO or peroxynitrite has
also been demonstrated. We tested whether DHODH could be inhibited
indirectly by NO via inhibition of complex IV. L1210 cells were
permeabilized with digitonin and incubated at 37C, with dihydroorotate
as a substrate. Consequent respiration was inhibited by antimycin A,
sodium cyanide or by A77 1726, a specific inhibitor of DHODH. These
inhibitors also completely inhibited orotate production. DEA-NO, a
chemical donor (tv2 = 2 lmin) induced a dramatic decrease in 02
consumption. This inhibition was more pronounced at low 02
concentration. It was fully reversible in a few minutes, corresponding to
NO disappearance. It could also be reversed by adding hemoglobin which
binds NO. The strongest inhibitions were correlated to a decrease in
orotate production. However the decrease observed was often lower than
expected from respiration inhibition. A decomposed NO donor could
inhibit neither respiration nor orotate production. We also observed
decreased pools ofpyrimidine nucleotides in LI210 cells incubated with
NO donors under low 02 conditions approaching tissue levels. These
results suggest that cytochrome c oxidase inhibition by NO decreases
indirectly DHODH activity. Since physiological levels of NO have been
proposed to efficiently inhibit cytochrome c oxidase activity, inhibition of
pyrimidine biosynthesis in tissues is expected.

(Mo/12.1/052)

Mitochondrlal structure and respiratory chain

of the fungus Armillarilla mellea (A.M.)
B. Ch. Behboudia, L. Feiza, E. Keyhanib
a Dept of Biology, Faculty of Siences and b Inst. Btochem
Biophys . Universiry o! Tehran. Tehran. lean

A. M. iS a world-wide pathogen of broad leaved trees and conifers.

Light and electron microscopic studies revealed that the rhizomorphe
of this fungus was composed of four distinct layers of cells, covered
by a thick layer of mucilagenous materials. The mitochondria of the
four layers showed the same typical structure as that found for the
mitochondria of other plant and animal cells: round, oval (diameter
0.7 ~m) and elongated up to 3 ~m. Moerover they were in both
orthodox and condensed configurations A method was worked out
to isolate mitochondria from the rhizomorphe. For rhizomorphe
grown in malt-agar-peptone medium with glucose as substrate, room
temperature reduced-minus-oxidized difference spectra of isolated
mitochondria showed absorption bands due to cytochrome (cyt.) aa3
at 605-630 nm and 444-450 nm, and to cyt. b and c at 560-575 and
550-540 nm respectively. The four complexes of the respiratory
chain were characterized. NADH was oxidized by isolated mitochondria. With succinate as substrate in the presence of rotenone,
cyt. aa3, b and c become reduced. Antimycin A inhibited succinate
oxidation while cyt. a a 3 and c were reduced by TMPD (tetramethylparaphenylenediamine) and ascorbate. For rhizomorphe grown in
liquid medium (peptone, vitamins, minerals) and with glucose as
substrate, the complex IV (cyt. c oxidase) was not detected even
though cyt. b c l were found. Moreover, pyridine hemochromogen
difference spectra showed also the lack of cyt. a a 3 m the cells grown
in glucose liquid medium. However rhizomorphe grown in liquid
medium with ethanol as substrate showed cyt. c oxidase as well as
cyt. b c l . Data showed that A M. contained mitochondria similar in
structure to the mitochondria of other cells and that in semi-solid agar
medium as well as liquid medium with ethanol as substrate, all four
complexes of the respiratory chain were present. However in liquid
medium with glucose as substrate, even though other respiratory
chain complexes were present, the complex IV was not detected,
suggesting an alternattve pathway of electron transport in these
mitochondria.
(Me/12.1/054)
Identification of IKB-o~ molecular partner
using two-hybrid system.
V. Bottero*. F. Rossi and J. -F. Peyron.
INSERM C.IF 96-03 Av de l alombro.*e F- 06107 NICE L'edex 2

The transcription factor NF-~cB is a conserved coordination element in the

organism response to situations of infection, stress, and injury. NF-~B dimers
are sequestered in the cytosol of unstimulated cells via non-covalent
interactions with a member of a class of inhibitory proteins called IB. I~B
molecules mask NF-~B nuclear localization site. preventing translocation of
the transcription factor into the nucleus. NF-,cB activation is a consequence
of bcB-c~ modification and two activating pathways have been proposed.
We exploited a two-hybrid system based approach to isolate cellular proteins
which interact with I~B-cc Using its N-terminal domain as the bait. we fished
out. as putative interactors, the C-terminal domains of two kno~,,n proteins:
- Cyclin GI: a member ofcyclin family implicated in G2/M arrest.
- ANT 1: a mitochondria protein, which exchanges cytosolic ADP against
ATP, produced by oxidative phosphorylation.
These interactions were confirmed by independent in vitro tests.
The entire open reading frames of Cyclin G1 and ANT ,,',ere sub-cloned. In
vitro experiments and two-hybrid system analysis confirmed the interaction
of the full-length proteins with boB-co
A model is proposed for the possible role of ANT and IKB-c~in the apoptosis
regulation: I~B-~, retained in the mitochondria by its interaction with ANT,
could be released alter apoptotic signals and, in this way. could block NF-~cB
dependent expression of survival genes. This model has to be confirmed by
the demonstration of the presence of bcB-e~in the mitochondria and its
relocalisation during apoptosis.
The possible role of Cyclin GI in G2/M arrest in Jurkat cells and its
relationship with I*:B-~J NF-~B alter DNA damage is under investigation.

s 180

(Mo/12.1/055)

Abstracts

Cooperation of F1-ATPase and ANT to maintain growth

and mitochondrial membrane potential of human p cells
K.Buchet and C. Godinot

(Mo/12.1/056)

Centre de Genettque Moldculatre et Cellulaire (UMR 5534 - CNRS),

Universit~ Lyon 1, 69622 Vgleurbanne eedex, f'rance

Two lines of human mitochondrial DNA-depleted rho cells were

studied in order to determine how they maintained a mitochondrial membrane
potential in the absence of a functional respiratory, chain. Indeed, the
mitocbondrial membrane potential of at least one of these rho ceil lines was
similar to that of the control rho cells, as measured by Rhodamine 123
fluorescence. Since, in these cells, oxidative phosphorylation cannot provide
A T P . their growth relies on glycolysis. F 1-AYPase assembly has been studied
in our cells. In spite of the absence of the mtDNA-coded F0 subunits 6 and 8,
rho cells possessed normal levels of F1-ATPase o: and 1~subunits. Moreover.
in rho cells, the F1-ATPase yeas functional and azide- or aurovertin-sensitive
but oligomycin-insensitive. However. this F1-ATPase was unstable at 4C,
showing that it was not normally protected from cold inactivation by
membrane-bound F0 subunits. In addition aurovertin decreased cell growth in
rho cells and also reduced their mitocbondrial membrane potential. Therefore.
a functional F1-ATPase was important to maintain the mitochondrial
membrane potential and the growth of these rho cells. Bongkrekic acid, a
specific adenine nucleotide translocator (ANT) inhibitor, also reduced rho cell
growth and mitochondrial membrane potential. In conclusion, rho ceils need
both a functional F1-ATPase and a functional ANT to maintain their
mitochondrialmembrane potentiak which is necessary for their growth. ATP
hydrolysis catalyzed by F1 must provide ADP 3" at a sufficient rate to
maintain a rapid exchange with the glycolytic ATP 4- by ANT, this
electrogenicexchangeinducinga mitochondrial membrane potential efficient
enough to sustain cell growth. However, since the effects of bongkrekic acid
and of aurovertin were additive, other electrogenic pumps should cooperate
with this pathway. In addition, the production of reactive oxygen species
(ROS), as measured by DCF fluorescence, was increased by azide in rho
cells. While this increase in rho" cells must be related to the azide-induced
inhibition ofcytochrome oxidase (COX). in COX-null rho cells, this increase
must be due to an inhibition of F1-ATPase. Therefore. F1-ATPase controls
both mitochondrial membrane potential and ROS production in rho cells.
(Mo/12,1/057)

Precursor processing is not linked to translocation in

plant mitochondriai protein import.
Patrick Dessi, Chartotta Rudhe and Etzbieta Gtaser.

Analysis of the targeting signal(s) of the outer

mitoehondrial membrane rat liver CPTI
I. Cohen, J. Baer, J. Girard and C. Prip-Buus
CNRS- UPR 1524, 9 rue ,Z Helzel, 92190 Meudon, France

Rat liver carnitine palmitoyltransferase 1 (CPT1) is an integral protein of the

outer mitochondrial membrane (OMM) which, in association with CPT2 and
carnitine-aeylcarnitine translocase, permits the entry of long-chain fatty acids
into mitochondria for ~-oxidation. CPT1 plays a key role in the regulation of
fatty acid oxidation, with malonyl-CoA acting as a physiological inhibitor. We
have recently demonstrated that the N-terminal domain of CPTI (1-147 amino
acids (aa)) contains all the intrinsic information to mediate its mitochondrial
targeting and OMM insertion and is essential to maimain an optimal
conformation for both catalytic function and malonyl-CoA sensitivity [1 ].
The N-terminal domain of CPT1 contains two hydrophoblc
transmembrane (TM) domains (47-75 aa: 103-122 aa) and three charged regions
(1-46 aa; 76-102 aa; 123-147 an). By contrast to OMM proteins harboring a
single TM domain, the mechanisms of OMM insertion of polytopic proteins are
still unclear. The aim of the present study was to determine the nature of the
mitochondrial targeting signal(s) of CPTI by using an in vitro import assay.
Deletion of 1-82 aa of CPT1 resulted in a protein that was still imported into the
OMM of freshly isolated rat liver mitochondria in a temperature- and
mitochondrial receptors-dependent manner. By contrast, removal of 83-147 aa
abrogated protein import, whereas fusion of these aa to a non-mitochondrialrelated protein, the cytosolic mouse dihydrofolate reductase (DHFR), mediated
specifically its mitochondrial targeting and OMM insertion. Thus, the targeting
signal(s) of CPTI is located within 83-147 aa (encompassing the second TM
domain of CPTI).
We then checked whether this TM domain could be a "signal-anchor
sequence" as described for the yeast Tom70p. 97-122-DHFR was not
specifically imported into the OMM, ruling out this hypothesis. Then, we tested
whether the two charged regions flanking the second TM domain of CPT1 (76102 an, t23-147 aa) could behave as a matrix-targeting signal. We showed that,
in the presence of mitochondria, 123-147-DHFR, and not 76-102-DHFR,
became protease-resistant in a temperature- and mitochondrial receptorsdependent manner. This study demonstrates that 123-147 aa function as a
matrix-targeting signal specifying the mitochondrial targeting of CPT 1, whereas
its second hydrophobic TM domain acts as a stop-transfer sequence that stops
and anchors the translocating protein into the OMM. In conclusion, OMM
import of CPT1 follows a "stop-transfer" model and not a "signal-anchor
sequence" model.
[ 1] References: Cohen et al., J. Biol. Chem., 273, (1998), 29896.

(Mo/12.1/058)

UV-A radiation reduces oxygen consumption and

ATP intracellular content in human keratinoeytes.
M.D.jacaheri-Mergly", CA4~sac b, C . M ~ r e ~, L . D u b ~ m d J Mazihe ~.
"Laboratolre Lie Dermatologle, lNSERM U312, Hg~pttalSt-Louis, Paris,
France. J'CHG'/~]~et; Part3. France. ~CHU d :4nliens.4mtens, France.

Department of Biochemtstry, Arrhemus Laboratories for Naturc~l Scwnces,

Stockholm Untverstty, 10691 Stockholm, Sweden

Upon import to the mitochondrial matrix, the N-terminal presequence is

cleaved from precursor proteins by the Matrix Processing Peptidase (MPP),
producing the mature protein that can then adopt its correct location and
conformation within the organelle. The MPP catalyses a remarkable function: it
is able to specifically cleave the presequence from several hundred precursor
proteins that show no consensus sequence, probably recognizing higher order
structure in the precursor protein. In yeast and mammals, the MPP heterodimer
is a matrix soluble protein and has been shown in yeast to be able to cleave the
presequence as soon as this enters the matrix. Surprisingly, in higher plants the
MPP is completely integrated within the inner membrane cytochrome bet
complex of the mitochondrial electron transport chain, thus the plant complex
is bifunctional. The location of the processing machinery in this location in
plant mitochondria raises the question as to whether processing occurs during
translocation of precursors to the matrix, or whether the protein must be
processed at a remote site following complete translocation. To investigate this
matter, we have used chimeric precursor proteins consisting of the yeast
cytochrome b, presequence with varying lengths of mature protein attached,
fused to a passenger protein, mouse dihydrofolate reductase (DHFR) which can
be tightly folded to prevent complete translocation of the precursor protein.
Thus. we can determine the length of polypeptide that must feed into the
matrix before processing by the MPP can occur. In yeast, previous studies have
shown that only about 50 amino acid residues attached in front of the folded
DHFR were required for the MPP cleavage site to reach the matrix and be
cleaved. In this study we have found that even much longer presequence/mature
segments (167 residues in length) fused to the folded DHFR domain are not
processed in plant mitochondria. This data suggests that the plant
MPP/cytochrome bc~ complex is not associated with the translocation complex
and that translocation and processing are independent events in plant
mitochondria.

FEBS'99

UV-A radiation (320-400nm) has been shown to be involved in

degenerative

processes

of

the

skin,

such

as

photoaging

and

photocarcinogenesis [1]. In this study, we investigated the effect of this

radiation on the mitochondrial functions. UV-A irradiation mediated a
dose-dependent decrease in oxygen consumption and ATP content in
NCTC 2544 human keratinocytes, one hour after light treatment.. This
effect was partially reversed when the irradiated cells were maintained for
24h m normal culture conditions. In parallel, by using malate or succinate
as substrates of mitochondrial electron transport, oxygen uptake of
digitonin-permeabilized UV-A irradiated cells was greatly inhibited.
Neverthless, the activities of different complexes remain unchanged upon
UV-A radiation. Under identical conditions, UV-A exposure did not
impair the mitochondrial transmembrane potential. Finally, the vitamin E
antioxidant inhibited UV-A induced lipid peroxydation, but did not
significantly prevent the UVA-mediated alterations in cellular respiration
nor the decrease in ATP content. These finding suggest that mitochondrias
may be the primary targets of UV-A radiation.
[I I Tyrell+ R.M. (1991) UV-A radiation as an oxidative stress. In Oxidative Stre.~s.
Oxidant and Antioxidants ( Edited by H. Sies), pp 57+83. Academic Press, London.

Abstracts FEB S'99

(Mo/12.1/059)

s 181

Imported tRNATM variants can function in yeast

mitochondrial protein synthesis
N. Entelis, O. Kolesnikova +, R. Martin & I. Tarassov

(Mo/12.1/060)

UPR 9005 CNRS, 21, rue Ren~ Descartes 67084 Strasbourg, France
*Molecular Biology Dept, Moscow State University, Russia

Saccharomyces cerevisiae mitochondria contain a single nuclearencoded tRNA, tRNALys(CUU), imported from the cytosol. Its organellar
function remains still unclear [1,2]. We have constructed a set of mutant
tRNA TM transcripts and studied their aminoacylation, import into isolated
yeast mitochondria and ability to hind to the mitochondrial lysyl-tRNA
syntethase precursor (pre-MSK), which is thought to act as a carrier for
translocation of the tRNA across mitochondrial membranes [2].
Most of the changes in the anticodon of tRNALys(cuu) led to
decreased lysine aminoacylation and increased misacylation. In vitro
import assays showed that misaeylated tRNA variants can be imported. A
tRNA Lysvariant with an anticodon CAU(Met) is efficiently aminoacylated
by yeast MetRS and is imported in the misacylated form. Incorporation of
3ss-Met into newly-synthesized mitochondrial polypeptides was detected
after in vitro import of 35S-Met-tRNALyS(CAU). That means that the in
vitro imported tRNA can participate in mitoehondrial translation.
To validate the above conclusion by in vivo data, we used another
mutant tRNA TM version which contains the amber CUA anticodon and a
strong AIaRS aminoacylation determinant, G3:U70. In fact, this tRNA is
imported and is able to suppress an Ala->amber nonsense mutation in the
mitochondrial gene coding for the 2nd subunit of the cytochrome c oxidase
(COX2).
Supported by INTAS (grant N96-1515)

Cloning of the gene for a nucleoside diphosphtte kinase -

novel mitochondrial phnsphoprotein
M L Eseobar Galvis, C Knorpp, K Alexeiev, and G I-I~ansson
Plant Cell Biology, Land University, Box 7007, S-220 07 Land, Sweden.

Partial purification and N-terminal sequencing of an autophosphorylated 17

kDa protein (detected in the intermembrane space of isolated pea
mitocbondria), revealed homology with nucleoside diphosphate kinases
(NDKs) from other organisms [1]. The 17 kD protein showed the highest
identity with NDK isolated from Avena and the haman tumor supressor
NM23. Two phosphorylation sites were detected in the mitochondrial NDK.
In humans phosphorylationof a secondary site plays a role in signal
transduction pathways [2].
Based on protein sequence data from the N-terminal region and a
characteristic conserved sequence in the internal region of NDKs from
several organisms, a probe was generated from pea leaves by RT-PCR. This
PeR product was used to screen a pea leaf cDNA fibrary and a positive
clone of 1.2 kb was isolated and sequenced. The deduced aminoacid
sequence is identical to the isolated protein [1] and showed high similarity
with another NDK isolated from A. thaliana (AF044265). In order to
confirm the intracellular location of our clone, in vitro import experiments
into mitochondria and chloroplasts will be carried out.
The results from the differential and developmental expression of this
mitochondrial NDK will be discussed. We are also investigating stress
regulated transcription of this NDK, as it has been demostrated for other
NDKs in other plant species [3].
[1] Stroglics and HLkansson (1999), Submitted to Ear J Biochem.
[2] MacDonald et al., J Biol Chem 268 (1993), 25780
[3] Harris et al., Plant Mol Biol 25 (1994), 739

[1] Martin R.P. et al., Biochemistry, 18, 1979, 4600; [2] Tarassov I.A. et
al., E M B O J . , 14, 1995, 3461.
(Mo/12.1/061)

A new mutation of E1 ~ gene associated with extreme

PDH deficiency.
F. Fouque',A. Kobetzb, P. de Lonlay',J.M. Saudubray',
C. Marsacband C. BenellP.
INSERMU31T, CERTO~, clinique gdndtique*, H6pital Necker, Paris

Pyruvate dehydrogenase (PDHc) deficiency is a nuclear-encoded

mitochondrial disorder and a major recognized cause of neonatal
encephalomyopathies associated with primary lactic acidosis. We report
biochemical and molecular analysis of PDHc deficiency in a patient which
had developed by 4 months of age a severe metabolic acidosis. Plasma
lactate and pyruvate were elevated (5 and 0.6 mmol/l respectively) while
lactate/pyruvate ratio was low (<10). He was referred for hypotonia, severe
visual impairment, areflexia and psychomotor retardation; brain MRI
showed abnormal corpus callosum, ventriculomegaly and cortical atrophy.
He was treated with a low-carbohydrate diet and thiamine.
PDHc activity in isolated lymphocytes and cultured fibroblast cells of
the patient were decreased by 99 and 90 % respectively. Lymphocytes of the
asymptomatic mother and father had normal PDHc activities. Immunoblot
analysis of fibroblasts showed that the level of Elc~ and EII~ PDHc subunits
were not detected in the patient, whereas the levels of both subunits were
normal in the parents. Direct sequencing of exons 10-11 revealed a C----T
point mutation at nucleotide 1243, resulting in a substitution of arginine for
cysteine in position 378 (R378C).
To date, this is a new mutation; another substitution (R378H) affecting
the same aminoacid has already been described in 6 unrelated patients which
presented a similar clinical and biochemical pattern (1,2) with differently
affected levels of E10~ and E1 ~ subunits. In this case,the presence of a new
cysteine residue, near the carboxyl terminus of the E let subunit, could allow
a new possibility of disulfide linkage that may reduce the accessibility to the
other subunit EI~ and actually destabilize the Elct protein.
References
1. Chun K et al. Am.J.Hum.Genet. 56, 1995, 558.
2. Wexler ID et al. Neurology. 49, 1997, 1655.

(Mo/12.1/062)

Functional conservation of proteins necessary for the

biogenesis of mitochondrial respiratory complexes
O.Groudinsky, N.Bonnefoy, B.Cardazzo, P.Hamel,
M.Kermorgant, C.Lemaire, G.Dujardin
Centre de GFn(tique Mol(culatre. CNRS, 91198 Gif sur Yvette, France

The biogenesis of the mitochondnal respiratory complexes depends on the

expression of both mitochondrial and nuclear genes. In addition to genes
encoding most of the subunits of the complexes, the nuclear genome also
encodes various proteins that do not belong to the complexes themselves but
play essential roles in their biogenesis. Namlp, Abclp and Oxalp belong to this
class, and their genes have been cloned in our laboratory by complementation ol
S. cerevisiae respiratory deficient mutants. Namlp is required for the
stability/processing of some mitochondrial transcripts. Abe lp is essential for the
conformation and activity of complex lII (bcl). Oxalp is an inner membrane
protein which plays a crucial role in the insertion and assembly of complexes IV
(cytochrome c oxidase) and V (ATP synthase). Functional complementation ol
natal, a b c l or o x a l mutants with heterologous genomic or cDNA libraries
allowed the identification of several functional homologs of these genes. A
functional homolog of Namlp was only found in the closely related yeast S.
douglasii. Functional homologs of Abclp were identified in S. douglasii, in the
highly divergent yeast S. pombe and in the plant A. thaliana. Oxalp is
functionally conserved in all eukal3~otes tested: S. douglasii, S. pombe, A.
tbaliana and H. sapiens. Furthermore sequence comparisons with databases
indicate that Abclp and Oxalp are also conserved in prokaryotes while Namlp
is not. The conservation of various mitochondriaI proteins through evolution will
be analysed in relation to their function within mitochondria.
N.Bonnefoy et ah, Proc. Natl. Acad. Sci. USA,91 (1994), 11978
P.Hamel et al., Plant J., 12 (1997), 1319
B.Cardazzo et al., Gene, 221 (1998). 117

s 182

(Mo/12.1/063)

Abstracts F E B S ' 9 9

Interpretation of recombination events in fungal mtDNAs

Zs Haman . B. Toth . A Juhasz. I. Ferenczy . F Ke\e~
a
bMICrob~ologtcal
,Research Group ~f MTA-JATE, Sze~ed, Hungar~.
Department of [~I~croblology Antla ~ozsef Un~verslt~'. Szeged, Hungar~

The mitochondrial genomes of the strains of A japomcus belonging to the

imperfect black Aspergdh display highly variable RFLP patterns. The 80
collection strains and field isolates investigated could be classified into eight
different mtDNA RFLP groups (one of the eight types represented by some A.
aculeatus strains which are closely related with A j a p o n i c u s ) . Applying a
mitochondrial oligomycin resistant mutant (oliR) strain transmissions of
mitochondria were carried out between fully incompatible oliR strain as
standard donor and sensitive recipient strains w~th different mtDNA RFLP
patterns by using protoplast fusion technique. These transfer experiments
resulted in ollR progeny with recombinant and/or unchanged donor mtDNA in
each case of intraspecific combinations, but it failed when A. aculeatus was
the recipient partner. For interpretation of recombination processes physical
and functional maps of mtDNAs of a recombinant and its parental strains were
constructed The mtDNA of recombinant s~rain consists of basically the do~or
mtDNA with additional recipient sequences. On the basis of sequence analysis
the sequences derived fi'om recipient mtDNA proved to be introns. It is
suggested, that the recombination events are due to the mobility of certain
group I introns Both intron acquisition and loss play role in development of
the recombinant character of examined strain.
This work was financially supported by OTKA grant T025849.

(Mo112.1/065)

UVA Induced MitochondHal Injury in Live Human

Dermal Flbroblasts - Assessment with Microplate Cytometry
C. Korwm-Zmijo~skaa, P Rat a'b. L Pascual le Tallcca, .M. Adolphca
a Laboratoh'e Pharmacofi~gte Cellolatre-EPHE-750(~6 Parle, b Untt~de PhaTvna'o
l'oxicologie Cellulaire (Pr J-NI Warrlet) .CHNO 15/20 - 75012 Parig Franl'e

Ultravlolct radmtton, particularly UVA, reduces to,~ic effccts and the skin is
~ts first target. It ~s well known that UVA causes considerable damage to skin
cells - The aim of this study was to evaluate the effects of UVA on
cellular prohfcrat,on assay and on the mltochondnal actl~lty of young and
aged human culturcd dermal fJbroblasts.
- For this purpose, Rhodammc 123 (Rh 123), a fluorcsccnt probe, was
u ~ d [~r the cvaluation of mxtcuzht~ndnaI actwlty. This spectflc Rh t23
fluorcsccncc depends on the nntochondrlal transmcmbranc potential. The Rh
1o_~ mcasurcment is gcncrally perlk)rmcd using flow cytometry (FCM), but
FCM is a slt)~ and cxpensl~e mcthtxl, not adapted to screening. Morcovcr,
Rh 123 can now bc detectcd dircctly m bye cells in 96-well mlcroplates [ ! 1
or Pctn dishcs (MiFALC tests) wJth a MIcroplate Cytofluorimeter (Huorolitc
I(X)O -DYNEX ) using cold hght fluorimctry (MCCM).I21 Thcrcfore,
MCCM was uscd to measure Rh 1 ~ fluorescence and then mitochondrial
actlvlt3 vanatlon after UVA trradlatlon m cultured human dermal flbroblasts.
-Resnlts : A decrease m Rh 123 fluorescence was observed m irradiated
cells as c~mparcd to sham-irradxatcd cells ~lth high significant dfffercncc
(p<_O.0()[). In our mtxlel, a Mtcrotitration Fluorimctnc Assa 3 on Live Celts
(MIFALC Tcst) [31 using Rhodaminc 1-3 probe, reveals UVA effects
whtch reduce an abt)ut 30c~ decrease c)l mJtochondnal activtty. Thts effect
appears lhr onl 3 after ~rradlatlon, ~ hereas 48 h are necessary to observed a
5()~ ccllular proliferation decrease. Moreover, a htgher scns~ttvtty of UVA
was obscr~ cd wtth aged fibroblasts compared to young ceils.
C o n c l u s i o n ' Then, mttochondrial actw~ty inJUry is a scns~twe and early
biomarkcr for UVA effects. Th~s new M~crotltratton Fluorimetrlc Assay on
Lwe Cells (MIFALC/ Rh 123 assay) can evaluate easily and directly in
m~croplate the early UVA effects in live human demaal fibroblasts.
[ 1 ]-Rat P. er al., Ceil B~ol. Toxicol , 1 0,(1994), 329
[ 2J-Rat P e r a l , Xlcth Enz.vmol, 252,(1995),331[ 3 ]-Rat P et al., Anirrt,'d Ahernati~ us, Welfare & Ethics, Elsevier, DAVS 27, (1997) 813

(Mo/12.1/064)

Nitric oxide inhibits mitochondrial creatine kinme

A.Kaasik"b, A.Minajeva~, E.Sousa ~, R.Ventura-Clapier ~, V~Veksler
"INSERAt U-446, Umverstt~de Paris-Sud, Facult~ de Pharmaie~ France
Departments of bPl'~nTnacologyand ~Pathoanatomy. Umversitp~t~/,Tartu

NO biosynthesis in cardiac muscle leads to a decreased,~oxygen

consumption and lower ATP synthesis. It is suggested that this. gfa~ect of
NO is mainly due to the inhibition of mitochondrial respiratory,t~hain
enzyme, cytochrome c oxydase. However, our work demonstratesithat NO
is able to inhibit also mitochondrial creatine kinase. Nitric oxideidonor Snitrosoglutathione inhibited concentration dependently the anti,lily of
mitochondrial creatine kinase. Half maximal inhibition was achieved~in at
30-35 ~tM donor concentrations. Creatine kinase was inhibite~aho in
skinned cardiac fibres that maintain the architecture and ,rlSpatial
relationships between the intracellular compartments. Since creatina~kinase
reaction eatalysed by the mitoehondrial isoform increases the availability
of ADP for oxidative phosphorylation, the sensitivity of mito~hondrial
respiration to ADP was used as an index of mitochondrial creatind kmase
functional activity to test whether the NO inhibit also the funtional
activity of mitochondrial creatine kinase. Indeed, NO donor d~pressed
significantly the functional activity ofmitochondrial creatine kinase.!~hese
results allow to suggest that the mitochondrial erentine kinase ihllibition
combined with the effect of NO on the respiratory chain may play, a very
important role in the mechanisms of inhibition of the mitochondri~i.energy
production by NO.

(Mo/12.1/066)

The influence of oncotic pressure on the outer

mitochondrial membrane permeability for ADP
J. Liobikas, D.M Kopustinskiene, A. Toleikis
Laboratory o f Biochemtstry. [nstztute for Bzomedwal Res'earch,
Kaunos AIedlcal Lrniverszty, Etvemu 4, L T-3007 Kaunas, Ltthuama

100 mg/ml (10%) of bovine serum albnmi, (BSA; Fraction V, Sigma) was
added to the physiological salt solution containing yeast hexokinase and
glucose, as external ADP regeneration system, to mimlc the uncofic
pressure of the cellular cytoplasm and to test for its effect on the external
ADP-dependent respiration of isolated rat heart mitochondria (MT) and
saponin- (SF) or sapunin plus crude collagenase-treated (SCF) cardiac
fibers. The medium with 2 mg/ml (0.2%) BSA was used as control. To
obtain the kinetic constants of oxidative phosphorylation the titration of
respiration was made by different ADP concentrations k,_ each separate
probe. It is assumed [1, 2] that apparent K~ of oxidative phosphorylation
for ADP reflects the outer mitochondrial membrane permeabi/ity for ADP.
We obtained that 10% BSA increased the apparent t ~ of oxidative
phosphorylation for ADP in MT and SCF by 70-90% (p<0.01) and had no
significant effect on AV,,~. However, the K~ value for ADP in SF in the
medium supplemented with BSA was even slightly lower '.hart in control
(by 20%; statistically not significant) and several times higher than that of
MT and SCF. AV,,~ was reduced by 23% (p<0.01). We assume that in
vivo the oucotic pressure plays little, if any, role in the regulation of the
outer mitochondrial membrane pores' permeability for ADP. It is more
likely that preserved intracellular structure or/end outer mitochondrial
membrane bound proteins (which remain in SF but not in SCF and MT
preparations) is respons~le for this property of membrane.
[ 1] Saks et al., Biochim Biophys Acta, I 144 ( 1993 ): 134.
[2] Gellerich et al., Eur J Biochem, 254 (1998)" 172.

Abstracts F E B S ' 9 9

(Mo/12.1/067)

s 183

Mechanisms of repear of mammalian

mitochondrial DNA
I. Majsterek, Z. Walter,

(Mo/12.1/068)

Equlpe lNSERM Ph~iologw Mitochondriale Uni~ersit~Bordeaua 2,

146. rue [12o8mgnat. 33076 Bordeaux-Cdde~ Fiance

Departament of Molecular Geneticist Umversi .t.tyo f Lodz

Banacha 12/16 90 237 Lodz, Poland

Mitochondrion possesses own DNA (mtDNA), which is only

extranuclear source of genetic information in mammalians' cells.
Mitochondrial genom because of lack of histones and non-histon proteins
is more susceptible on influence of factors damaging DNA structure.
Mitochondrial myopaties and process of growing old, are considerably
dependent on existence and efficiency of repair-mechanisms [1]. Till
now however one did not clearly show possibility of working in
mitochondrion mechanisms of repair o f D N A Mitochondrion is potential
place of activity antieancer platinum compounds (cisplatinum,
carboplatinum) [2]. The properties of platinum compounds applied in
chemotherapy depend on their interaction and joining with DNA,
however creating ofPt-DNA adducts provoke local changes and damages
of native structure of DNA [3]. Proof possibilities of repair of Pt-mtDNA
adduets enabled to answer the question: Can mammmalian
mitochondrium repair own DNA?
In passed experiment sign of ability of repairing of the adducts
was decrease in time of concentration of platinum bounded with "lively"
mitochondria's (Sus scrofa domesticus) DNA incubated with examined
platinum compounds. Using method of AAS (atomic absorption
spectroscopy) the mesurment of concentration changes of platinum
bounded with DNA contained in organellums was preceded by
estimation of ability of platinum binding with native structure of mtDNA.
These measurements with reference to examined total concentration of
platinum linked in "lively" mitochondria clearly point to their actively
participation in removing of Pt-DNA adducts. Mark of activity of
respiratory enzymes (cytchrome c oxidase and ATP synthetase) in
mitochondria exposed to influence of platinum showed statystically
significant changes additionally proving actively participation of
organellums in energy-consuming mechanisms of repair.
[1] Linnane W., et al , Mutation Research, 275, 1992, 195.
[2] Olivero O , et al , Mutation Research, 346, 1995, 221.
[3] Vrana O., et al , Anti-Cancer Drug Design, 1, 1986, 95.
(M0/12.1/069)

Nm23-H4, a human mitochondrial NDP kinase

L. Milon a, A. Munier a, I. Lascu b, J. Capeau a, M.-L. Lacombe a.
alNSERM U402 CHU St-Antoine, Paris, b IBGC-CNRS, Bordeaux, France.

Nucleoside diphosphate (NDP) kinases which are responsible for the

synthesis of triphosphate nucleosides from diphosphate nueleosides, are
involved in the regulation of cellular functions, such as development, normal
and tumoral proliferation, differentiation and apoptosis, by unknown
mechanisms [1]. Up to now, five human sequences corresponding to or
homologous to NDP kinases have been identified: NDP kinases A and B,
encoded by the nm23-Hl and -H2 genes, respectively, DR-Nm23, Nm23-H4
[2] and Nra23-H5 [3]; the latter two were isolated in our laboratory.
Nm23-H4 is a protein of 187 residues (Mr: 20650) which presents 55 to
60 % identity to the other human NDP kinases and possesses a NH2 terminal
extension, characteristic of a mitochondrial target. The full length protein,
expressed in E. coli, is inactive whereas the truncated protein, lacking the NH 2
extension, possesses NDP kinase catalytic activity. The activity is highly
thermolabile (Tin 40C), which is due to the presence ofa serine (position 129)
instead ofproline conserved in most NDP kinases, since the enzyme is greatly
stabilized (Tin 65C) by the reverse mutation (Ser--*Pro). Interestingly, this
substitution corresponds to a Drosophila NDP kinase mutation (K-pn),
involved in developmental alterations. Localisation of Nm23-H4 to
mitochondria has been demonstrated: 1) by fluorescent microscopy of human
HEK 293 cells transfected with a vector expressing Nm23-H4 fused to GFP
("green fluorescent protein") and 2) by Western blot analysis of subcellular
fractions using specific anti-Nm23-H4 polyclonal antibodies. Protein targeting
to mitochundria is accompanied by cleavage of the NH2 terminus, a process
which is also required for NDP kinase activity. The analysis of the submitochondrial localisation and the mechanism of activation of Nm23-H4 should
improve the understanding of its role in nucleotide metabolism within
mitochondria and its possible relationship to physiological processes controlled
by mitochondria, such as apoptosis.
[1] De La Rosa A. et at., BioEssays, 17, (1995) 53.
[2] Milon L. et al., Hum. Genet., 99, (1997) 550.
[3] Munier, A et al., FEBS Let., 434, (1998) 289.

Threshold effect and tissular specificity

Different behaviours and implication for mito:hondrial cytopathies
J.-P. MAZAT, R. ROSSIGNOL, M. MALGAT and T.LETELLIER

Mitochondrial cytopathies present a tissutar specificity characterized by the fact

that even if a mitochondrial DNA mutation is present in all tissues, only some
will be affected and induce a pathology. Several mechanisms have been proposed
to explain this phenomenon such as the appearence of a sp?radic mutation in a
given stem cell during embryogenesis or mitotic segregation, giving different
degrees of heteroplasmy in tissues. We propose an additional mechanism
contributing to tissular specificity, based on the biochemical threshold which can
be characterized by the following: (i) a low activity of a given oxidativephosphorylation (OXPHOS) complex is able to maintain a normal level of
oxidative phosphorylation, but (ii) a minute decrease in this activity may make
the respiration and ATP synthesis of mitochondria collapse. We have shown that
this biochemical threshold can be predicted in the framework of the metabolic
control theory.
The threshold effect for a given OXPHOS complex can vary according to the
tissue, thus changing its sensitivity to a defect in the complex. To verify this
hypothesis and to illustrate the pathological consequences of the variation in
biochemical thresholds, we studied their values for seven OXPHOS complexes
in mitochondria isolated from five different tissues. Two types of behaviour in
the threshold curves can be distinguished corresponding to two modes of
OXPHOS response to a deficiency. We therefore propose a classification of
tissues according to threshold values and type of OXPHOS response to a
complex deficiency.
(1) Letellier, T. et. al., Biochem. J. 302, (1994) 171;
(2) Korzeniewski, B. and Mazat, J.-P., Biochem. Z 319, (19%)143-148 .

(Mo112.1/070)

Exploration of the topography of the mitochondrial

membrane-embedded ADP/ATP carrier of S.cerevisiae.
F. Noel d A.C. Dianoux", C. Fiore~,V.Tr~zrguet b,
G.Brandolin" & G.J.M. Lauqu n .
DBMS-BBSI, CEA-Grenoble, 38054 Grenoble Cedex 9, France.
b IBCG-CNRS. 33077 Bordeaux Cedex, France.

The adenine nucleotide carrier (Anc) is a nuclear encoded protein, located

m the inner mJtochondrial membrane. It exclusively catalyses the exchange of
adenine nucleotides between the matrix and the cytosolic compartments and can
be inhibited by two classes of inhibitors with strong affinity : the atractyloside
family (CATR and ATR) which interacts with Anc from the cytoplasmic side and
the bongkrekic acids (BA) which reach the carrier from the matrix side. Intrinsic
fluorescence studies of the detergent-isolated Anc show that addition of ADP (or
ATP), CATR and BA induces different responses. These tryptophanyl
fluorescence variations reflect conformational changes of Anc most probably
related to binding and/or transport processes.
The yeast Saccharono,ces cerevisiae contains three Anc isoforms. Stable
strains expressing the sole Anc2 isofocm, tagged or not with a poly-histidine
extension, have been constructed and characterised.
In order to map the binding sites of substrates and inhibitors of Anc2,
photoactivable and radioactive derivatives of these coumpounds have been
synthesized.
Incubation of mitochondria with [32p] 2-azido-3'-O-naphthoyl-ADP (ANADP), a substrate derivative, leads to the labelling of a protein of about 30 kD in
the whole lx/sate. This protein was identified as Anc2, due to the lack of labelling
in presence of CATR. Labelled Anc2 was purified and submitted to chemical
cleavages by CNBr, or NH,OH or N-bromosuccinimide. The released peptides
were separated by SDS-PAGE, detected by radioactivity, identified by aminoacid
sequence analysis and immunochemical reactivity. Two regions, located in the
middle and at the C-terminal parts of Anc2, have been labelled with the probe
and are therefure involved in the recognition and the binding of AN-ADP.
Two atractyloside derivatives, the azido-nitro-phenyl-amino-butyryt-ATR
and the azido-nitro-phenyl-amino-peptidyl-ATR are actually under investigation.
The binding of CATR or BA or adenine nucleotides leads to formation of
complexes with different conformations of the carrier in the membrane. Lirruted
proteolysis of these complexes w~th various enzymes can give informat~ons
about the topography of Anc2. Preliminary results confirm that this approach is a
valuable tool.

s184

Abstracts FEBS'99

(Mo/12.1/072)

Identification of mitochondrial abnormalities, in unrelated patients

with multiple deletions, suggests different molecular mechanisms
R. Paula, J. Pougetb, J.F. Pellissierb, C. Richelmec, M.F. Monfortb,
C.Butori~, A. Sauni~resa,V.Paquis-Flucklingera, C. Desnuellea
a Laboratozre de Neurobiologie cellulaire,06107 Nice, France
b CHU Timone, 13385 Marseille, France; c CtfU Nice 06202 Nice, France

Multiple mitochondrial DNA (mtDNA) deletions have been described in

numerous neurological diseases like progressive external ophtalmoplegia
(PEn), ptosis, muscle weakness or encephalomyopathy. In affected families,
the transmission is in an autosomal dominant or recessive mode. Linkage
studies indicated several nuclear loci predisposing to mtDNA deletions,
suggesting a genetic heterogeneity.
We studied two affected members of a family presenting a late-onset central
neurological disorder. None of them had a PEn, unlike the other patients
previously described. We also studied one young boy with no familial
history. He had a P E n with a cerebellar ataxia.
By using the "large fragments" PCR method, we obtained in addition to the
15.6kb amplification product expected, numerous shorter mtDNA fragments
in muscle but not in circulating leucocytes of the 3 patients. Southern blot
analysis confirmed the presence of multiple deletions in the 3 cases.
Mapping of the deletions was performed using a PCR-based analysis and
sequencing.
In the first family, repeated sequences were present in the deletion
boundaries. We also evidenced numerous nucleotide transitions and a 9-bp
motif triplication of the intergenic COII/tRNA lys. In the young patient's
case, direct repeats at the breakpoint junctions or point mutations were
extremly rare and no triplication of the 9-bp motif was found.
Our results suggest that multiple deletions observed in the 2 pedigrees result
in different molecular mechanisms and point out the role of repeated
sequences in the first pedigree. No mtDNA repair system has been described
in mammals so far, but the molecular abnormalities found in the first family
suggest that a defect in a mtDNA repair system homologous to the E . c o l i
MutHLS pathway could be responsible for such a phenoype

(Mo/12.1/073)

Identifying new mutations of

mitochondrial DNA in mitnchondrial myopathies.
D.Perucca, V.Paquis. JB Hernandez, A.Sauniere~,B.Vialettes,C.Desnuellc
Laboratoire de Neurobiologie Cellulaire, CNRS UMR 6549. Faculte de
Medecme, avenue de Valombrose, 06107 NICE Cedex 2, France

Several mitochondrial DNA (mtDNA) mutations have been linked to

human diseases. However, the complexity of these mitochondrial
syndromes suggests genetic heterogeneity. To identify new mutations of
mtDNA, we investigated molecular analysis in two different
mitochondrial myopathies : Myoclonic Epilepsy with Ragged-Red
Fibers (MERRF) and Non Insulin-Dependent (typelI) Diabetes Mellitus
(NIDDM).
MERRF is characterized by mutations in the tRNA lysine at positions
8344 and 8356. These mutations arc not observed in 20% of the cases.
Sequencing of the complete mitochondrial genome of a MERRF patient
without established mutations revealed a homoplasmic missense
mutation in the cytochrome b gene (A 14793G). This A-to-G substitution
at position 14793 leading to an histidine for an arginine is located in a
conserved region of the cytochrome b genc and was absent in 100
controls. The same mutation has been identified in two different
mitochondrial diseases with ragged-red fibers.
Patients with NIDDM in combination with sensorineural hearing loss
and maternal inheritance were selected on clinical findings. Point
mutations in tRNA lcucine at position 3243 and in the IRNA glutamic
acid at position 14709 were excluded. The mt DNA of one patient was
totally sequenced and the 22 tRNA genes of 7 others patients were
analysed. Different substitutions GI888A in the RNA 16S and A8381G
in the ATPase 8 gene were identzfied. Various substitutions are currently
under analysis. Possible clinical significance of these substitutions is to
be determined.
The pathological eftects of the identified mutations are investigated by
cellular analysis allowing c o m p l e m e n t a t i o n s between patients
fibroblasts and human mtDNA-less cell lines.

(Mo/12.1/074)

Missense mutations of SURF-I in Leigh syndrome

with cytoehrome oxidase defieiene)
A Poyau. K Buchet. MF Bouzidi. MT Zabot and C Godinot
Centre de G~ndnque Mot~culawe et Celhdaire (G3IR 5534 - CNRS),
Umversttd Lyon L 69622 l)/leurbanne cedex, krance

Cytochromc oxidase (COX) deficiency is one of the most common

disorders in pathologies involving mitochondria. The deficit of this enzyme
can be of mitochondrial or nuclear origin because the COX consisted of 3
subunits encoded b3, the mitochondrial DNA and 10 subunits encoded by the
nuclear DNA. We have studied three patients suffering from the Leigh
syndrome with COX deficiency. This progressive neurodegcnerative disease is
a subacute necrotizing encephalomyopath 3 that dcx elops in infancy.
Fusion of thc patient cnucleated fibroblasts ~ith mtDNA-dcpleted rbo
cells restored the normal COX activity in cybrids indicating that the paticnt
mtDNA was functional and, therefore, that the deficiency was of nuclear
origin in our 3 patients. To find the nuclcar gene responsible for this
syndrome, we have analysed the COX gene expression in patient fibroblasts.
The amount of several COX subunits obser,,ed by western-blot ~ere strongly
decreased in patients while the transcript expressions were similar to those of
controls. A mutation in a nuclear COX subunit could explain a complex
instabilitF. Howexer. the sequences of all nuclear COX subunit cDNAs were
normal in our patients. Moreover, cytochrome c which seems to act on COX
stability in yeast, also had a normal eDNA sequence.
Very recently, several nmtations ha,,e been identified in the SURF-1
gene of patients with Leigh syndrome by the groups of Shoubridge [1] and of
Zeviani [2]. This gene is localised in tire 9q34 region but the SURF-1 protein
function is unknown. All mutations gaxe a truncated protein. Scquencingof
the 9 SURF-I exons and of the intron-cxon junctions in our 3 patients revealed
5 new heterozygous nmtations a 4-bp insertion, a splicing mutation, a
nonsense mutation and two missense point mutations. One of the latter
modified a Gly very well conserved exen in bacteria, the second one changed
an lie essential t\~r a ~-sheet alwa 5 s conser~ ed in the structure predicted for all
known eukar3,otic SURF-1 proteins. These missense mutations provide new
elements to identity, important sites lbr SURF-1 function.
111 Zhu et al., Nat Genet. 20. (1998) 337,
[2] 1 iranti et al.. Am J 1lum Genet. 63. ( 1~)t)8) 1609.

Abstracts FEBS' 99

(Mo/12.1/075)

N u c l e a r - D N A origin of mitochondrial Complex I

in fatal infantile lactic acidosis
v. Procaecioa, R. Beugnota, B. Mousson b, J.P. Issartel a, J. Lunardia

s 185

(Mo/12.1/076)

alz~boratol 3' qf Bioenergetics BECP-EA 2019-UJF CEA Grenoble France

bpedlatric Btochemtstty Debrousse Hospital Lyon France

NADH-ubiquinone oxidoreductase (Complex I) is the first component

of the mitochondrial respiratory chain. In mammalia, this complex is
composed of at least 43 different polypeptides. Seven are encoded for
by the mitochondrial genome, the other subunits are encoded by nuclear
genome. In human, defects in Complex I have been described as a
major cause of mitochondrial encephalomyopathy or neuromuscular
diseases. In comparison to the mitochondrial genes, the nuclear genes
coding for the subunits of the human Complex I are poorly known.
However, these numerous genes can be the target o f deIeterious
mutations in relation with mitochondrial diseases with a mendelian
transmission. We started a genetic study to clone and sequence the
genes coding for the major nuclear encoded subunits. In the first part of
this work, we have characterized 2 genes coding for the 23 and 49 kDa
subunits which are very important for the function of complex I (1) (2).
The second part represents functional and genetic characterisation for
patients with an isolated defect of complex I. The knowledge of the
structure for some genes and availability of specific antibodies directed
against some subunits allows the study of the molecular defects of the
patients.
Distinction of the nuclear or the mitochondrial origin of a detect is a
preliminary step. This step is realized by complementation experiments
between cells without mitochondrial DNA (rho cells) and patient
fibroblasts. This strategy showed that 2 patients do suffer from a
nuclear defect responsible for complex 1 deficiency, lmmunoblots have
shown a decrease for all the complex I subunits for one patient, and a
selective decrease for the 24 kDa subunit for another patient. We are
now in the way to identify molecular abnormalities in relation with these
functional defects.
(l) Procaccio V. et al. Biochim. et Biophys. Acta 1351 (1997) : 37-41
(2) Procaccio V. et al. Mammal. Genome 9 (1998) :482-484

(Mo/12.1/077)

Synthesis of yeast cell wall constituents : role of the

mitochondria and action of respiratory chain inhibitors
A. Rakotoarivony-Iung, J. Ngondi-Ekom6, J. Coulon,
R. Bonaly
U M R 7564 L C P E -Biochimie Microbienne- 54000 Nancy - France

Structure of the complex between cytochrome c and

cytochrome c oxidase by the use of EPR techniques
J. Pyka, B. Turyna, W. Fronc~sz
l ~ t a u t e ot Moleculat B~olog~, Jag~ellonian Uml ei ~tt~, Krakdu, Poland

EPR spectroscopy has the potential of being a very valuable tool in

investigations on cytochrome c - cytochrome c oxidase complex. The manl
advantage is to obtain structural and motlonal reformation directly from
the place of interest by the use of spin labels. The purpose of these studtes
was to test an application of CW EPR to study the structure of the
cytochrome c - cytochome c oxidase complex.
A set of spin-labeled monoderivat~\es of cytochrome c from horse
heart (lysine-specific spin label, succininddyl-2.2,5,5-tetra-methyl-3pyrroline-l-oxyl-carboxylate), and cytochrome c
from
Sacharomyces
c e r e v t s i a e (cysteme-speclfic spin label, methanethiosulfonate spin label)
was used for the studies. Spin-labeled monoderivatives were characterized
by the use of EPR techniques: CW EPR and CW EPR power saturatmn,
saturation recovery EPR. Computer slmulauons were performed to gain
informations from CW EPR spectra.
Cytochrome c oxidase was isolated from the bovine heart.
Complex [i)rmation between spin-labeled cytochrome c and bovine
cytochrome c oxidase were carried out in 5 mM MOPS pH 7.4, 0 . 1 %
Triton X I00. CW EPR spectra were recorded for different ratio of the
complex components.
Comparison of the EPR spectra of the free spin-labeled
cytochrome c to these of the spin-labeled cytochrome c bound to
cylochrome c oxldase demonstrated clearly that the complex formation
can be detected by EPR. The EPR changes depend also on the posmon of
the spin label attachment to the cytochrome c molecule. The CW EPR
spectra exhibit only small changes in the case of the high ionic strength
(0.5 M NaCI).
These results demonstrate the apphcabdlty of EPR to study the
cytochrome c - cytochrome c oxidase complex.

(Mo/12.1/078)

UVA Induced Mitochondrial Injury in live lluman

Dermal Fibroblasts - Assessment ~*ith Mieroplate Cytometr)
P Rat .gb. C. Korwm-Zin(iowskaa, L Pa'.cual Ic "Fal/cc<M Adolphea
~t L a b o l l l l o t l e
Phalmal o

The yeast cell wall has a dynamic structure. Its chemical composition and
structure change according to the strains, the culture medium and the
growth conditions. Phosphopeptidomannans (PPM) are the most peripheral
polymers of the cell, they participate in cell-cell recognition phenomena.
PPM are the ligands of lectins, the both components beeing involved in the
phenomenon of flocculation which is a reversible cell aggregation process.
In this study we show the possible influence of the mitochondrial
function in the synthesis of the cell wall constituents. Two strains of
S a c c h a r o m y c e s d i a s t a t i c u s were studied, a hight flocculent wild type strain,
and a less flocculent [rho ] mutant strain, obtained by ethidium bromide
treatment. We also studied the action of antimycin, a respiratory chain
inhibitor. By culture of the wild type strain in the presence of antimycin, the
cells became less flocculent.
To determine the respiratory chain influence on the biosynthesis of the
cell wall, yeasts were grown under aerobiosis, anaerobiosis and in the
presence of antimycin. We observed changes in the PPM chemical
composition of the respiratory chain deficient ceils compared to PPM of the
wild type cells. Moreover by treatment with mercaptoethanol, [rho ] PPM
showed a lower stability than wild type PPM. Alteration of the PPM leads
to a modification of the cell wall structure and in this case change in the cellcell aggregation phenomenon.
Mituchondria could be directly or indirectly involved in yeast cell wall
synthesis, m t D N A products may intervene in the expression of nuclear
genes which encode proteins involved in cell wall biosynthesis or in the
secretory pathway necessary for the biosynthesis of cell wall constituents.

dc Phalmll~ ologtc CeHtdao~ -LPIIE.751n16 t',~ i~ h ( 'row dt

Totuolo~lc Cl,lhdatre .CH~vO l~/2a . 7~(tl2 Purt.~ l'J~lm e

Llltraviolet radiation, particularly UVA. reduces toxic effects and the skin is
its first target. It is well known that UVA causes considerable damage to skin
cells. - The aim of this study was to evaluate the effects of UVA on
cellular proliferation assay and on the mitochondrial activity of young and
aged human cultured dermal fibroblasts.
- For this purpose, Rhodamine 123 (Rh 123), a fluorescent probe, was
used for the evaluation of mitochondrial activity. This specific Rh 123
fluorescence depends on the mitochondrial transmembrane potential. The Rh
123 measurement is generally performed using flow cytometry (FCM), but
FCM is a slow and expenswe method, not adapted to screening. Moreover,
Rh 123 can now be detected directly in live cells in 96-well microplates [ 1 ]
or Petri dishes (MiFALC tests) with a Microplate Cytofluorimeter (Fluorolite
1000 -DYNEX ) using cold light fluorimetry (MCCM).[2] Therefore,
MCCM was used to measure Rh 123 fluorescence and then mitochondrial
activity variation after UVA irradiation in cultured human dermal fibroblasts.
-Results : A decrease in Rh 123 fluorescence was observed in irradiated
cells as compared to shamqrradiated cells with high significant difference
(p_<0.001). In our model, a Microtitration Fluorimetric Assay on Live Cells
(MIFALC Test) [3] using Rhodamine 123 probe, reveals UVA effects
which induce an about 30% decrease of mltochondrial activity. This effect
appears lhr only after irradiation, whereas 48 h are necessary to observed a
50% cellular proliferation decrease. Moreover, a higher sensitivity of UVA
was observed with aged fibroblasts compared to young cells.
Conclusion : Then, mitochondrlal activity injury is a sensitive and early
biomarker for UVA effects. This new Microtitration Fluorimetric Assay on
Live Cells ( M I F A L C / R h 123 assay) can evaluate easily and directly in
microplate the early UVA effects in live human dermal fibroblasts.
[l]-Ral P. et a l , Cell Blol, Toxtcol, 10,(1994), 329
[2J-Rat P e t al.. Meth Enzyrnol.. 252,(1995), 331[3J-Rat P. et a l . Ammal Alternatives. Welfare & Ethics, Elsevier, DAVS 2%(1997) 813

s 186

(Mo/12.1/079)

Abstracts FEBS'99

A biophysical characterization of
human mitoehondrial ereatine kinase isoforms
U. Schlattner and T. Wallimann

(Mo/12.1/080) Importation in plant mitochondria of a protein involved

in cytochromes c biogenesis
N. Spielewoy, J.M. Grieuenberger, G. Bonnard
lBMP/CNRS-Universiti Louis Pasteur, 12. rue du Gdndral Zimmer,
67084 Strasbourg, France

Inst. of Cell Biology, Fed. Inst. of Technol. (ETH), Z~irich, Switzerland

Creatine kinase (CK) isoenzymes, catalyzing the reversible transphosphorylation between ATP and phosphocreatine, play an important role in energy
buffering and energy transport of cells with high and fluctuating energy
demands [1,2]. Tissue-specific mitochondrial CK isnforms (Mia-CK and
Mib-CK), located in the mitochondrial intermembrane space, form homodimers and homooctamers. The octameric state was shown to be essential for
strong membrane binding [1], which in turn seems to be important for MiCK functions like: (i) metabolic channeling during "energy export" from
mimehondria into the cytosol [2,3], (ii) regulation of the mitochondriaI
permeability pore possibly involved in apoptosis [4] and (iii) crosslinking of
mitochondrial membranes (structural role).
We show here a functional comparison of human Mi-CK isoforms. A
pronounced difference between both isoforms was the low K m of Mia-CK
for Cr (1.0 mM) and ATP (0.1 mM), which may constitute an adaptation to
lower Cr/PCr levels in Mia-CK expressing tissues like brain. Dissociation of
octamers and reassociation of dimers was much slower for Mia-CK as
compared to human Mib-CK. Analysis of the thermodynamic octamer/dimer
equilibrium revealed that octamers of both human Mi-CKs are more stable
than e.g. octamcrs of chicken Mib-CK. Thermodynamic data also indicated
ciiffercoccs al the dimer/dimer interaction face of Mia-CK a,, compared to the
known Mib-CKs. Membrane binding kinetics of Mi-CK ~as monitored by
surface plasmon resonance (BiaCore/ using biotinylated cardiolipin-containing vesicles immobilized on an avidin-coated surface. Association and
dissociation kinetics of octameric Mi-CK fitted well to a model for heterogeneous interaction suggesting two independent binding sites [1]. Further
analysis revealed that both binding sites have similar association equilibrium
constants (affinity) and differ only in their rate constants (binding velocity).
Mia-CK showed slightly higher membrane affinity than human Mib-CK,
which is possibly due to an additional positive charge at the C-terminus.

Three different model systems have been proposed to describe

cytochromes c biogenesis : yeast mitochondria, Gram- bacteria, and
chloroplasts. This metabolism includes transport and covalent attachment
of haem to the apocytochromes in specific cellular compartments
(intermembrane space, periplasm and lumen). Land plant mitochondria
seem to follow a separate way from yeast mitochondria, but very similar
to that described in Gram- bacteria.
A nuclear gene of A r a b i d o p s i s thaliana (AtccmE) shows high
similarity with a bacterial gene involved in cytochromes c biogenesis. By
different approaches, we clearly demonstrate that AtCcmE protein is
present in the mitochondria.
Polyclonal antibodies raised against AtCcrnE surexpressed in E. coil
have been purified by immunoaffinity and used in immuno-detection
analysis on Arabidopsis thaliana's mitochondria purified from protoplasts.
AtCcmE is located in the mitochondrial inner membrane and its
hydrophilic C-terminal part faces the intermembranc space.
In vitro experiments show that A. thalkTna protein could be, after
traduction in an acellular system, imported into mitochondria of potatoes.
Its importation needs membrane potential tAW), a characteristic of
mitochondrial importation system. So we could postulated the presence of
a 50 residus long presequence, which would be excised at a clivage site
consensus in plants.We have caracterised the first nuclear gene involved in
steps specific of plant mitochondria during cytochromes c biogenesis.

[1] Stachowiaket at., Mol. Cell. Biochem. 184 (1998). 141; [2] Schlanneret al., Mol. Cell,
Biochent 184 (1998). 125; [3] Khuchua et al., J. Biol. Che~ 273 (1998), 22990; [4]
O'Gorman et aL, FEBS Lett. 4t4 (1997) 253

(Mo/12.1/081)

Transmission of mitochondrial DNA in cloned cattle

R. Steinborn~, V. Zakhartchenko~. E. Wolfb, M. MUller~, G Brem~
"lnstttute of Ammal Breeding & Genettcs, I'MU,, A-1210 Vtenna.
hDept Molecular Amrnat Breeding & Genetics, LMU D-80539 Muntch

The cloning of livestock species yields genetically identical copies

which can be used in research, agricultural practice, and human
medicine. Our research investigates the mode of inheritance of
mtDNA in cattle produced by different cloning protocols [1, 2[.
These protocols include the fusion of blastomeres, embryonic
fibroblasts, primordial germ cells, and somatic ceils into enucleated
recipient oocytes. Allele-specific quantification of parental mtDNA
in these clones is performed by Allele-specific (AS-) TaqMan TM PCR
[2]. This method uses a fluorogenic TaqMan TM probe, specific for the
mitochondrial control region, in combination with allele-specific
oligonucleotide primers for the mtDNA type to be followed. Quality
parameters for AS-TaqMan TM PCR like dynamic range, accuracy,
sensitivity and discrimination range are presented.
Quantifying parental mtDNA in clones produced by fusion of
somatic cells can show to what extent somatic mitochondria are
reprogrammed. It can also be shown whether the increase in body
weight observed in some cloned animals at birth correlates with a
deviation from neutral transmission of parental mtDNA into the
cloned offspring.
References' l 1] Steinborn R et al FEBS Len 426 (1998) 352.
[2] Steinborn R et al FEBS Len 426 (1998) 357.

(Mo/12.1/082)

Establishing a system of tRNA import in human

mitochondria
I. Tarassov, N. Entelis + & R. Martin
UPR 9005 CNRS, 21, rue Ren~ Descartes, 67084 Strasbourg, France.
"Molecular Biology Dcpt, Moscow State University, Moscow, Russia.

In budding yeast, mitoehondrial import of a cytoplasmic tRNA Lys

(tRK1) requires both the integrity of the pre-protein import machinery and
the presence of cytosolic import-directing proteins. We thought to use our
knowledge on tRNA import mechanisms in yeast to develop an artificial
tRNA import system in human cells. The main interest of such a system is
to attempt to complement mutations in mitoehondrial tRNA genes known
as sources of various myopathies and neurodegenerative diseases [1] by
tRNAs expressed in the nucleus and imported from the cytoplasm.
As a first step towards this goal, we have shown that tRK1 can be
imported into isolated mitochondria from HeLa or HepG2 cells providing
that yeast import-directing factors are supplied. The import was further
stimulated by addition of a HeLa cell extract, which suggests that at least
some of the import factors are present in human cells. A panel of mutant
tRK1 derivatives [2] was assayed for import into isolated yeast and human
(HeLa) mitochondria. We found that import selectivity was comparable
but not identical in the two systems. All the transcripts which are not
imported in yeast mitoehondria are also not imported in the human ones.
The best import efficiency for human mitoehondria was observed for the
native ttLK1 and its in vitro synthesized transcript. Another tRKI variant
with an anticodon CAU(Met) can be aminoacylated with MetRS by
methionine and is imported into isolated human mitochondria. Preliminary
results suggest that this tRNA can function in mitochondrial protein
synthesis in isolated human organeUes. The obtained results open the
possibility to create a system of suppression of mutations in human mittRNAs by mutagenized tRNAs imported fi,om the cytoplasm.
supported by INTAS grant N 96-1515

[1] Wallace D.C. Proc. Natl. Acad. Sci. USA 91, 1994, 8739; [2] Entelis
N.S. et al., Proc. Natl. A c a d Sci. USA 95, 1998, 2338.

Abstracts FEB S' 99

s 187

(Mo/12.1/083) Activity of mitochondrial respiratory chain complexes in

peroxisome deficient mice
I. Vanhorebeek, M. Baes, P. Declercq
Laboratory of Chmcal Chemistry, Katholieke Umversiteit Leuven.
3000 Leuven. Belgium

Beside the fact that patients with the cerebro-hepato-renal syndrome of

ZeUweger lack functional peroxisomes, there is also evidence for the
presence of mitochondrial abnormalities.
Structural mitochondrial
malformations as well as functional deficiencies, at the level of the
electron transfer chain, have been identified in these patients [1]. The
generation of peroxisome deficient mice or P e x 5 / mice, as a model for
the syndrome of Zellweger [2], allows a thorough examination of these
abnormalities.
We compared the individual activities of the respiratory chain complexes
and citrate synthase, as a mitochondrial marker enzyme, in liver and heart
of newborn P e x 5 -/- versus control mice. We obtained a 55 % reduction in
complex I and a 100 % increase in complex IV activity in the liver of
Pex5 / mice. In heart tissue, the differences were less pronounced, i.e. a
35 % reduction in complex I and an increase of about 20 % in complex
IV activity. There was no difference in the activities of complex II,
II+III, III, V or citrate synthase in either tissue. Whether other tissues
show the same abnormalities remains to be examined. The possible
significance of the impaired complex I activity in the pathogenesis of
peroxisome deficiency disorders will need to be further investigated.

[1] Goldfisher et al., Science, 182, (1973) 62.

[2] Baes et al., Nature Genetics, 17, (1997) 49.

Supported by Biomed grant BMH4-CT98-3569

(Mo/12.1/084)

Abnormalities
Mitochondrial

of Mutant tRNAs
Encephalomyopathies

Responsible

for

T. Yasukawa"-',T. Suzthki',T. Ueda~,S. Olla: and K. Watanabe'

'D ~ ofChon andBiote~ carl'De~ ~ h ~ t a 4 B i ~ i ~
Burdgz~ Tolffo113,-'FAVMS 19~ ofBiochemCffl Bid., ~

Univq["Td~ 7-3-1
~,a
211,.lc~oan

Mitoehondrial DNA (mtDNA) mutations at either nucleotide position (np)

3243 or 3271 in the tRNAL~u(UUR) gene and at np 8344 in the tRNA Lys genes
have been proven to be responsible for MELAS and MERRF, respectively,
which are major subgroups of the mitochondrial encephalomyopathies caused
by mitochondrial dysfunction. From mass cultures of cybrid cells in which
mutant mtDNAs derived from patients were intercellularly transferred into
human cells lacking mtDNA, we succeeded in purifying the above three
mutant tRNA molecules and determined directly their primary sequences
includingmodificationx We found that these three pathogenic mutant tRNAs
show differences in a single nucleotide modification compared to the wild-type
IRNAs. We also examined the stability of mutant tRNA in cybrid in the
presence of ethidium bromide (250ng/ml) which inhibits only mitoehondrial
transcription, because we observed substantial reduction of the steady state
amounts of the mutant tRNAs. This new degradation assay clearly showed
the instability of patient-derived mutant tRNAs. These f'mdings suggest that
quantitative and qualitative defects of mutant tRNAs are responsible for
mitochondrial diseases caused by tRNA mutations.

s 188

Abstracts FEBS'99

14.1 Ionic channels

(Mo/14.1/085) Expression and distribution of sodium and potassium

voltage-gated channels in rat embryos motoneurons.

N.Alessandri-Haber% M.Gola ~tJ P.Gtraud <21,C.Manrique <2~
C.Paillart~z)" F.Couraud~2~and
M.Crest m. ~ICNRS UPR 9024
(2

(Mo/14.1/086)

Marsedle, France." INSERM I M64, Morsedle, France.

Sodium (Na) and potassium (Kv) voltage-gated channels are involved

in the genesis, the conduction and the regulation of action potentials m
motoneurons. However, little is known about their pharmacology, their
molecular structure and their distribution within the different
anatomical compartments
Na and Kv currents were studied in cultured spinal motoneurons
purified from E l 4 rat embryos during the first two weeks m wtro. After
18 h in culture, a TTX-sensitive Na current was detected, ~t was strtctly
restricted to the axon. It's amplitude is multiplied by 4 during the first 8
days in vitro (DIV1-DIV8). Two types of Kv currents with (IA) or
without (IDR) inactivation were activated one hour after platting The
DR current increased gradually without kinetics modification, whereas
characteristics of the 1A current were modified in the D1V5-DIV10
period corresponding to the establishment of a new component
RT-PCR experiments showed that the Na current resulted mainly
from the expressmn of cdI, a l l l and the 131 auxihary subumts mRNAs.
otl and cdV mRNAs were also detected but underwent alternative
sphcmg resulting in truncated translated products. Kv currents were the
result of the expression of various etKv subunits mRNAs: KvI 3, Kvl.4,
Kvl.5, Kvl.6, Kv3 3, Kv4 I, Kv4.2, Kv4.3 and auxiliary subumts 131
and/32.
The study m immunochemistry of Kvl.4, Kvt 5, Kvl.6 showed a
differential distrtbution of these subunits within the different
motoneurones compartments.
Co-culture of motoneurons and Schwann cells is currently under study
in order to determine if the expression and the distribution of the Na
and Kv subunits are regulated by ghal cells

Modulation of CIC-2 chloride current by TGFc~ in an

intestinal cell line.
M. Bali~, J. Lipecka~, F. Broaillard ~, A. Edelman~ & J. Fritsch~
INSERM U.467, Faeultd:de M~.decineNecker.75015 Pans, France,
b CNRS UPR 1524, Hdpttal S'-Vmcentde Paul, 75014 Pans, France.

The ubiquitously expressed CIC-2 chlonde channel belongs to the CIC

family, whose first member was initially cloned from Torpedo marrnorata
by Thiemmann et al. [1]. Using the nystatin-perforated configuration of
the patch clamp technique, we investtgated the modulation of CIC-2 current by Transforming Growth Factor o~ (TGFo0, an agonist of the Epidermal Growth Factor receptor (EGF-R), in an intestinal cell line (%84).
External application of TGFe (1 ng.ml-~) mdueed an inhibition of CIC-2
current amphmde by 48.3 _+ 10.4 %. This effect was prevented by preincubating the cells with PD153035 (lp.M), a potent inhibitor of the EGF-R
tyrosine kinase activity. Preincubating the cells with BIM (10.6 M), an inhibitor of PKC, also prevented TGFot inhibitory effect on CLC-2 current.
At a higher dose (50 ng.ml-I), TGFot induced a less pronounced inhibition
of CIC-2 current, suggesting that the current inhibition was masked by a
concomitant current activation. Since it has been described that EGF-R can
activate the Na+/H + exchanger (NHE), we checked whether cellular alcalinisation could activate CIC-2 current by submitting the cells to 15 m M
NHaCI. Indeed, CIC-2 current was markedly activated at higher cytoplasmic pH values. The alcalinisation of the cell induced by NH4CI and TGFo~
(50 ng.ml t ) was verified using a pH-sensitive fluorescent probe (BCECF).
Applicatton of the amiloride derivative, EIPA, a potent inhibitor of NHE,
reversed the current activation reduced by 50 ng.ml-~ TGFcc, demasking a
concomitant inhibition.
We demonstrated that TGFa modulates CIC-2 current amplitude in a dual
manner, depending on the concentration used: inhibition, involving activation of PKC, and stimulation, revolving cellular alcahnisation. The latter
effect needs a high dose of TGFc~. whereas the inhibitory et'tbct occurs
even at low doses.
I.Thiemann et al., Nature,. 356 (1992) 57

(Mo/14.1/087)

Influence of length and secondary structure on antibacterial

activity of linear membranotropicpeptides
L Beven"*, S Castanob, J. Dufourcqb, K Btittner~& H. Wrdblewski"
" UPRES.4 CNRS 6026 Umv Rennes i, France,~Centre de Recherche Paul
Pascal, C.VR5] Pessae, Francef Fournwr Pharma GmbH, Sulzbach, Germany.

The most widely used defense system in nature involves membranepermeabilizing peptides Biological activity of these molecules depends on
both their intrinsic properties (length, net charge, secondary structure,
hydrophobicity) and the characteristics of the target membrane (composition,
thickness, electrical potential) To elucidate their mechanism of action, a
minimalist approach consists in studying synthetic peptides differing in length
and sequence, but containing only Leu and Lys residues. We examined the
antibacterial effects of strongly hemolytic amphipathic Leu/Lys peptides on
bacteria devoid of a cell wall (mollicutes or mycoplasmas) The peptides used
in this work varied in length from 9 to 22 residues and adopted either a 13
sheet or an oc helical secondary structure. Finally, we studied the influence of
the target membranes on the activity of these molecules The antibiotic effects
of the ot helical peptides varied with their length, but not monotonously.
Peptides shorter than 12 or longer than 19 residues were quite ineffective As
for natural defense peptides, the primary effect of the most potent peptide (15
residues) was due to its ability in abolishing the proton-motive force. This
dose-dependent phenomenon was followed by the loss of motility, shape
change and splitting of the cells into rounded vesicles. This peptide remained
the most active one, independent of the bilayer thickness and of the presence
or absence of cholesterol Furthermore, 13 sheeted peptides were far less
potent than the ct helical ones Thus, as for the hemolytic activity, an cL helical
structure and a positive net charge proved to be sufficient to obtain a strong
antibacterial activity. The shorter peptides having weaker hydrophobicity and
the longer ones forming aggregates in buffer, the 15-residue peptide
constitutes the best compromise for an optimal association with the
membrane The first limiting step of the mechanism of action is probably an
accumulation of monomers on the membrane surface, followed by structural
changes (aggregation, orientation) which modify membrane permeability and
also membrane fonction
This work is performed in the frame of the GDR/CNRS 790 "Pepudes & Proteincs
Membranotropes"
* Present address : Laboratoire de TechnologicEnzy_matNue, UTC, Compl+gne, France

(Mo/14.1/088)

Mutational analysis demonstrates that CIC-4 and

CIC-5 directly mediate plasma membrane currents
T. Breiderhoff, T. Friedrich, and T.J, Jentsch
ZMNH, Hamburg University.Mammstr. 85, D-20246 Hamburg

C1C-3, CIC-4, and CIC5 form a distinct branch of the CLC chloride
channel family. Although C1C-5 was shown to be mainly expressed in
endocytotic vesicles, expression of CIC-5 in Xenopus oocytes elicited
chloride currents [1,2].
Like CIC-5, CTC-3 and C1C-4 predominantly localize intracellularly after
heterologous expression in COS-7 cells. We could show that CIC-4 gives
rise to strongly outwardly rectifying anion currents when expressed in
oocytes. They closely resemble CTC-5 currents with which they share a
NO3-> CI- > Br- > I- conductance sequence that differs from that
reported for the highly homologous CIC-3. Both CIC-4 and CIC-5
currents are reduced by lowering extracellular pH. We could also
measure similar currents after expression of each channel in HEK293
cells.
To demonstrate that these currents are directly mediated by the channel
proteins we introduced several point mutations that change channel
characteristics. In CIC-5, several point mutations alter the kinetics of
activation but leave macroscopic rectification and ion selectivity
unchanged. A mutation (N565K) equivalent to a mutation reported to
have profound effects on C1C-3 does not have similar effects on CIC-5.
By contrast, a mutation at the end of D2 (S 168T in C1C-5) changes ion
selectivity, and a mutation at the end of D3 (E211A in CIC-5 and E224A
in CIC-4) changes voltage-dependence and ion-selectivity. This shows
that CIC-4 and CIC-5 are indeed chloride channels that directly mediate
chloride currents [3].
[1] Steinmeyer et a1.(1995) J. Biol. Chem. 270, 31172-31177
[2] Gianther et al. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8075-8080
[3] Friedrich et al. (1999) J. Biol. Chem. 274:896-902

Abstracts FEB S' 99

(M0/14.1/089)

s 189

Characterization of the Ca 2 site inhibiting lnsPs binding

to cerebellar membranes
J.F. Coquil, L. Picard, J.P. Mauger

(Mo/14.1/090)

1NSF.RM U-442, Universit~ Paris-Sua~ Bdt.443, 91405 Orsay. France

Cytosolic Ca 2. stgnals. generated

. by cell
. actavatton
.
frequently d~splay a
complex pattern involving repetitive spikes. The mobilisation of Ca2 from
endoplasmic retieulum caused by stimulation of inositol 1,4,5-trisphosphate
receptor (InsP3K) is a key process in this cellular response. Current evidence
supports a mechanism in which Ca2 spikes reflect biphasi feed-back control of
InsP3R by Ca2+. Different binding sites are thought to mediate inhibitory and
activatory effects of Ca 2+, but the molecular basis of the biphasic Ca2
dependence of the InsP3R is poorly known.
In cerebellum and other tissues, InsP3 binding to type 1 isoform of
InsP3R is inhibited by Ca2. In this work, we addressed the question of the role
of this effect in the InsP3R1 function by investigating its mechanism and the
selectivity of the inhibitory site in sheep cerebeilar microsomes. InsP3 binding
was measured at equilibrium on a short period (3 s) using a perfusion protocol.
Microsomes adsorbed to a glass fiber filter were perfused with a binding
medium at 20 C, consisting of cytosolic like medium (pH 7.1) supplemented
with 0.5 nM [3H]InsP3 and various unlabelled InsP3 concentrations and adjusted
for free Ca2+ concentration.
We observed partial inhibition by Ca2 of [3H]InsP3 binding which could
be explained by the conversion of InsP3R1 to a state with a lower affinity for
InsP3 (Ks = 146 + 24 nM at pCa9 and 321 + 56 nM at pCa 5.3). Conversely,
increasing [3H]InsP3 concentration from 30 to 400 nM tripled the IC~0 value for
Ca2+ and reduced maximal inhibition by 63 %. This ability of each ligand to
reduce the affinity of the other is similar to the negative interactions between
InsP3 and cytosolic Ca2 previously described for the Ca2+-dependent inhibtion
oflnsP3R1 channel activity.
Mn2+and Sr2~ partially inhibited [3H]InsP3 binding to about the same
extent as Ca2 but their affinities were 10 and 200 times lower than that of Ca2,
respectively. No inhibition was observed with Ba2 up to 1 raM. The order of
potency for the inhibitory site (Ca2 > Mn2 > Sr2 >Ba2) was identical and the
relative affinities very similar to those reported for the site inhibiting InsP3R1
activity, suggesting that these sites are identical. This result and the similarity in
the mechanisms of the two inhibitory effects of Ca2indicate that the conversion
of the InsP~R1 to the lower affinity state by Ca2 is a process intimately
connected with the Ca2 inhibition of InsP3R1 channel activity.
(M0/14.1/091)

Hypo-osmotic shock stimulated K+(SrRb+) efflux in primary

cultures of seawater fish (Dicentrarchus iabrax) gill
' C. Duranton, E. Mikulovic, M. Avella, M. Tauc & P. Poujeol
U M R - C N R S 6548, Nice, France

Recent patch-clump studies performed on apical membranes of

seawater fish gills in primary culture have demonstrated the existence of
stretch-activated K+ channels having a conductance of 125 pS (1). The
present report examines the involvement of these K + channels in the
mechanisms of ionic transport and control of membrane potential. SrRb+ was
used as an isotopic tracer to indicate potassium movements on gill
respiratory-like cells in culture b~rowingon permeable supports.
Apical and basolateral ~Rb effluxes were stimulated by exposure to
hypotonic medium (30 % decrease of the osmotic pressure) and to
extracellular ATP (50 laM) and were insensitive to 8-br cAMP (1 raM).
During this hypotonic shock, the stimulation of SrRb efflux (764-20% ;
n= 15) on the apical side of the cell monolayer is inhibited by the addition of
500 [aM quinidine (20-~2% ; n=4) and of 100/aM gadolinium (423% ; n=4)
but remains insensitive to the addition of LQH venom. Similarly, the increase
of SrRb efflux on the basolateral side (28333%) is also reduced by the
addition of quinidine (433%) and of LQH venom (406% ; n=4) but is not
modified by exposure to gadolinium.
This kinetic study confirms the presence in cultured gill cells of a K+
conductance which has already been described and characterized by
electrophysiological techniques. There is convincing evidence for two
populations of K+ channels differing by their cellular localization (apical or
basolateral membrane) and by their functional and pharmacological
characteristics. The further step will be to understand how the functions of the
K+ conductance channels are integrated into the volume regulation
mechanisms of gill cells.
(1) Duranton C., Avella M., Tauc M. and Poujeol P. (1998). J. Physiol. Lond.
513P, 136P.

Differential regulation of two K + voltage-gated current

from Jurkat T cells by HIV-I gpl60.
O. DellisLb, F. Bouteaub, R. Lenaour', J.-P. Ronab, M. Guenounoua
Lab. d'lmmunologte, Univ. de Resins. F-51100 Relms.
b Lab. d'Electrophystologle des membranes, Univ. Par~s 7, F-75005.

During HIV-1 infection, there is an early impairment of T CD4 cell

functions, followed by a decrease of the T CD4 lymphocyte number, altough
only a small number of these cells appears to be productively infected. The
HIV-1 envelope giycoprotein gpl20/gpl60 is shed from infected cells, and
can cross-link the CD4 receptor of uninfected cells, leading to apoptosis,
anergy or decreased proliferative response after TCR stimulation. K+
voltage-gated ("Kv") currents control the membrane potential value and
were implied in the T CD4 lymphocyte activation process.
Our aim was to study the effect of gpl60 on Kv currents and
membrane potential.
For this study, we used Jurkat T cells and JCaMI.6 cells (p56 I~kdeficient), two CD4 + T cell lines.
We show that Jurkat and JCaM1.6 cells possess two type of Kv
current, one inhibited by charybdotoxin CKv1.3"), the other not ("Kv CTXinsensitive").
Jurkat cells show a Kv1.3 current intensity decrease after five days of
culture with gp 160. PKC inhibitor prevents gp160-induced current decrease.
PKC activators (TPA and PDBu) and protein phosphatases inhibitor
(okadaic acid) decrease Kv1.3 current intensity of control cells, by a manner
similar to gpl60. These results show that gp160 reduces the Kvl.3 current
by a PKC-dependent process. In contrast, the Kv CTX-insensitive current is
not modified by Jurkat cell incubation with gpl60.
Gpl60 has no effect on Kv currents of JCaM1.6. This shows that
gp 160 needs a fonctionnal p5@ k to decrease the Kv current
Jurkat cells are depolarized by gp 160-incubation.
In conclusion, we show that HIV-1 can induce membrane disorders
(Kvl.3 current decrease, cell depolarization) in addition to cytoplasmic
disorders (modified PKC activity2, altered Ca2- concentration variation). Cell
depolarization decrease the Ca + driving force. Thus, by decreasing the
Kvl.3 current, HIV-1 could disturb the entrance of Ca ~- during T CD4
lymphocyte activation.

(Mo/14.1/092)

Pore-forming properties and antibacterial activity of a

63 kDa protein from mucus of trout (Oncorhynchusm y k i s s )
N. Ebrana, S. Julien a, N. Orangea, B. Auprrinb and G. Mollea
a UMR 6522 - 1FRMp 23 - Universitd de Rouen, 76821 MS AJgnan, France
bINRA, Physiologm des pmssons, 35 042 Rennes, France

Among several biological functions, the epidermal mucus of fish may play an
important role in host defense, particularly in the prevention of colonization
by parasites, bacteria and fungi. After a first step of purification, watersoluble and hydrophobic material from lyophilized epidermal mucus, were
separated. When reconstituted in planar lipid bilayers, the hydrophobic
component induced a pore-forming activity, well correlated to a strong
antibacterial activity against several bacteria strains [1]. These results suggest
that fish mucus possess antibacterial proteins able to permeabilize
membranes of target cells and thus act as a defense barrier. A protein of 63
kDa was isolated from the hydrophobic part by SDS-PAGE and reconstituted
into planar lipid bilayers induced large ion channels. To test the potential role
of this protein in the defense process, the antibacterial activity of this 63 kDa
protein was determined against several bacteria (Gram + and Gram -).
Finally, secondary structure assessment using circular dichroism indicated
this protein is mainly composed of helix and unordered structures.

[ll N. EBRANet al. (1999). Comp. Biochem.

PhystoL

(In press)

s 190

(Mo/14.1/093)

Abstracts F E B S ' 9 9

The role of the N-terminal domain in channel-forming

properties of OprF from Pseudomonasfluorescens MF0
C. E1-Hamela, E. D6", E. Forestb, N. Orange= and G Molle"

(Mo/14.1/094)

~UIvIR6522, IFRMP23, FacultOdes Sciences, 76821Mt-St-Aignan, France.

nlBS. 41, ruedes Martyrs, 38027 Grenoble,France.

The psychrotrophic bacterium Pseudomonas fluorescens undergoes

modifications of the outer membrane permeability depending on the growth
temperature [1] We previously reported that the major outer membrane
protein OprF displays differences in channel-forming properties depending
on whether they were purified from cultures grown at 8C or 28C with
conductance values of 80 pS to 250 pS, in 1M NaC1 respectively [2]. It is
suggested the pore structure of these OprFs may vary with growth
temperature. In an attempt to explain this phenomenon, we purified the OprF
from 8C and 28C cultures and compared their structure by mass
spectrometry, circular dichroism analysis and measurement of pronase
digestion kinetics. If the primary and secondary structures were similar, the
difference of kinetic constants indicated a modification of the protein
folding Resulting proteolytic fragments of 18 kDa, resistant to further
pronase digestion, were obtained from 8C and 28C OprF forms and
identified as N-terminal half of native protein (177 amino-acids). Both
fragments (OprF177) form ion channels with similar conductance values
about 65-75 pS in 1M NaCI. This result shows that the C-terminal domain
seems to confer the ability to modulate the P. fluorescens OprFs ionophore
activity with varying growth temperatures.

[1] Orange, N (1994)Microbiol. 140, 3125-3130.

[2] De, E. etaL (1997)Microbiol. 143, 1029-1035.

(Mo/14.1/095)

Yeast Geflp Golgi chloride channel is functionally

complemented by Arabidopsis thaliana AtCLC-d
M. Hechenberger,B Schwappach,S. Stobrawa,WN. Fischer,WB,
Frommer,TJ. Jentsch and K. Steinmeyer
ZMNH, HamburgUniversity,Martinistr. 85, D-20246 Hamburg

Effects of Nucleotide Triphosphates on the

Sarcoplasmic Reticulum Anion Channel
S. Frankenbach, A. Schuster, H.G. Baumert
]nst~tutfur Biochemie, .Z I~ Goethe-Un~versttttt,
Marie Curie-Str. 9, 60439 Frankfurt/M., Germany

Muscle contraction and relaxation is triggered and accompanied by large

amounts of Ca 2~ crossing the sarcoplasmic reticulum (SR) membrane. To
prevent osmotic effects and long-term membrane potentials this current is
shunted by the movement of ions through CI-- (and K'-) channels. Thus
chloride plays an important role in charge balancing during Ca 2+accumulation and Ca~+-release.
We investigated possible regulation mechanisms by studying the effects of
various nucleotide triphosphates, their analogues, and several inhibitors on
35SO42"-effiux from preloaded SR-vesicles and by single-channel measurement. Anion efflux is inhibited with varying efficiency by all NTPs applied.
The presence of mM Mg z+ leads to drastic changes in rate constants. The
effect of purine and pyrimidine nucleotides is comparable, and even
polyphosphates and derivatives exhibit similar effects. Application of a series
of synthetic inhibitors leads to inhibition stronger than, comparable to, or
weaker than that of the universally used anion transport inhibitor DIDS
For the detection and identification of the SR anion channel protein(s) we
investigated radiolabelling by reactive NTP-derivatives and VDAC/porinspecific monoclonal antibodies.
Isolation of the voltage-dependent anion channel (VDAC) was attempted by
affinity-purification of solubilized SR proteins with an amine-linked 4acetamino-4"isothiocyanato-stilbene-2 2-disulfonic acid (SITS) sepharose
4B column and elution with 50 mM KCL

(Mo/14.1/096)

Relationships between Na+/K + pumps activity and

2ITIuptake during skeletal muscle contraction
N.Khider a, C.Nemoz b, B. Issauratb ,M. Fanchet b, D. Garnet a
~UMR CNRS 6600, Univ. Tech Compi~gne, 60200 Compibgne. France
bCentre d'lma.~erie M~dicale Avanc~ , 60200 Compi~gne, France

The CLC-famlly of chloride channels is an evolutionarily old family with

members from eubacteria, archaebacteria, yeast, plants and mammals. By
cloning four novel CLC-genes (AtCLC-a to AtCLC-d) we have extended
this family to the plant model organism Arabidopsis thaliana [1].
All four AtCLC-genes were expressed in every tissue examined. These
results, obtained by RT-PCR, were confirmed by Western blot with an
antiserum specific for AtCLC-d. Heterologously expressed AtCLCproteins can be detected in Xenopus oocytes, however we could not
detect currents induced by these proteins either by single or pairwise
expression.
We used the yeast Saccharomyces cerevisiae to functionally characterize
the AtCLC-proteins. S. cerevisiae contains a single CLC-gene named
GEF-I. Growth of mutants in this gene on nonfermentable carbon
sources are iron limited (Glycerol/Ethanol Fe-requiring). By expression
of AtCLC-d in a gef-1 strain we were able to fully restore the ability to
grow on iron limited plates. A C-terminal GFP-fusion protein of AtCLCd shows in the gef-1 strain the same dot-like staining as Geflp. The other
AtCLC-proteins are localized to a different compartment, presumably the
ER.
In costaining experiments the dot-like Geflp staining overlaps with the
Mntlp staining from the medial, but also with marker enzymes from the
early and late Golgi [2]. gef-1 strains showed a reduced growth on plates
with neutral pH. This may suggest a similar role as found with hCLC-5 in
assisting a H+-ATPase in acidifying intracellular compartments [3].

Thallium-201 (21T1) is a radiopharmaceutic product used in nuclear

medicine, especially in the cardiac function testing and less in other domains
as testing of muscular function. In its hydrated state, 2~Tl is considered as a
biologic analogue of potassium ion (K), because its ionic radiuses are
equivalent. During a sustained muscle contraction, changes in K + gradient are
due to K effiux from the intracellular compartment. In these conditions
Na/K+ pump activity increases, in order to restore the K + gradient [ 1,2].
The purpose of this study is the analyse of the activity of Na/K* pumps by
following 2IT1 radioactivity during hindlimb muscle contraction recorded on
sixteen anaesthetised rats.
The muscle activity was evoked by supramaximal electrical stimuli directly
applied on the right sciatic nerve at frequencies of 2, 5 and I0 Ha. After 15
rain of stimulation, lmCi in lml 2~T1solution was injected via penis vein.
Cardiac and muscular radioactivity areas were determined on scintigraphic
images, obtained every minute during 30 rain. The initial value of 2~Tl
uptake was defined as the uptake level in the cardiac and muscular areas at
the end of the first minute following injection. During image recording, a
decrease in radioactivity in the myocardic area, which is classically reported
[3], was observed. Muscular radioactivity levels of stimulated leg (sl) and non
stimulated leg (nsl) tended to stabilised, sl/nsl ratio at stabilisation were
computed and showed a curvilinear increase with stimulation frequency. This
relative increasing in 2roT1 uptake is interpretable in terms of increase in
Na+/K+ pump activities.
This investigation by 2IT1 uptake will be used to attest changes in Na+/K+
pump activity and density as a result of modifications in muscle functional
demand.

[1] Hechenberger etal. (1996) J. Biol. Chem. 271, 33632-33638

[2] Schwappach et al. (1998) J. Biol. Chem. 273, 15110-15118
[3] Giinther et al. (1998) Proc. Natl. Acad. Sci. U S A. 95, 8075-8080

[1] Nagaoka R. etal., Comp. Biochem. Physiol. 109A, (1994), 957.

[2] Clausen T. et al., Acta Physiol. Scand. 152, (1994), 365.
[3] Llaurado J.G. etal., Eur. J. Nucl. Med. 5, (1980), 217.

Abstracts FEBS'99

(Mo/14.11097)

s191

A new Arabidopsis thaliana Shaker-like K channel that

probably contributes to stomatai movements.

(Mo/14.1/098)

B. Lacombe, G. Pilot, F. Gaymard, 1. Ch6rel, H. Sentenac, J.-B. Thlbaud

Bioch. & Physiol. Mol. Plantes, UM2/INRA/ENSA-M/CNRS URA 2133,
place Viala, 34060 Montpellier cedex 1, France

In plant cells, vohage-gated non-inactivating Shaker-like K channels, which

rectify either inwardly (e.g. AKT1 and KAT1) or outwardly (SKOR) [1], are
involved in sustained K transports. These types of transporters, and their
controls, have been most extensively investigated in guard cells. Rapid
influx/efflux of K + ions into/from these cells through inward/outward K +
channels result in turgor changes that trigger stomatal opening/closing,
controlling the rate of gaz exchanges (CO2 fixation, H20 loss) between the
photosynthetic tissues and the atmosphere. Based on expression data [2], it is
commonly thought that KAT1 channel activity is responsible for K + influx in
guard cell in the plant model Arabidopsis. However, whereas the Cs+-block
of the Arabidopsis guard cell inward K + current is essentially voltageindependent, that of KAT1 (expressed in xenopus oocytes) is clearly voltagedependent [3]. We have cloned a new Arabidopsis cDNA, which shares
strong homology with KAT1, and have named it KAT2. KAT2 expression
(GUS reporter gene in transgenic plants) is Iocalised in guard cells (and in
other tissues of the leaf). Functional properties of KAT2 (in xenopus oocytes)
are similar to that of KATI (inward rectification, voltage-gating, high K +selectivity) but the Cs+-block of KAT2 channel is voltage-independent like
that observed m Arabidopsis guard cell. Surprisingly, mutational analysis
revealed that the difference in Cs + sensitivity between KAT1 and KAT2 is
not due to the single amino acid exchange in the pore domain between the 2
channels, indicating that this P-domain is not the only determinant of Cs +sensitivity. Co-expression of the two channels in xenopus oocytes and two
hybrid experiments in yeast indicated that KAT2 is able to co-assemble with
KATI, the cytoplasmic C-terminus domain of the channels being involved in
the interaction. These findings indicate that in vivo hetero-multimerisation
between different cx-subunit can occur in plant like in animals. In conclusion,
KAT2 gene product may significantly contribute to K + uptake by guard celts,
by forming either homo- or hetero-tetrameric channels with KAT1.
Regulation of KAT2 expression by plant hormones is under investigation.
[1] Gaymard et al., Cell, 94, (1998) 647
[2] Nakamura et al., Plant Physiol, 109, (1995) 371
[3] Ichida et al., Plant Cell, 9, (1997) 1343

(Mo/14.1/099)

Activation of Na channels by ciguatoxin-lB produces a

Iocahzed increase of mtracellular Ca m chromaffin cells
C. Mattei~, E. Benoit ~, F. Darchen b, A.M. Legrand*, J. Molgo ~

2 + .

~Neurobtologie Cell. et MoL, CNRS UPR 9040, Gtf-sur-Yvette, F~ance.

I~Instttut de Btologte Ptwsico-Chimtque, CNRS ERS 575, Parts, F)'ance,

Ciguatoxin-lB (CTX-IB) is a liposoluble cyclic polyether involved in a

human seafood poisoning, named ciguatera, caused by the consumption of
poisonous coral reef fish. This toxin, which binds to the receptor-site 5 of
neuronal voltage-dependent Na t channels, produces a permanent opening of
these channels at physiological potentials. CTX-1B has been reported to
increase spontaneous release of acetylcholine at the flog neuromuscular
junction [1], that could result from Ca :~ mobilization from intraeellular
stores. Indeed, CTX-IB causes an increase in intracellular Ca 2" concentration
in rat neuroblastoma cells bathed in a Ca2+-free medium. In the present study,
we have characterized the action of CTX-1B on intracellular Ca ;* of bovine
chromaffin cells loaded with the Ca 2~ indicator fluo-3/AM, using confocal
laser scanning microscopy. We observed that CTX-IB (10-30 nM) produces a
transient and localized Ca 2' increase either in a standard external medium or
in a Ca2*-free medium supplemented with the Ca2+-chelator EGTA In the
presence of external Ca 2+, such CTX-1B-induced Ca 2+ increase was still
detected when chromaffin cells were pre-treated with dimethyl-benzamide, a
Na~-Ca2+ exchange blocker. However, no Ca2- increase occurred during
CTX-1B action when external Na + was replaced by lithium or when
chromaffln cells were pre-treated with the Na ~ channel blocker tetrodotoxin.
The present results suggest that CTX-1B, by activating Na + channels of
chromaffin cells, produces an increase in intracellular Na + which in turn
evokes a localized elevation of intracellular Ca 2+ In Ca2--free medium, this
increase results from Ca 2+ mobilization from intracellular stores. In the
presence &external Ca 2~, Ca 2+ entry also contributes to the intracellular Ca 2~
increase Finally, the fact that no intracellular Ca 2~ increase is detected when
external Na + is replaced by lithium, suggests that CTX-1B-modified Na +
channels are not permeant to lithium or that this cation is unable to mobilize
intracellular Ca :~ More experiments are necessary to precise the effects of
CTX-1B on intracellular Na and on catecholamines secretion in chromaffin
cells.
[1] Molgo et al, Br J Pharmacol, 99, (1990) 695
* Present address, Oceanographic Aled, Inst. Malardd, Papeete, Tahm, Po(vnesle Fean~atse

Identification of a region important for the ion selectivity

of Acid-Sensing Ion Channels (ASICs)
S. Coscoy, J. R. de Weille, E. Lingueglia* and M. Lazdunski
lnstitut de Pharraacologie MoldculaJre et Cellulaire (CNRS-UPR 4l 1),
660 route des Lucioles, 06560 Valbonne, France. * presenting author

ASICs are proton-gated channels expressed in the central nervous

system and in sensory neurons where they are thought to play an
important role in pain accompanying tissue acidosis. They belong to
the superfamily of amiloride-sensiuve Na + channels and degenerins
(NaC/DEG). The two splice variants ASIC2 and ASIC2b differ only
in the first 185 N-terminal amino acids of ASIC2 but display very
different properties. In particular, ASIC2b is not active on its own
but can modulate the properties of other ASICs. In sensory cells, it
associates with ASIC3 and confers a nonselective character to its
sustained component. Chimeras between ASIC2 and ASIC2b
allowed identification of a 9-residue region preceding the first
transmembrane domain that participates in the ion selectivity and the
pH dependence of ASIC2. The role of three amino acids in this
region (Ile-19, Phe-20 and Thr-25) appears particularly important
since their mutations cause a 3- to 7-fold reduction in the Na+/K +
permeability ratio. A corresponding mutation in ASIC3 (T26K)
induces similar changes in ion selectivity and suggest that the preTM1 domain of ASIC2b may be one of the structural elements
responsible for the loss of Na selectivity of the ASIC3/ASIC2b
sustained component. These results indicate the important role of the
region preceding the first transmembrane domain in the ion pore
formation of ASIC channels.

(Mo/14.1/lO0)

Potassium channels in rat prostate epithelial cells

H. Ouadid-Ahidoucha, F Van Coppenolle ~, X. Le Bourhls b,
N. Prevarskayaa ,

~Lab. Physto. Cellulaire et bLab. Btologte de d~veloppement,

USTL, 59655, Vtlleneuve d'Ascq ('edex, 1+ante

These experiments were conducted to determme the membrane K +

currents and channels in rat prostate cells in vitro. Keratin-positive
epithelial cells were dissoctated by collagenase and hyaluromdase from
adult rat prostates. K + currents were assessed by the whole-cell patch-clamp
technique. A voltage-dependent, inactivating K + channel was the most
commonly observed ion channel both in lateral and dorsal cells. The K +
current exhibited a voltage threshold at -40 mV Averaged half-mactwation
potential (Vt:) and the slope factor (k) values were -26 mV and 6
respecttvely. It showed a monoexponential decay with an mactwatton time
course of about 600 ms at +60 inV. The deactwaUon time constant at -60
mV was 30 ms and the potentml d'invers~on was estimated at -80 mV. The
channel exhibited cumulative inactivation and was reduced by increasing
the cytoplasmic Ca 2+ concentration. The Scorpion venom pepttdes,
charybdotoxin (5 riM) and Margatoxm (1 nM) inhibited K + channel activity
at all membrane potentials wtth a rapid and a slow reversibility respectively.
Moreover, tetraethylammonium (10 mM) reduced the K* channel activity
by 40 % only
We conclude that plasma membranes of lateral and dorsal rat prostate
epithelial cells contain the Kv K channels with biophysical and
pharmacological properties consistent with a Kv 1.3 family

s192

(Mo/14.1/101)

Abstracts FEBS'99

The S4 segments of Shaker potassium channel rotate and

move towards each other during channel activation

(Mo/14.1/102)

DSCI is the major sodium channel expressed in the

peripheral nervous system of adult Drosophila

Chris J. Partridge, Qadeer Aziz and A. Sivaprasadarao

D Pauron, C. Castella, M. Amichot, J.-B Berge

School of Biochemtcal Sciences, Leeds University, Leeds LS2 9JT, U.K.

1.N R.A., UX VE., B.P 2078, 06606Antibes cedex, France

DSC1 is one of the two genes encoding a voltage-sensitive sodium
channel o~-subunit cloned in Drosophila melanogaster so far. Its low

Voltage-gated K channels have the remarkable ability to sense changes

in potential difference across the cell membrane and transmit the signal to
the pore region; the pore opens when the membrane is depolarised and
closes when hyperpolarised. The fourth transmembrane segment, $4,
which contains the positively charged arginine or lysine residues is
primarily responsible for voltage sensing. Recent studies have shown that
when the membrane potential is depolarised, the N-terminal end of the
segment physically moves out of the lipid bilayer into the extracellular
phase by about six residues. In an attempt to identify the accompanying
changes that lead to channel opening, we have introduced cysteines, one
at time, at various positions of $4 of the Drosophila Shaker potassium
channel and expressed the mutants in Xenopus oocytes. We measured K
currents elicited by these mutants by two-electrode voltage clamp and
studied the effect of disulphide inducing agents, iodine and copper
phenanthroline on channel currents. The amplitudes of the mutant, but
not the wild-type channel, currents were inhibited by the reagents,
suggesting that the engineered cysteines form disulphides. By
systematically knocking out other native cysteines in the channel, we
show that cysteines from $4 segments of neighbouring channel subunits
of the tetramer rotate and move towards each other when the membrane
is depolarised. This is the first report to demonstrate that rotation and
lateral movements of voltage sensing transmembrane segments underlie
channel activation.

level of homology with other sodium channel genes identified in

insects as well as in other organisms and the lack of any related mutant
make this gene a particular case in the sodium channels family Some
clues about its function were brought by Hong and Ganetzky [1] who
showed that the pattern of expression of DSC1 is similar but not
identical to the one of para, the other Drosophila sodium channel
gene. Our works brings new insights in the characterization of the
expression product of DSC1 by analyzing the distribution of this
channel protein. The polyclonal antibodies used throughout this study
were designed using the pEX fusion protein technique. These
antibodies were used in immunoblotting experiments where a 270 kDa
protein has been identified in adult flyheads membrane preparation.
The pattern of regional localization of the protein was also investigated on cryosections performed on adult flies. DSC1 is expressed in
both the central and the peripheral nervous systems In the brain,
DSC1 is widespread in several parts of the neuropil and in the main
tracts which connect various nervous centres. A weak signal is also
observed in the compound eyes. Several structures of the PNS are also
strongly labelled with the antibody such as the antennal nerve, leg
nerVes and the innervative system of the dorsal median muscles in the
thorax. None of these structures were recognized by anti-para antibodies. Results obtained both on wild-type strains and on strains where
the sodium channel functionality is altered are presented
[1] Hong, C -S and Ganetzky, B., J. Neurosci., 14 (1994) 5160.

(Mo/14.1/103)

I d e n t i f i c a t i o n o f a m i n o acids c o n t r i b u t i n g to the
p o r e o f the F M R F - a c t i v a t e d s o d i u m c h a n n e l

M. Poet,M. Tauc,E Lingueglia*,P. Polar, M La:~nsla*andL COunllloa

UMR CNRS 6548, UNSA. Part" Valrose, 06108 Nice, France, and
*UPR-CNRS 41l, 660 route des lucioles, 06560 Valbonne, France

The cation channel FaNach, a member of the ENach/degenerin gene

family, is a homotetrameric membrane protein activated by the
cardioexitatory peptide FMRF-amide. Each subunit possesses a simple
topological organization, consisting in two transmembrane segments
connected by a large extmcellular loop.
To gain access into the structure of the ionic pore of this channel, we
used the following strategy :
- introduction of individual cysteines at various positions of the
transmembrane segments;
- test of the surface accessibility of these cysteines, using water-soluble
thiol specific reagents (MTS), the covalent reaction of MTS with an
accessible cystein resulting in the inhibition of the channel activity.
The wild-type and the mutated channels were transiently transfected
into HEK 293 cells, and the response to FMRF amide was measured using
the whole cell clamp technique. Following the characterization of the FMRF
amide-evoked currents, MTS reagents were applied to the cells, and the
changes in the FMRF-amide evoked currents were monitored. Under these
conditions, MTS application did not modify the electrophysiological
properties of the wild-type channel.
Nine amino acids of the first hydrophobic segment were individually
mutated to cysteines and tested with MTS. Three classes of mutants were
generated.
Mutants in which the cysteine substitution leads to a loss of function of the
channel; mutants in which extmceUular application of MTS inhibits currents
evoked by FMRF-amide; and mutants possessing the same
electrophysiological properties than the wild type channel, with no effect of
the thiol reagents.
The pattern of accessibility of the mutants generated in the first
transmembrane segment will be presented and discussed.

(M0/14.1/104)

CI- conductances in primary cultures of renal tubules

from CFTR knock-out mice.

P. Poujeol, H. Barri~re, I. Rubera, C. Pou]eol, L. Hass6ine,

B Curlier and M. Tauc
U~,[R-CNRS6548, Umversit~de Nice,06108 Nice, France

The aim of this work was to study the implication of CFTR expression
in the Cl- handling in mouse renal epithelium. For this purpose we performed
primary cultures of proximal convoluted (PCT), distal convoluted (DCT) and
cortical collecting (CCT) tubules from wild type (cftr +/+) and CFTR knockout (cftr -/-) mice. Tubules were microdissected from collagenase treated
kidney, seeded on collagen coated Petri dish and grown in hormonally defined
medium supplemented with 1% SVF. CI" conductances were analyzed by the
whole cell clamp technique with 140 mM NMDGC1 in the pipette and in the
bath.
In cultured DCT and CCT from cftr +/+ mice, forskolin (10 ttM)
activated a conductance with a linear I/V relationship.This conductance was
inhibited by I", insensitive to DtDS (1 raM) and shared the characteristics of
the CFTR-CI" current. Occasionally, forskolin induced a very weak linear
current in cultured PCT from ct~r +/+ mice. On the contrary, the addition of
forskolin did not elicited significant currents in PCT, DCT and CCT cell
cultures originating from cftr -/- mice. In all cultures obtained from cfir +/+
and -/- mice, ionomycin stimulated outwardly-rectifying CI" currents in the
presence of 100 nM free calcium in the pipette solution. These currents
increased during depolarized voltage pulse and exhibited the kinetic
characteristics of the Ca2+- activated CI" conductances found in other epithelia.
A third C1- conductance was identified in PCT, DCT and CCT cultures from
cftr +/+ and cftr -/- mice. It was activated by a hypo-osmotic shock. These
large, outwardly-rectifying currents showed time-dependent inactivation at
depolarizing potentials.
In conclusion, 1) the inactivation of the gene encoding CFTR induced
the disappearance ofa forskolin-activated CI" conductance localized mainly in
DCT and CCT tubules. 2) the genie inactivation of CFTR did not modify
significantly Ca ++and volume- activated CI" conductances.

Abstracts F E B S ' 9 9

(Ma/14.1/105)

s193

Na regulates Ca 2+ entry in glioma C6 cells.

K Przybylek, J. Barafiska

(Mo/14.1/106)

Nencki Institute of Experimental Biology 3 Pasteur St,

02-093 Warsa*~; Poland

D~partement de Btologle ('elhdatre et Motdclduwe / SBCe

CEA Saclco'. Bdt. 532. 91191 Gtf-sur-Yvette Cedex, France

We have previously shown that glioma C6 cells belong to the

type of nonexcitable cells and demonstrated that ATP and thapsigargin
m~txated Ca response consLstent wath the capacttauve model of Ca
entry. This was specifically shown in a rise in response to ATP and
thapsigargin after addition of Ca 2 to the Ca 2. free medium
(Neurochem. Int. 31, 55, 1997). The same effect we observed
presently when the cells were perfused in Ca 2+ free buffer solution
(with 500 jaM EGTA) for 30 min and then reperfused with buffer
containing 1.5 mM [Ca2+]. This effect was studied with Fura-2 video
imaging technique After such slow depletion of the pools, addition of
Ca z+ to gliema C6 cells resulted in a dramatic elevation in intracellular
Ca 2 (from 50 nM to 700 nM). We found that this effect was strongly
prevented when the perfusion, reperfusion, or both of them, were
performed with a Na + free solution (sodium-free solutions were made
with equimolar substitution of choline chloride for sodium chloride)
However, when such cells were treated with thapsigargin (100 nM)
and then re-exposed to the control buffer solution containing Ca 2,
there was a rapid rise in [Ca2], (up to 700 nM). These findings indicate
that the absence of external Na diminishes the release of Ca ~* from the
endoplasmic reticulum stores and suggest that the capacitative Ca 2+
entry may be regulated by external Na most probably by modulating
Ca 2 content of intracellular stores.

Aquaporin selectivity by chimera

expression in Xenopns oocyte
N. Roud~er. J. M4rot. A. Bazureau, MB. Barrault, P. Ripoche

2+

Aquaporms are members of the famdy of major intrinsic proteins (MIP) that
transport water. Some of them are selective for water (AQP1, AQP2...) but others, as
AQP3 and AQP7, also transport small solutes such as glycerol and urea AQPI has
been shown to form a tetramer even if monomers alone are able to transport water.
The pore for water is formed reside the monomers, but we don't know ff water and
solutes are sharing the same pore or a distract one. In order to characterize the
segments involved m the solute permeability, we used chimeras of aquaponns
presenting differences in their selectivity. Their function was tested in Xenopus
oocyte. Chimeras whom hydrophlhc C-terminal sequences were exchanged between
AQP2 and AQP3 (AQP2-CT3 and AQP3-CT2), had a lower water permeabihty than
wild aquaporms (PfAQP2 = 9.4 l0 -3 + 1.1 10-3 crd/s, PfAQP2-CT3 ~ 37 %
PfAQP2, PfAQP3 = 9.9 10-3 _+ 0.9 l0 "3 crrds, PfAQP3-CT2 -= 15 % PfAQP3).
Moreover, AQP3-CT2 chimera showed 69 % of AQP3 glycerol permeabihty. These
results suggest that the C-terminal could take part in the formation of the water pore
but would not be essenttal for the glycerol pore. Chimeras with exchanges from the
second NPA box (AQP2-NPA'3 et AQP3-NPA'2) showed absent or reduced water
permeabihty (PfAQP2-NPA'3 = 1 % PfAQP2, PfAQP3-NPA'2 = 30 % PfAQP3).
Glycerol permeabihty of AQP3-NPA'2 yielded only 7 % of that of AQP3. Part of E
loop and/or the last transmembrane segment could be involved m glycerol transport.
Expression of chimeric aquaporms m oocytes was tested by western-blot on total
membranes and by lmmunofluorescence for some of them.

We analysed the results according to the hourglass model, proposed by Jung et al. in
1991 (represented above) in which B and E loops fold back into the membrane and
together form the water pore. In this hypothesis, chimeras hawng heterologous B and
E loops could constitute an inefficient pore, due to the low sequence identity of these
two loops m AQP2 and AQP3 The construction of AQP2 with loops B and/or E
from AQP3 and of AQP3 with loops B and/or E from AQP2 are now m progress.

(Mo/14.1/107)

Chloride conductance activated by extraeellular ATP

in immortalized rabbit distal tubule cells in culture.
I. Rubera, M. Bidet, G. De Renzls, M. Tauc,
C. Verheecke-Mauze and P. Poujeol.
UMR-CNRS 6548, Universtt~ de Nwe-Sophla Antspohs. Nwe. Franc~

We characterized ATP-activated chloride conductance in an

immortalized cell line derived from rabbit distal bright convoluted tubule.
Using the whole cell patch-clamp technique, we showed that ATP (100 jaM)
activated outwardly rectifying chloride currents which exhibited delay
activation at depolarizing voltages even in the presence of DPCPX excluding
the involvement of P I receptors. 125 I- efflux experiments performed on cells
grown on Petri dishes showed that ATP increased ,25I" loss in a concentrationdependent manner (ECso=31aM). ATPyS (10 jaM) or UTP (50 ktM) also
activated a 125I-effiux. Suramin (500 jaM) was found to inhibit ATP-induced
~2sI- effiux consistent with the existence of P2 purinoceptors on the cells.
Chloride channels inhibitors, DPC (10"JM) or NPPB (10-4M) abolished the
~25I" effiux. The ATP-evoked increase in 12sI effiux was abolished in
thapsigargin-loaded cells suggesting that this response is dependent upon the
intracellular Ca 2+ concentration, [Ca 2+ ],. 125 I- efflux from cells grown on
permeable filters suggest that ATP induced an apical effiux mediated through
apical P2 receptors. In another series of experiments, we investigated the
effects of extracellular ATP on [Ca2+], by a fura-2 microfluorescence
technique. We showed that ATP and various ATP derivatives raised [Ca2+][ in
distal tubule cells. The rank order of potency of various agonists on [Ca "~
was ATP=UTP>ATP~S>>ADP>UDP consistent with the pharmacology of
the P2Y2 purinoceptor subtype The [Ca~+], responses to ATP were strongly
inhibited by suramin, and blocked by U73122, a phospholipase C inhibitor.
These results demonstrate the presence of a purinergic regulatory mechanism
involving ATP, P2Y2 receptors and Ca 2+ mobilization for chloride
conductance stimulation in distal tubule cell line.

(Mo/14.1/108)

Thesigmaligand( + ~ e
O. ~ ,

depressesa M-cnrrentm mdanoU'ophs

F. Le Follb,L. GalaS,F. Rcra,~, H. V ~ ,

L Gaziff

'qnst. Recherche Jouveinal/Parke-Davis, 3-9 rue de la Loge, 94265 Fresnes,

France, blab d'Ecotoxicolog~e, Umv. du Havre, 76058 Le Havre, France,
ClNSERM U 413, IFRMP 23. Univ. Rouen, 76821 Mt-St-Aignan, France

Sigma receptors are widely represented in nervous, immune and endocrine

systems. However, the functions and the transduction pathways associated
to sigma receptors still remain mysterious. In previous studies, we have
demonstrated that sigma receptors stimulate pituitary cell electrical activity
through a Gs-dependent reduction of both the delayed rectifier and the
transient outward potassium conductances [1,2]. We have also shown that
sigma ligands reduce a tonic outward K + current [1]. Herein, gramicidinperforated patch clamp experiments were performed in primary cultured
frog (Rana ridibunda) melanotrophs to study this latter conductance. In the
presence of calcium and sodium channel blockers, the sigma ligand
(+)-pentazoeine (50 p_M) depressed a sustained outward K current. The
kinetic properties of this K + component investigated by analyzing tail
currents were reminiscent of those of M-current (IM) with an activation
threshold close to -60 mV, a -21-mV half-maximal activation potential and
a two-component exponential deactivation kineties at -90 inV. (+)pentazocine (20 ~M) produced a 12-mV rightward shift of the activation
curve and accelerated the deactivation rate of the tail current. Altogether
these results demonstrate for the first time the existence of the IM current in
melanotrope cells. The inhibition of this current by (+)-pentazocine explains
the depolarizing effect of sigma ligands occurring in melanotrope cells.
[1] : Soriani et al., J Pharmacol. Exp. Ther. 286, (1998), 163.
121 : Sonani et al., J. Pharmacol. Exp. Ther. (in press).

s194

(M0/14.1/109)

Abstracts FEBS'99

Control mechanisms of airway transepithelial

t r a n s p o r t for sodium and chloride ions
T. Tyrakowski, B. Banach, M. Bartiomowicz, I.
Greczko, A. $ediaczek, M. Wojciechowska
Pomeranian Nedicai University, Szczecin, Poland

Transient opening of ionic channels after mechanical or

chemical stimulation of airways epithelial surface was evidenced
as hyperpoiarization. The role of "sensory" neuropeptides
(SP, NKA, GGRR) from C-fiber ending in this reaction was
indirectly evidenced (T. Tyrakowski et al., FEBS'98 Abstract,
p. 106).
This s t u d y was performed to show how the transepitheliai ion
t r a n s p o r t pathways for sodium and chloride ion are
interdependent.
The macroscopic transepithelial ionic c u r r e n t s of isolated
rabbit tracheal wall were measured in Ussing a p p a r a t u s as
electrical potential difference changes (dPD) after
stimulation.
The modification of the reaction was accomplished by the
addition to the bathing fluid following s u b s t a n c e s (in
parentheses concentrations in micromoles are given): amiloride
( 100 ), bumetanide ( 100), t r a n s - 4 - ( 2-amino-3,5
dibromobenzyl)-aminocyclohexanol hydrochlorlde (ambroxol,
5), atropine sulfate ( 10), benextramine tetrahydrochloride
( 10), hexamethonium dichloride ( 10), nicotine d i - d - t a r t a r a t e
( l ) . S(-)-timolol maleate (10).
It is possible to open or close different ionic channel
populations (to change transiently the component ionic
c u r r e n t s ) by influence on the extracellular control system.
The overall control system for transepithelial t r a n s p o r t seems
to optimize the transepithelial potential difference
hyperpolarization, so sodium ion and chloride channels open or
close synergically during stimulation, but when one of those
transepithelial pathways is inhibited the other is activated to
compensate for the resulting diminution of hyperpolarlzation.

Calcium permeability activated by nifedipine in newborn

rat renal distal tubules in primary culture.
L. Valencia b, M. Bideta, J.D. Troadec ~, F. Lafuente a, C.
Poujeol a, E Sanchez~', M. Tauc", J.L. Reyesb and P. PoujeoP

(Mo/14.1/110)

CNRS UMR6548 06108 Ntce, France. bCINVESTA~ IPN, Mextco.

At early stages of life, high amounts of calcium are required for the
rapid growth. This calcium may result from kidney reabsorption particularly
in the distal portion of the nephron where a hormonal regulation occurs.
Segments of the rat proximal and distal tubules were microdissected from 7
day-old Wistar rat. Proximal convoluted tubule (PCT) and cortical
collecting duct (CCD) cells were grown on collagen coated Petri dishes in a
hormonally defined medium. The intracellular calcium concentration was
measured in 3- to 5-day-old CCD or PCT monolayers using fura2/AM and
fluorescence video microscopy technique. CCD cells showed an increase in
[Ca], in response to AVP. This effect, specific for CCD cells, indicated that
these cultured cells retained same physiological properties of the original
tissue
To characterize the AVP reduced [Ca ], increase we used mfedipme, that m
adult rat &stal tubules inhibits the [Ca ], increase secondary to a hormonal
stimulus. Surprisingly we found that nifedipine (20~tM) produced an
increase in [Ca2+], from 87.54.0 nM to 402.828.5 nM in 65% of the CCD
cells. Similar effect was observed with other dihydropyridines (BayK 8644,
isradipme). Verapamil did not induce [Ca2+], rise. Experiments in presence
of EGTA showed that external calcium required for nifedipine effect.
Dihydropyridines did not induce any [Ca2], increase in cultures obtained
from PCT. In CCD cells the nifedipine effect was observed more frequently
in newborn than in adult rat Electrophysiological experiments performed by
the whole cell clamp technique showed an inwardly directed calcium current
activated by 201aM nifedipine. Unitary channel clamp technique also
showed an important exit of K* after nifedipine addition. In Fura2
experiments, clamping the membrane potential with valinomycin did not
modify the increase m [Ca ]i reduced by mfedipme.
We conclude that newborn rat CCD cells in primary culture exhibit a
nifedipine-activated calcium permeability. Experiments are now undertaken
to precise the nature of this permeability (i.e. calcium leak channels).

2 +

2+

2+

Abstracts FEBS'99

s195

18.1 Molecular mechanisms of cell adhesion

(Mo/18.1/lll)

Cyclophilin B Binding to Platelets Supports CalciumDependent Adhesion to Collagen

F. Allain, S. Durieux, A. Denys, M. Carpentier, G. Spik

(Mo/18.1/112)

UMR nl l l du CNRS, UST Lille. 59655 gdleneuve d'Ascq. France

We have recently reported that cyclophilin B (CyPB), a secreted

cyclosporin-binding protein, could bind to T lymphocytes through
interactions with two types of binding sites. The first ones, referred to
type I, involve interactions with the conserved domain of CyPB and
promote the endocytosis of surface-bound ligand, while the second type
of binding sites, termed type II, are represented by glycosaminoglycans.
Here, we further investigated the interactions of CyPB with blood cell
populations. In addition to lymphocytes, CyPB was found to interact
mainly with platelets. The binding is specific, with a Kd of 9 + 3 nM
and the number of sites estimated at 960 + 60 per cell. Platelet
glycosaminoglycans are not required for the interactions but the binding
is dramatically reduced by active cyclosporin derivatives. We then
analyzed the biological effects of CyPB and found a significant increase
in platelet adhesion to collagen. Concurrently, CyPB initiates a
transmembraneous influx of Ca 2+ and induces the phosphorylation of
the P-20 light chains of myosin. Taken together, the present results
demonstrate for the first time that extracellular CyPB specifically
interacts with platelets through a functional receptor related to the
lymphocyte type I binding sites and might act by regulating the activity
of a receptor-operated membrane Ca 2" channel.

(Mo/18.1/113)

A minimized integrin 0t5~ I that retains ligand recognition

L-Louis Ban~res a, F. Roquet b and J. Parello a
aESA CNRS 5074 and b UMR 9921 CNRS, FacultOde Pharmacie. 15 Av F]ahault.
34060 Montpellier COdex02. France

We have produced two recombinant fragments from human integrin w5[3I

encompassing the III to VII N-terminal repeats from ct5, i.e. ~-(190-476),
and the I-type domain from 131, i.e. 131-(121-329). Both fragments display a
well-defined structural organization as assessed by circular dichroism
(CD). The ~-(190-476) fragment, with 30% t~-helical and 25% [3-stranded
residues, includes the integrin EF-hand type domain with four divalent
cation-binding sites. Both e~-helical and [3-stranded residues (35% and 30%,
respectively) occur in 131-(121-329), in agreement with the known tertiary
structures of c~ I-domains. Divalent cation binding to these c~ and
fragments, taken separately, induces a conformational adaptation of the
protein, as monitored by CD, that is achieved upon binding of Ca2+, Mg2+
or Mn2+ to ~. -(190-476), whereas it only occurs with Mg2+ or Mn2+, but
not with Ca24, for 131-(121-329). The ~ and [31 fragments, taken separately,
bind the same recombinant fibronectin ligand FNIII7-10 in an RGDdependent manner in the presence of divalent cations. The selectivity
observed with divalent cations with regard to the conformational
adaptation of these recombinant fragments also occurs with regard to
ligand recognition, i.e. ~-(190-476) binds FNIII7-i0 in the presence of
either Ca2+, Mg2+ or Mn2+ whereas [31-(121-329) binds FNIIIT_I0 only in
the presence of Mg2+ or Mn2+. This suggests that the divalent-induced
conformational adaptation of these integrin fragments is required for
ligand-recognition, c~5-(190-476) and [31-(121-329) foian a stable
heterodimer in the presence of Mg2+ with no apparent cnnformational
change of the components making up the dimer. The ~-(190 476):J31(121-329) dimer recognizes the RGD-containing FNIII7-10 ligand to form
a ternary 1: 1: 1 complex. The formation of this ternary complex involves a
conformational adaptation of one or several of the protein components that
is visualized in the near-UV CD spectrum.

Functional analysis of laminin-5 demonstrates the pivotal

role of the 440kDa extracellular form of the protein in cell
adhesion. L. Gagnoux, M. Allegra, F. Spirito, J. Vailly, J.-P.
Ortonne, and G. Meneguzzi. INSERM U385, Nice, France.

Laminin-5 is the main epithelial adhesion ligand which is involved in cell

migration, wound healing, tumoral invasion and morphogenesis. In the
extracellular matrix (ECM) deposited by keratinocytes, this laminin e~3[~3T2
heterotrimer is detected as a 440 kDa protein and a more abundant 400 kDa
form resulting from the proteolytical cleavage of the y2 chain (155 kDa) into
a polypeptide (105 kDa) with a truncated N-terminal domain. To analyze the
role of the two extracellular forms of laminin-5, we have transfected human
~2-nnll keratinocytes (cell line LSV5) with eukaryotic vectors expressing
mutated laminin 72 cDNAs. Keratinocytes LSV5-NC and LSV5-C were
obtained that restore the assembly of recombinant laminin-5 molecules with
either the intact 155 kDa or the truncated 105 kDa -/2 chain, respectively.
Analysis of monolayer and organotypic cultures of LSV5-NC and LSV5-C
cells showed that: -i) Integrity of the putative cleavage site of the laminin ~'2
chain by protease BMP1 is required for the extracellular processing of
laminin-5; -ii) Only the 440 kDa form of laminin-5 incorporates the ECM,
where it acts as an adhesion ligand. In addition, transfection of normal and
laminin -/2- keratinocytes using a eukaryotic vector expressing the short arm
of laminin 72 chain demonstrated that the N-terminal domain of the
polypeptide undergoes incorporation into the ECM. Moreover, our study
reveals that integrity of the fibulin-2 binding site is necessary to cell adhesion.
Our results show that integrity of the short ann of the ~t2 chain is essential
to a correct integration of laminin-5 in the ECM, where the protein is
proteolitically cleaved. They also reveal that the unprocessed 440 kDa form of
laminin-5 plays an active role in cell attachment.

(Mo/18.1/114)

"CaMKII and ICAP-1 control the ct5~l

integrin dependent inside-out signaling"
D. Bouvard, C. Marie, L. Vignoud, M.R. Block
LEDAC UMR CNRS 5538 lnstitut Albert Bonmot Domatne
de la Merci 38706 La Tronche FRANCE

Fibronectin binding on c%13~integrin is strictly dependent on intra

cellular calcium. Using an in vitro assay, we previously found that either
calcineurin inhibitors or a blocking calcineurin monoclonal antibody added
to cell lysates completely abolished the fibronectin / integrin interaction.
This suggested that the activity of calcineurin, a calcium calmodulin
dependent phosphatase, was required to counteract some kinase activity
and maintain the high affinity state of %[3~ integrin. Here, we show that
blocking of the calcium calmodulin kinase II (CaMK II) activity with the
specific inhibitor KN-62 or with its pseudosubtrate Autocamtide II
preserves the high affinity state of the integrin even under experimental
conditions that inhibit calcineurin. Conversely, the addition of purified
CaMKI1 in the cell lysate inhibits cql3~ integrin binding to fibronectin in
vitro. Consistent with these results, cell adhesion on fibronectin is
stimulated by KN-62. Moreover, Scatchard analysis of fibronectin binding
on CHO cells reveals that KN-62 decreased the Kd value from 0.3 to 0.05
p-M. Finally the expression of exogenous constitutively active CaMKII
results in a dramatitc defect in cell adhesion with no significant
modification in %13~ integrin cell surface expression. In summary our
results demonstrate that CaMKII controls the affinity state of the integrin
cql3~ integrin in vitro and in living cells. Recently, we have shown that the
effect of CaMKII involves the protein ICAP-1 (Integrin Cytoplasmic
domain Associated Protein). Indeed, the mutation of the well conserved
putative CaMKII phosphorylation site into ICAP results in cell adhesion
effect similar to what is observed when the CaMKll activity is modified.
Furthermore the ICAP dependent effect on cell adhesion was not KN-62
sensible. Altogether our results suggest that ICAP controls CaMKII
dependent inside-out signaling pathway.

s196

Abstracts FEBS'99

(Mo/18.1/115) A new epithelial cell line to study tight junctions

A. Choquet', E. Blanc', G.S. Baldwin , R.H. Whitehead ~,
J.-P. BalP & F. HollandC

(Mo/18.1/116)

~CNRSEP 0612, Montpellier, France; r'Dpt of Surgery, Austin H',

~LudwigInstitutefor Cancer Research, Melbourne. Australia

This study describes a novel non-tumoral gastric epithelial cell line

(IMGE-5) isolated from transgenic mice harboring a temperature- and "tinterferon-sensitive mutation of the SV 40 large T antigen.
IMGE-5 cells constitutively expressed some epithelial markers
(cytokeratinl8) and membrane receptors (HGF receptors), while others
(gastrirdCCK-B receptors) are preferentially expressed under nonpermissive conditions (39(2). Similarly, confluent IMGE-5 cells
displayed a temperature- and v-interferon-sensitive expression of
adherens (E-cadherin, [~-cateuln) and tight (ZO-1/occludin) junction
proteins. Furthermore, their respective position along the membrane
(assessed by confecal fluorescence microscopy) was compatible with
their involvement in adherens and tight junctions, suggesting a
polarisation of this cells line under non-permissive conditions. The
expression of functional tight junctions was also confirmed by
measurement of a stable transepithelial resistance across a confluent
monolayer of IMGE-5 ceils.
Under non-permissive conditions, IMGE-5 cells showed reduced and
serum-dependent growth, and their proliferation was stimulated by factors
such as EGF, HGF, and Gastrin, as assessed by measurement of BrdU
incorporation and X T r assay. Furthermore, they did not form any
colonies in soft agar and did not induce tumors 9 months after being
injected into nude mice.
In conclusion, the ability to modulate the phenotype of IMGE-5 cells
represents an important advantage in order to study in vitro the regulation
of the proliferative and adhesive functions by the gastro-intestinal
epithelial cells. Finally, IMGE-5 cells should prove very useful to study
in vitro the pharmacological regulation of paxacellular absorption in the
gastric epithelium.

(M0/18.1/117) Homotypie interactions of type I and type II classical

eadherins. Comparison of kinetic parameters
H. Feracci 1, A.M. Benolie] 3, V. Delmas2, A.W.Koch 4,
A.Pierr~s 3, J. Engel 4, P.Bongrand 3 and J.P.Thiery 1
JUMR144 [nstitut Curie, Paris; :UMR 146 Institut Curie
Orsay; sINSERM U387, Marseille; 4Biozentrum, Basel

Cadherins are fundamental determinants of tissue organization in

developing and adult organisms. They are single pass transmembrane
glycoproteins which mediate calcium dependent ceil-cell adhesion by
homophilic interactions. Classical type I cadherins (including E-, N-, P-,
M-cadherin) share common structural features but moderate partial
homology with type II cadherins (as -5, -6, -7, -11). The prototype of
the type I family member, E-cadherin, participates to the formation and
cohesiveness of epithelia, although eadherin 11, prototype of type II
cadherins, is found to be expressed in dispersed and migrating
mesencbymal cells.
Since NH2-terminal fragments are crucial for the adhesive function
of cadherins, we recombinantly expressed domain pairs consisting of the
two N-terminal domains of E-cadherin (type I) and cadherin 11 (type
II). Trypsin digestion and CD spectroscopy have been used to evaluate
the correct folding as well as structural changes upon calcium binding of
these two fragments.
Laminar flow chamber has been used to determine the kinetic
parameters of these homophitic interactions. Our results show a
stronger molecular interaction between E-cadherin than cadherin 11
fragments and that this difference resides in the dissociation step.

L a n i ~ 5 ~sinvavm m human fetal W a d ~ .epiah~. edl

C. Coraux ' J.M. Zanm 'a o"' ~vxeneguzz~,
aJ r t3rt
E. Puchel]e and D. (3-adlard

e ,

alNSERM U514," Remls bINSERM U385; Nice, France.

Interactions between the epithelial cells and the basement membrane play a
key role during airway development, by modulating cell proliferation,
migration and differentiation. These interactions are mediated by integrins,
cell surface receptors composed of non-covently linked 0t and ,[3 subunits. In
order to assess the role of lamimn 5 (LNS), a basement membrane adhesion
ligand composed of the 0`3, ~3 and 72 chains, and its receptors ot3131, o6131
and 0`6]34 integrins, during branching morphogenesis, we developed an in
vitro culture model. Human fetal tracheal explants were seeded on a type I
collagen matrix. Epithelial cells grew and proliferated on the matrix around
the explants (2D cultures). These cultures were then overlaid by a second
collagen layer to form a sandwich configuration (3D cultures). This
overlaying induced branching morphogenesis and tubule formation. By
immunocytochemistry, we demonstrated that the epithelial cells of the
outgrowth produced the 0`3, [33 and y2 chains of LN 5 on the collagen matrix
in 2D cultures. In contrast, LN5 was not detected around the tubules formed
in 3D cultures. In 2D cultures, epithelial cells in close contact with the
collagen matrix expressed the 0`3, 0.6, ]31 and ]34 integrin subunits while
these cells in the tubules formed in 3D cultures only expressed 0.6, [31 and [34
subunits. Incubation of 2D cultures with blocking anti-LN5 antibodies
significantly decreased the migration speed of the outgrowth peripheral cells.
Inhibition of the branching morphogenesis was also observed in cells
overlaid by collagen to form 3D cultures. Our results suggest that LN5 plays
a major role in epithelial cell migration, specifically during branching
morphogenesis, and that ~6131 and e~6!34 integrins are involved in
tubulogenesis in vitro.
C. Coraux is a fellowship funded by MESR (Minist~re de L'Enseignement et
de la Recherche).

(Mo/18.1/118)

Expression of the platelet integrin 0`Iib[33 in talin

deficient cells.
P. Frachet, V. Martel, C. Albig~s-Rizo and M.R. Block.
LEDAC, UMR UJF-CNRS 5538, lnstina Albert Bonniot,
facult( de Mddecbw de Grenoble, 38700 La Tronche, France

In an attempt to define more about the role of talin on integrin expression and
function, we decided to examine the effect of de novo expression of an
exogenous integfin by the talin deficient cells that we have previously
charactefised [1]. In these cells the level of talin is reduced down to 20-40 %
of that normally found in wild type cells, cDNAs coding the platelet integrin
~IIb[33 which functions as receptor for fibrinogen and fibronectin were
transfected in talin deficient cells (AT22) and we examined the effects of this
de novo expression on cell adhesion and focal contact formation. Our major
finding are as follow: (a)The exogenous integrin is produced with
conformationat change similar to that we have previously reported on the
endogenous
[31 class of integrin. This result according with our previous
observations on the/31 class of integrins focus our attention on a potential role
of talin on the processing of the integrin heterodimer that will be normally
associated with it. (b) The cxlIb[33heterodimer is abnormally localised to focal
adhesion when the cells are plated on fibronectin and this recruitment is ligand
independent. This abnormal recruitment was already observed by others with
the non activable ~llb(A996)/~3 mutant. Alternatively, our results may suggest
that the lack of interaction between talin and cytoplasmic domains of the
integrin influences its recruitment to focal contact independently
to the
extracellular ligand activity.
1. Albigbs-Rizo et al, J.Cell Science(1995) 108, p 3317

s196

Abstracts FEBS'99

(Mo/18.1/115) A new epithelial cell line to study tight junctions

A. Choquet', E. Blanc', G.S. Baldwin , R.H. Whitehead ~,
J.-P. BalP & F. HollandC

(Mo/18.1/116)

~CNRSEP 0612, Montpellier, France; r'Dpt of Surgery, Austin H',

~LudwigInstitutefor Cancer Research, Melbourne. Australia

This study describes a novel non-tumoral gastric epithelial cell line

(IMGE-5) isolated from transgenic mice harboring a temperature- and "tinterferon-sensitive mutation of the SV 40 large T antigen.
IMGE-5 cells constitutively expressed some epithelial markers
(cytokeratinl8) and membrane receptors (HGF receptors), while others
(gastrirdCCK-B receptors) are preferentially expressed under nonpermissive conditions (39(2). Similarly, confluent IMGE-5 cells
displayed a temperature- and v-interferon-sensitive expression of
adherens (E-cadherin, [~-cateuln) and tight (ZO-1/occludin) junction
proteins. Furthermore, their respective position along the membrane
(assessed by confecal fluorescence microscopy) was compatible with
their involvement in adherens and tight junctions, suggesting a
polarisation of this cells line under non-permissive conditions. The
expression of functional tight junctions was also confirmed by
measurement of a stable transepithelial resistance across a confluent
monolayer of IMGE-5 ceils.
Under non-permissive conditions, IMGE-5 cells showed reduced and
serum-dependent growth, and their proliferation was stimulated by factors
such as EGF, HGF, and Gastrin, as assessed by measurement of BrdU
incorporation and X T r assay. Furthermore, they did not form any
colonies in soft agar and did not induce tumors 9 months after being
injected into nude mice.
In conclusion, the ability to modulate the phenotype of IMGE-5 cells
represents an important advantage in order to study in vitro the regulation
of the proliferative and adhesive functions by the gastro-intestinal
epithelial cells. Finally, IMGE-5 cells should prove very useful to study
in vitro the pharmacological regulation of paxacellular absorption in the
gastric epithelium.

(M0/18.1/117) Homotypie interactions of type I and type II classical

eadherins. Comparison of kinetic parameters
H. Feracci 1, A.M. Benolie] 3, V. Delmas2, A.W.Koch 4,
A.Pierr~s 3, J. Engel 4, P.Bongrand 3 and J.P.Thiery 1
JUMR144 [nstitut Curie, Paris; :UMR 146 Institut Curie
Orsay; sINSERM U387, Marseille; 4Biozentrum, Basel

Cadherins are fundamental determinants of tissue organization in

developing and adult organisms. They are single pass transmembrane
glycoproteins which mediate calcium dependent ceil-cell adhesion by
homophilic interactions. Classical type I cadherins (including E-, N-, P-,
M-cadherin) share common structural features but moderate partial
homology with type II cadherins (as -5, -6, -7, -11). The prototype of
the type I family member, E-cadherin, participates to the formation and
cohesiveness of epithelia, although eadherin 11, prototype of type II
cadherins, is found to be expressed in dispersed and migrating
mesencbymal cells.
Since NH2-terminal fragments are crucial for the adhesive function
of cadherins, we recombinantly expressed domain pairs consisting of the
two N-terminal domains of E-cadherin (type I) and cadherin 11 (type
II). Trypsin digestion and CD spectroscopy have been used to evaluate
the correct folding as well as structural changes upon calcium binding of
these two fragments.
Laminar flow chamber has been used to determine the kinetic
parameters of these homophitic interactions. Our results show a
stronger molecular interaction between E-cadherin than cadherin 11
fragments and that this difference resides in the dissociation step.

L a n i ~ 5 ~sinvavm m human fetal W a d ~ .epiah~. edl

C. Coraux ' J.M. Zanm 'a o"' ~vxeneguzz~,
aJ r t3rt
E. Puchel]e and D. (3-adlard

e ,

alNSERM U514," Remls bINSERM U385; Nice, France.

Interactions between the epithelial cells and the basement membrane play a
key role during airway development, by modulating cell proliferation,
migration and differentiation. These interactions are mediated by integrins,
cell surface receptors composed of non-covently linked 0t and ,[3 subunits. In
order to assess the role of lamimn 5 (LNS), a basement membrane adhesion
ligand composed of the 0`3, ~3 and 72 chains, and its receptors ot3131, o6131
and 0`6]34 integrins, during branching morphogenesis, we developed an in
vitro culture model. Human fetal tracheal explants were seeded on a type I
collagen matrix. Epithelial cells grew and proliferated on the matrix around
the explants (2D cultures). These cultures were then overlaid by a second
collagen layer to form a sandwich configuration (3D cultures). This
overlaying induced branching morphogenesis and tubule formation. By
immunocytochemistry, we demonstrated that the epithelial cells of the
outgrowth produced the 0`3, [33 and y2 chains of LN 5 on the collagen matrix
in 2D cultures. In contrast, LN5 was not detected around the tubules formed
in 3D cultures. In 2D cultures, epithelial cells in close contact with the
collagen matrix expressed the 0`3, 0.6, ]31 and ]34 integrin subunits while
these cells in the tubules formed in 3D cultures only expressed 0.6, [31 and [34
subunits. Incubation of 2D cultures with blocking anti-LN5 antibodies
significantly decreased the migration speed of the outgrowth peripheral cells.
Inhibition of the branching morphogenesis was also observed in cells
overlaid by collagen to form 3D cultures. Our results suggest that LN5 plays
a major role in epithelial cell migration, specifically during branching
morphogenesis, and that ~6131 and e~6!34 integrins are involved in
tubulogenesis in vitro.
C. Coraux is a fellowship funded by MESR (Minist~re de L'Enseignement et
de la Recherche).

(Mo/18.1/118)

Expression of the platelet integrin 0`Iib[33 in talin

deficient cells.
P. Frachet, V. Martel, C. Albig~s-Rizo and M.R. Block.
LEDAC, UMR UJF-CNRS 5538, lnstina Albert Bonniot,
facult( de Mddecbw de Grenoble, 38700 La Tronche, France

In an attempt to define more about the role of talin on integrin expression and
function, we decided to examine the effect of de novo expression of an
exogenous integfin by the talin deficient cells that we have previously
charactefised [1]. In these cells the level of talin is reduced down to 20-40 %
of that normally found in wild type cells, cDNAs coding the platelet integrin
~IIb[33 which functions as receptor for fibrinogen and fibronectin were
transfected in talin deficient cells (AT22) and we examined the effects of this
de novo expression on cell adhesion and focal contact formation. Our major
finding are as follow: (a)The exogenous integrin is produced with
conformationat change similar to that we have previously reported on the
endogenous
[31 class of integrin. This result according with our previous
observations on the/31 class of integrins focus our attention on a potential role
of talin on the processing of the integrin heterodimer that will be normally
associated with it. (b) The cxlIb[33heterodimer is abnormally localised to focal
adhesion when the cells are plated on fibronectin and this recruitment is ligand
independent. This abnormal recruitment was already observed by others with
the non activable ~llb(A996)/~3 mutant. Alternatively, our results may suggest
that the lack of interaction between talin and cytoplasmic domains of the
integrin influences its recruitment to focal contact independently
to the
extracellular ligand activity.
1. Albigbs-Rizo et al, J.Cell Science(1995) 108, p 3317

Abstracts FEBS'99

(Mo/18.1/119)

A new function for C M A R :

a cellular adhesion regulator.
S. Gout, L. Rouard-Talbot, M. Block and M. Jacquier-Sarlin.
LEDAC. UMR 5538, lnstitut Albert Bonniot,
F-38706 La Tronche eedex, FRANCE.

The
gene
CMAR
(" Cell
Matrix
Adhesion
Regulator "),localized in 16q24.3, codes a protein consisting of 82 amino
acids which have been described to modulate cellular adhesion via the
integrin cc2131. A polymorphism in the CMAR locus resulting from a
CACA insertion produces the ancestral smaller CMAR protein called
"Variant-C ". In human, the two mRNA can coexist in the same cell.
These proteins share the same N-terminus but differ by their C-terminus
with loss of a putative tyrosine phosphorylation site. However, the precise
role of these two proteins remains to be determined.
Comparative analysis of adhesion capacity of the different clones
established from the colorectal HT29 cells transfected by the fusion GFPCMAR or GFP-Variant C indicate that, while only CMAR increases the
cellular adhesion on collagen matrix (type I and IV), both CMAR and
Variant C favors the cell-cell aggregation. These effects on cellular
adhesion were correlated with the number of CMAR transcripts which are
very stable compared to the protein. According to these observations, we
propose that CMAR could be a regulator of adhesion process, with two
different functional domains. The N-terminus favors cell-cell contacts
which correspond to the ancestral function, while acquisition of the Cterminal tyrosine phosphorylation site regulates the adhesion of cell to the
extracellular matrix.
Preliminary experiments with lysophosphatidic acid (LPA), an
activator of the small GTPase protein Rho, suggest that CMAR could
participate in this transduction pathway already described to modulate
cellular adhesion processes. Further investigations concerning the
signaling pathway involving CMAR could be determinant for the
involvement of this protein in tumor progression and embryogenesis, two
process where cellular adhesion is often perturbed.

(M0/18.1/121)

TGF-131dependent regulation of adhesion receptor and intracellular signalin~ molecule expression in HT-144 melanoma cells
B. Janjia, V. Gouon, C. Melchior~, and N.Kieffer~. ~LaboratoireFraneoLuxembourganis(CRP-Santt/CNRS), Luxembourg.bDepartmentof
Molecular Biology, Albert EinsteinCollege of Medicine, Bronx,NY, USA.

We have recently shown that two human melanoma cell lines, the
metastatic HT-144 and the non-metastatic SK-Mel-2 ceils, exhibit
marked in vitro heterogeneity with respect to integrin expression,
migration and invasion potential. Here we provide evidence that HT-144
melanoma cells, but not SK-Mel-2 ceils, undergo a reversible transition
to a fibroblastoid morphology following treatment of the ceils with either
their own serum-free acidified conditioned medium, or with biologically
active exogenous TGF-131, thus identifying TGF-131 as an autocrine
regulator of the spindle shape morphology of HT-144 melanoma cells.
The fibroblastoid phenotype correlated with up-regulated 131 and 133
integrin expression and down-regulated E-cadherin expression, as shown
by flow cytometry, Western blot and RT-PCR analysis, as well as upregulated expression of the metalloproteinase MMP-9, as demonstrated
by zymography. Our data further demonstrate TGF-131 dependent upregulation of ILK and nuclear translocation of 13-catenin suggesting that
in the metastatically competent HT-144 cells, ILK acts downstream of the
autocrine TGF-131 loop as a common intracellular regulator responsible
for the metastatic phenotype of HT-144 melanoma cells. [1] Gouon et al.,
Int. J. Cancer, 68, (1996) 650.

s197

(Mo/18.1/120)

Low integrin clustering (point contact) is strongly

efficient for Caco-2 cell adhesion and spreading.
S Honore, V Piehard, C. Penel, C. Pr6v&, H. Kovacic, J.
Marvaldi, C. Briand, and J-B Rognoni.
LTRESA-CA'P,S 6032, Faculte de Phannae~e, 27 Bd J Moulin 13005 Marsedle.

We sought in this work to investigate the modalities of cell adhesion

and spreading of a spontaneous differentiated colic adenocarcinoma cell line
(Caco-2 cells).
Caco-2 cell adhesion was optimal on collagen IV (COL IV),
vitronectin (VN), fibronectin (FN), and laminin (LM) via otl[31, o.2131
integrins, ecv[31, etv135 integrins, ct5131, etvfl6 integrins, and ct6131/134 integrins
respectively. Cell spreading efficiency was better on COL IV as compared
with spreading on FN (54 % of cells spread in 1 h vs. 10 % ) After 5 h, this
process was highly increased on COL IV and LM (88 % and 82 % of spread
cells) and moderately increased onto FN and VN (60 %). Some integrins did
not seem to recognize their known ligands, i.e. txll31 vs. COL I and LM,
ot2131 vs. LM, ct3131 vs. COL I, FN and LM, and et9131 vs COL I and LM.
However, ct6fll and/or ct6134 integrins were shown to be partially efficient in
cell adhesion to F N
Using immunofluorescence (IF) and confocal microscopy (CM)
studies, we were able to show that in Caco-2 cells low integrin clustering
(point contacts) of t~2131, ccv136, and ct6131/134 are strongly implied in cell
adhesion to COL IV, FN, and LM respectively. Otherwise spreading process
which was highly efficient when cells were plated on COL IV and LM was
mediated by this integrin molecular organization. It must be noted that also
poorly aggregated ctvJ36 was implicated in this process which was low on FN.
However, poor efficacy in spreading process was observed for highly
clustered ctv135 integrin when cells were plated onto VN. Moreover, IF and
CM data suggested that low ct2131, ctvfl6, and ct6131/134 integrin clustering
activated by their respective substrates could induce the actin polymerization
and trigger the F- actin organizing i.e radial and transverse stress fibers and
cortical microfilaments.
Together these results demonstrate the preponderant role of point
contacts in cell adhesion and spreading and in structuring the actin
eytoskeleton in Caco-2 cell line.

(Mo/18.1/122) Human Endothelial Cell Lines : Tissue Specific

Adhesion molecules Phenotypes
C. Kieda', M. Paprecka ~, M. Monsign3?, C. Radzikowski b', D.Du~ b
aCBM. UPR CNRS 4301, rue Charles Sadron, 45071 Orldans France
bLudwik Hirszfeld last. lmmwt Er.ptl Ther., PAN, 53114 Wroctaw, Poland

Vascular endothelial cells recognize blood borne circulating ceils that they
allow to extravasate in a tissue-specific manner. This property determines
the selectivity of lymphocyte homing, which is fundamental in physiological
(immune response) as well as pathological processes (inflammation,
autoirnrnune diseases, metastasis). To study these phenomena, eight
microvascular endothelial cell lines were established. Endothelial cells
(ECs), isolated from infiarnmed lymphoid tissues (lymph nodes and
appendix) [1] and from non lymphoid immune effector sites: intestine,
lung and skin, were immortalized. Their general endothelial
characteristics, such as the presence of vonWillebrand factor (wWf),
angiotensin converting enzyme (ACE) as well as the intracellular Eselectin, were preserved [2]. Furthermore, this paper describes the distinct
phenotypic characteristics of these cell lines in relation to their tissue
origin [3]. Hence, endothelial cells from lymph nodes expressed GlyCAM1, PNAd.JG-I, sLeX and CD34 antigens, but did not express P-selectin.
ECs from non lymphoid immune effector sites were ICAM-1 and CD49e
positive, all but one were P-selectin positive. ECs from mucosal sites were
strongly MAdCAM-I positive. Endothelial cell lines were shown to
selectively bind lymphoblastoi'd T cells, in a specific manner according to
their tissue origin and in relation to the expressed cell adhesion molecules.
These data demonstrate the potential of such a panel of endothelial cells in
deciphering the molecular mechanism involved in cell-cell interactions. [1]
Bizouarne, N. et al., Biol Cell, 79 (1993) 27; [2] Bizouarne, N. et al., Biol
Cell, 79 (1993) 209 ; [3] Denis, V. et al., J L e u k o c BioI, 60, (1996) 744.

s 198

(Mo/.18.1/123)

Abstracts F E B S ' 9 9

Expression of integrins and cadherins in human melanoma

and bladder cancer cells.
PLaidlera,DKsia,2ek~,D Gil,A.Pituch-Noworolska,ALityfiska

(Mo/18.1/124)

aInstitute of Zoology, bInstttutofMedtcal Biochemist~ CoIL~k[ed

Jagiellonian Universtty,Krakdw, Poland

aInstitutMed. Biochem., Jagielloman Umverssty, Krak6w, Poland

Integrins and cadherins are transmembrane adhesion receptors that

mediate crucial cell-cell and cell-matrix interactions. The adhesive changes
occuring in tumorigenesis undoubtly involve changes in expression and
properties of these proteins [ I-3].
The expression of various integrin subunits (alpha2, alpha3, alpha5,
betal) and cadherins rE, N, Pan) was studied in human melanoma (WM9,
WM35, WM239, WM946, A375), human bladder normal (Hu609, HCV)
and carcinoma (T24, Hu465, BC3726) cell lines by flow cytometric
analysis using commercially available monoclonal antibodies (Dako,
Genosys, Boehringer, Zymed, Sigma).
High expression of alpha2 (80-95% and 54-98%), alpha3 (80-95% and
83-98%), alpha5 (60-95% and 95%) and betal (60-85% and 70-98%)
integrin subunits was observed in bladder and melanoma cells respectively
In case of melanoma WM35 and WM239 the expression of alpha5 subunit
was low (18% and 20% respectively). No statistically significant differences
between expression of a given integrin subunit in normal and cancer bladder
cells as well as between various subunits in a given bladder cell line were
found.
All studied cell lines did not show expression of E-cadherin (Zymed or
Boehringer anti E-cadherin mouse monoclonal antibodies). Additionally
melanoma cells were N-cadherin negative (Sigma anti N-cadherin mouse
monoclonal antibody). However, the presence of cadherin molecules was
detected in all studied cell lines (bladder and melanoma) using anti Pancadherin mouse monoclonal antibody (Sigma) after initial perforation and
permeabilization) of cell mambrane. The expression was in the range of 5592% and 59-86% for bladder and melanoma cells respectively.
1. Syrigos KN. e t a l , Int. J. Cancer 64 (1995) 367
2. Zheng M. et al., J.Biol Chem., 269 (1994) 12325
3. Sanders R.J. et al., Cancer Invest., 16 (1998) 329.
Thts work was supported by K]3Ngrant 4389/P04/97/08.

Characterization of N-glycans of or3131integrin

A. Lityfiska, M. Przybytoa, P. Laidler

Integrins are a family oftransmembrane adhesion receptors that mediate

cell-cell and cell-matrix interactions. These membrane glycoproteins consist
of noncavalently bound ~and 13subunits that associate in various
combinations to form integrin heterodimers with different ligand-binding
specificity The recent results indicated that 13tintegrins play an important
role in the tissue attachment, migration, invasion and metastasis of human
bladder carcinoma cell line T-24. Cell adhesion regulated by integrins seems
to be strongly modulated by N-glycosylation (1-3).
We analysed the carbohydrate moiety ofa313i integrins present in human
bladder carcinoma cell lines (T-24, Hu 456, BC 3726) and human normal
ureter epithelium (Hu 609, HCV). Glycan analysis was performed with the
use of a Glycan Differentiation Kit (Boehringer) on protein transferred onto
nitrocellulose after polyacrylamide gel electrophoresis of cell homogenate in
the presence of SDS under non-reducing condition. Glycan Differentiation
Kit included the following digoxigenin-labelled lectins: Galanthus nivalis
agglutinin (GNA), Sambucus nigra agglutinin (SNA), Maackia amurensis
agglutinin (MAA), Datura stramonium agglutinin (DSA), Aleuria aurantia
agglutinin (AAA) and Phaseolus vulgaris agglutinin (PHA-L). The ct3 and
~31 subunits were identified separately using monoclonal antibodies: P1B5
(Dako) and 2A4 (Genosys)
It was shown that glycans of 131 integrin subunit are three or
tetraantennary complex type oligosaccharides with sialic acid (T24, HCV,
BC3726) or fucose (Hu609, Hu456, BC3726). Additionaly oligomannose
structures were identified (Hu609, Hu456). In contrary no glycans were
found in c~3 integrin subunit (Hu609, HCV, BC3726). Only oligomannose
glycans were identified in c~3subunit of Hu456 integrins and three or
tetraantennary glycans with sialic acid were detected in T24 integrins.
1.Akiyama S K , etal., Cancer and Metast.Rev 14, (1995),173.
2.Fujita S., et al., Jpn.J.Cancer Res. 83, (1992), 1317.
3. Nakagawa H., et al., Eur.J.Biochem 237, (1996), 76.
This work was supported by KBN grant 4389/P04/97/08.

(Mo/18.1/125)

Analysis of the P-Glycoprotein activity on secretion of

proteases and cytokines by spheroids of cancer cells
Laurent Mougel. A4oncef Guenounou, Jean-Claude Jardillier
Laboratoire de Btochimte, Laborato:red'lmmunologte, F~ 2063, IFR 53
Biomol~cules, UFR Pharmacte, 51096 Reims Cedex

Development of multidrug resistance (MDR) in tumors during

treatment is a major cause of chemotherapy failure. To study the relation
oftbe P-170 glycoprotein (P-Gp) activity on the secretion of proteases and
cytokines by cells, we used as a first step monoclonal antibodies (clone
UIC2 and MRK16) specific to P-Gp and inhibitors of its activity. The
human breast cancer cell lines MCF-7 sensitive (MCF-7 s) and multidrug
resistant (MCF-7DxR) cultured in spheroids of 250,000 cells were used.
The proteases activity (collagenase IV, cathepsin D, elastase) and the
cytokine secretion (II-lct, -113, I1-6, It-8, II-10, I1-12, TNF-a) were
quantified in the supernatant of the spheroids formed with cells pretreated
or not with mAbs. The results obtained indicate an increase in proteases
secretion of resistant cells as compared to sensitive and different cytokines
profile between both cell hnes. These results are similar using cells treated
with anti P-Gp mAbs. In conclusion, blockage of P-Gp function by mAbs
doesn't modify the profile secretion of resistant cells. Other modulators of
P-Gp activity could be tested.

(Mo/18.1/126)

The calcium- and integrin-binding protein CIB does not

regulate the affinity state of integrin ctIIb133 in CHO cells
S. Plan~ona, L. Vallar a, E. Friederich, C. Melchiora, N. Kieffera
aLaboratotre Franco-Luxembourgeois(CRP-Santd_/CNRS), Luxembourg,
blnstitut Curie, UMRI44, 26, rue d'Ulm, Paris, France

Integrins are noncovalently associated ct13 heterodimers that mediate ceUcell or cell-matrix interactions. Integrin function is regulated through
eonformational changes mediated by the interaction of the integrin
cytoplasmic tails with cytoskeletal proteins and signaling molecules. The
platelet ~xlIb133 integrin functions as an inducible fibrinogen receptor, and
promotes platelet aggregation at the site of vascular injury, but its regulation
is still not clearly understood. The newly identified calcium- and integrinbinding protein CIB, present in platelets, interacts specifically with the
cytoplasmic domain of ctlIb. In order to address the question whether CIB
acts as a regulatory molecule modulating ~xlIb133 function, we determined
the intracellular localization of CIB, fused to the C-terminus of the green
fluorescent protein GFP and transiently transfected in CHO cells expressing
ctlIb[33. In cells adherent on fibrinogen, GFP-CIB was found both in the
cytoplasm and the nucleus, but no association with focal adhesions could be
demonstrated, in contrast to the cytoskeletal protein GFP-Zyxin that clearly
colocalized with cdlb133 in adhesion plaques. To test whether CIB could
modulate the ctllb!33 affinity state, we investigated the binding of mAb
PAC I, a fibrinogen-mimetie, to transfected cells by flow cytometry. Cells
coexpressing ~xlIb[33 and GFP-CIB did not bind PAC1, suggesting that CIB
may not be involved in MIb133-mediated inside-out signaling. Finally, using
reverse transcriptase-polymerase chain reaction performed on total RNA
from various cell lines, we detected CIB mRNA in non-megakaryocytic cell
lines, showing that CIB might be expressed in the absence of the ~xlIb
subunit. Taken together, these results question the regulatory role of CIB in
integrin etIlb133 activation, and suggest its implication in integrinindependent processes.

Abstracts FEBS'99

(Mo/18.1/127)

s199

Convergent effect of collagen IV and EGF on adhesion

and spreading of a colic adenocarcinoma cell line (HT29-D4)
V Piehard, S Honor~ H Kovaeic, J Marvaldi, C Briand and JIB Rognom

(Mo/18.1/128)

LT~RES-A CNRS 6032, Facu lte de Pharmacie 27 Bd J Mouhn 13 3 3 5 5/tRS EILLE

Effects in vitro of human growth hormone o n

osteoclastic resorption in rabbit hone cell cultures
Rousselle A.V., Damiens C., Guicheux J., Pilet P., Padrines
M., Heymann D.. UPRES EA 2159, Groupc de Ph~.slopathoh)~lc de la
RtsorptlOrl Osgctl~.c, Nantes, France.

Cell adhesion is governed by integrins, heterodimeric glycoprotein

receptors, which play an important role in cell-extracellular matrix (ECM)
interactions, and in cytoskeleton orgaruzatton and signahng protein activation.
The aim of the present work was to study the convergent effect of ECM
protein, collagen IV (COL IV) and growth factor (EGF) m adhesion (short
term) and cell spreading (long term) of a colic adenocarcinoma cell line (HT29D4),
EGF (10 ng/ml) increased cell adhesion at 5, 15 and 30 minutes in dosedependant manner on COL IV, no EGF effect was observed in cells attached to
poly-L-lysme. Using immunofluorescence, a recruitement sequence at the cell
basal site of mtegrin subunit 131, FAK, paxillin, vinculin and other tyrosinephosphorylated proteins constitutwe of focal contacts, was observed during cell
adhesmn and spreading on COL IV whether the cells were stimulated by EGF
or not. All the proteins investigated were transloeated to the basal site of the
plasma membrane after 15 hours, which induced focal contact organizing except
for 131. Simultaneously, integrms remained weakly clustered (point contacts). At
24 hours , actin filaments (stress fibers) converged towards focal contacts.
Using immunoprecipitation followed by m'anunoblot with an anti-tyr-P mab,
FAK appeared tyrosine-phosphorylated (tyr-P). As compared to COL IV, EGF
stimulated weakly FAK tyrosine~phosphorylation, process which was increased
only after 15 hours of cell spreading. This activation may be maplied in increase
of HT29-D4 cell spreading, suggesting a functional link wtth the
neoorganmation of focal contacts.
In conelusmn, EGF effect on HT29-D4 cell adheston seems not directly
mediated by FAK whereas this kinase is hkely revolved in cell adhesion basal
level. Other signaling pathways e.g Ca2"/PKC or Ras pathways could intervene
in a2131 integrin (expected COL IV receptor) membrane expression or
activation. However FAK activation together with the other focal contact
proteins intervene more strongly in organizing the actin cytoskeleton (corneal
cytoskeleton, stress fibers) and thus in triggering cell spreading Moreover low
azf31 integrin chistermg (point contacts) in opposition to the so-called focal
contacts seems to mediate HT29-D4 adhesion and spreading
(Mo/18.1/129) Involvement of the 133 cytoplasmic domain in the
regulation of LIBS epitope exposure on integrin av133
E. Schaffner-Reckinger and N. Kieffer
Laboratoire Franco-Luxembourgeois de Recherche Biorn~dicale / 162A. av,

de la Fai?ncerie / L-1511 Lltxembourg / Grand-Duchy of Llecembourg

We have investigated allosteric changes of 133 integrins during the process

of outside-in signaling and determined the structural requirements of the 133
cytoplasmic tail necessary for LIBS (ligand-induced binding site) epitope
neoexpression on the avl~3 ectodornain, by analyzing a series of 153
cytoplasmic tail mutants which have been described previously [1]. When
associated with etv, all 133 mutants were able to bind RGD containing
peptides; however deletion of the C-terminal NITY 759 sequence or
disruption of the NPLY 747 motif by a Y747A substitution prevented LIBS
exposure on av133 following RGDS or echistatin binding, while the
substitutions Y747F or Y759F allowed normal LIBS exposure. Defective
fibrin clot retraction

paralleled

defective LIBS epitope expression.

Truncation of the entire IB3 cytoplasmic tail resulted in constitutive LIBS

exposure on otv133, similar to that observed for aIIb133. Our data
demonstrate that the structural integrity of the 133 subunit cytoplasmic tail,
rather than potential phosphorylation of Tyr747 or Tyr 759, is necessary for
LIBS epitope expression on integrin aviS3. These results suggest that LIBS
exposure following ligand analog binding to av133 relies on a bidirectional
transmembrane signaling process, transmitted from the ligand binding
pocket to the cytoplasmic face of the receptor and back to the ectodomain.
[1] E. Schaffner-Reckinger et al., J. Biol. Chem., 273, (1998) 12623.

This study investigated the effects h~ vitro of human growth hormone (hGH) on
osteoclastic resorptmn in an unfractionned rabb~t bone cell model. We also
investigated the mechanisms implicated in the osteoclastic resorption like the
proteasic activity and the role of bone matrix proteins on these mechanisms.
After four days of culture on denUne slices, hGH (1, 10, 50 ng/ml) and human
insulin growth factor I (hlGF-1 : 1, 10, 50 ng/ml) stimulated significantly the
resorption activity induced by rabbit bone cells in term of the percentage of
dentine slice surface resorbed, the number of lacunae per surface unit and the
mean area of lacunae as compared to the control. When neutralizing anti-serum
against hIGF- 1 (4 gg/ml) was added at the start culture, the stimulator3,' effects of
hlGF-I and hGH on osteoclastic resorption activity were totally abolished.
These results indicate that the effects of hGH sttmulatior on osteoclastic
resorption in vitro are mediated via local hlGF- 1 secretion by stromal cells such
as osteoblasts. As fonctionnally specific hIGF-1 receptors have recently been
reported on rabbit mature osteoclasts, a direct action of hlGF- 1 on the activation
of osteoclast could be envisaged. The experiments also showed that hlGF- 1 and
hGH stimulated the production of cathepsins and matrix metalloproteases MMP9 and MMP-2. Similar to the resorption acuvity, hGH stimulated the proteasic
activity via production of hIGF-1 by stromal cells. In a second time, the
influence of organic matrix protems, such as vitronectln, on osteoclastic
resorption and proteasic activity was examined. When dentine was precoated
with vitronectin, the proteasic and resorption acuvity was significantly stimulated
in presence of hGH and hlGF-1 compared to the activity on alone dentine shoes.
When antiserum against vltronectin receptor (ctv[~3 integrin) was added, the
metalloproteasic activity was reduced. This results indicate that the stimulation of
proteasic and resorption activity are dependent of the cell adhesion to vitronectln.
This study suggest that the proteastc activity and the resorptlcm actlwty in our
rabbit bone cell model are the work of the osteoclasts. To confirm this
hypothesis, a new model using purified osteoclasts will be developed.

(Mo/18.1/130)

LAMC2 gene mutations associated with H-JEB in patients

immunoreactiveto laminin-5 and presentingmature hemidesmosomes.
F. Spiritoa, S. Chavanasa, L. Pulkinenb, J. Uitgob, J-P. Ortonnea, G.
Mcneguzzia. a,INSERM U385, Nice. France; bJefferson Medical College,
Philadelphm. PA.

Herlitz's junctional epidermolysis bullosa (H-JEB) is a recessive

inherited skin blistering disorder, characterized by dermal-epidermal
separation within the lamina lucida of the basement membrane zone of
squamous epithelia. H-JEB has been associated with genetic mutations that
hamper expression of laminin-5, and hinder nucleation of hemidesmosomes
(HD). We characterized a H-JEB family in which the severe clinical
phenotype of the affected members was associated with a reduced synthesis
of laminin-5. Ultrastructural analysis of non involved H-JEB skin revealed a
well-structured basement membrane zone with mature HD, while extensive
cleavage within the lamina lucida of the dermal-epidermal junction was
noted in involved skin and the papiltar dermis. The patients' skin displayed
markedly reduced reactivity to monoclonal antibody GB3 and polyclonal
antibodies to each chain of laminin-5.
Analysis of the cDNAs encoding laminin-5 detected a nonsense
mutation (Q895X) in exon 18 of the paternal allele of LAMC2 gene and an
intronic transversion (84%10 T->G) in the maternal allele. This mutation
causes misplicing of intron 6, out of frame deletion of 23 nt of exon 7, and
premature termination codon (PTC) at nt 897 of the laminin ,12 mRNA.
Northern blot analysis of the patients' keratinocytes indicated that the
aberrant '/2 transcripts undergo PTC-mediated decay. However long-range
allele-specific RT-PCR demonstrated that scants amounts of LAMC2 premRNA from the maternal allele undergo normal splicing.
Our results underscore the multifunctional role of laminin-5 in cell
adhesion, because we provide evidence that exiguous deposits of this
adhesion ligand are sufficient to induce nucleation of morphologically
mature HD, but not to provide firm anchorage of the HD to the underlying
dermis. They also demonstrate the pivotal role of laminin-5 in the
structuration of the dermal-epidermal junction.

s200

(Mo/18.1/131)

Abstracts FEBS'99

SPR analysis of integrin (xl~ cytoplasmic domain

interaction with intracellular proteins.
L Vallara, C. Melchior~, G. Lippensb, and N. Kieffera
Laboratoire Franco-Luxembourgeols (CRP-Santb/CNRS), Luxembourg,
blnstitut de Bzologie de Ltlle, Laboratozre de RAIN, 59000 Ldle, France.

Integrins are ct/13 heterodimeric cell surface receptors that promote adhesion,
and also transfer information into and out of a cell. Integrin cytoplasmic
tails play a crucial rote in these processes, presumably through
modifications of their own structural and spatial organization, and/or
through interactions with specific cytoplasmic components. We have used
recombinant or synthetic t~IIb and 133 integrin cytoplasmic peptides to study
their in vitro complexation and ligand binding capacity by surface plasmon
resonance. J13 heterodimerization occurred in a 1:1 stoichiometry with a
weak KD in the laM range resulting in a transient binding. Divalent cations
were not required for this association, but stabilized the u/13 complex by
decreasing the dissociation rate. tx/13 complex formation and stabilization
were both impaired by the R ~ A substitution or the KVGFFKR deletion in
allb, but not by the 133 $752P mutation, indicating that the ctlIb membraneproximal sequence is necessary to support these processes, whereas the [~3
C-terminus is not Recombinant calcium-and integrin-binding protein (CIB),
an ~Ilb-specific ligand, bound to the (tlIb cytoplasmic peptide in a Ca2+- or
Mn2+-independent, one-to-one reaction with a Kr) of 12 ~tM. In contrast, m
vitro liquid phase binding of CIB to intact cdlb133 occurred preferentially
with Mn2+-aetivated tllb133 conformers, as demonstrated by enhanced
coimmunoprecipitation of CIB with PAC-l-captured Mn2+-activated
cdlb133, suggesting that Mn2+ activation of intact o~IIb133 induces the
exposure of a CIB binding site, spontaneously exposed by the free ctllb
peptide. Since CIB did not stimulate PAC-1 binding to inactive cdlb133, nor
prevented activated ctllbj33 occupancy by PAC-1, we conclude that CIB
does not regulate ctllb133 inside-out signaling, but rather is involved in an
o~Ilbl33 post-receptor occupancy event.

(Mo/18.1/132)

P E C A M - I F U N C T I O N A L ASSOCIATION
TO PI-3K IN HUMAN NEUTROPHILS.
M.R. Zocchi 1, A. Poggi 2 and F. Pellegatta 1.
lS, Raffaaele Scientific Institute 1.20132 Milan.
2National Cancer Institute-ABC, 1-16132 Genoa.

The platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31)

participates in leukocyte transendothelial migration both directly, as an
adhesion molecule, and indirectly as a modulator of integrin functions [1].
Herein we show that the engagement of PECAM-1/CD31 up-regulates the
adhesion of human neutrophils to the EA.hy926 endothelial cell line through a
phosphoinositide-3 kinase (PI-3K)-dependent pathway. Indeed, LY294002
and wortmannin [2] prevented the effect of PECAM-1/CD31 cross-linlctng, at
concentrations known to inhibit PI-3K without affecting other kinases. Both
compounds blocked neutrophil binding to murine fibroblasts transfected with
human ICAM-1, to purified ICAM-1 protein or fibronectin, suggesting that
PECAM-1/CD31-mediated up-regulation of [~2 and 151 integrin-mediated
adhesion is PI-3K-sensitive. We also provide evidence for the association of
PECAM-I/CD31 to PI-3K, as PI-3K was detectable in neutrophil lysates after
PECAM- 1/CD31 cross-linking and immunoprecipitation. PECAM- I/CD31dependent recruitment of PI-3K was suggested by the finding that the
serine/threonine kinase p70 $6 kinase (S6K), a signaling protein downstream
of PI-3K [3], is activated in neutrophils upon PECAM-1/CD31 cross-linking,
based on the appearance of serine phosphorylation in S6K
immunoprecipitates. In turn, S6K is not directly involved in the up-regulation
of integrin function, since rapamycin did not inhibit PECAM-I/CD31-muucea
adhesion of neutrophils to 151 and [~2 integrin substrates. In conclusion,
PECAM-I/CD31 appears to be one of the molecules functionally coupled to
PI-3K, suggesting that this enzyme may represent a common pathway of
integrin and adhesiveness regulation in leukocytes.
1. Newman P.J. 1997 J.Clin.lnvest. 99:3-8.
2. Shimizu Y. and Hunt III W. 1996. lmmunol. Today 17:565-573.
3. Ward S.G. June C.H. and Olive D. I996. lmmunoI. Today 17:187-195.

Abstracts FEBS'99

s201

1.2 Genome analysis and evolution

(Mo/1.2/133)

ORGANISMAL VERSUS MOLECULAR APPROACHES

TO EVOLUTION : CONFLICT OR AGREEMENT ?
R. Acher, J. Chauvet.
Lab. Biol. Chem., Univ. Paris VI, 75006 - Paris (France)

Whereas the idea of selective competition between species dates back to Greek
philosophers such as Aristotle, the concept of transformism or filiation of species
was suggested for the first time on a scientific basis by J.B, Lamarck
(Philosophic Zoologique, 1809). During the XIXth century, comparative
anatomy, physiology, embryology, paleontology and genetics have progressively
imposed the evolution of species at the organismal level. The XXth century,
through biochemistry, biophysics, molecular biology and structural biology, has
set up all aspects of life on the molecular scene. In this transposition, a single
species such as man is roughly represented by about 100.000 distinct
genes/proteins and a confrontation between organismal evolution and multiple
molecular evolutions arises. Important disagreements appear between evolutionary clocks based on fossil records and RNA/protein sequences, respectively,
as well as in the branching of several groups in phylogenetic trees.
As the driving force for species evolution, Lamarck proposed adaptation to the
medium (heredity of acquired features) whereas Darwin, half a century later,
suggested natural selection. Modern neo-Darwinism couples random gene
mutations with molecular selective pressure. Despite adaptationism has never
been experimentally proved at the organismal level, introduction into the genome
of some retro-information coming from the medium is no longer a proscribed
concept. In addition to rico-Darwinism and neo-Lamarckism, new theories have
emerged, based on knowledge of genome and proteome sequences, In neutral
evolution or genetic drift proposed by Kimura, most of nucleotide or amino acid
substitutions are fixed without selection. In genomic drive, DNA turnover is
determined by its own physico-ehemical laws (selfish DNA). The duplication
propensity of DNA explains the progressive increase of genome sizes from
bacteria to vertebrates with creation of multigene families and therefore multiple
paralog protein lineages. Polypeptide chain self-folding and folding by
association build up protein shapes (conformations) crucial for specific
interactions involved in cascades leading to the physiological functions. Whatever
the driving force of evolution, it must first act on conformations through
variations of nucleotidedamino acid sequences.
Using the 12 neurohypophysial preprohormones we have characterized in vertebrates, implications of the above evolutionary mechanisms have been searched
for. Amino acid substitution rates greatly vary between the 3/4 domains of the
precursors, suggesting distinct domain subevolutions.Two ortholog lineages can
be traced from bony fishes to mammals. Duplications giving rise to additional
paralog lineages can be observed in marsupials and in cartilaginous fishes. Botb
neutral and selective evolutions seem to have operated on nonapeptide hormon
sequences, although a common gross conformation was preserved in all neur
hypophysial hormones.
(Mo/1.2/135)

Remote query of biological Web server from the protein

sequence analysis software MPSA
C. Blanchet, C Geeurjon,O. Del~age
PBIL. IBCP, 7 passage du Vercors, 69367 Lyon, France

Protein sequence analysis represents the current challenge in

retrieving most information contained in data generated by genome projects.
It has to face with the handling of databanks with increasing size, and
methods with considerable complexity. MPSA [1] answers to these
constraints with a friendly graphic user interface to several methods from
different biological or biophysical fields. In this way, parametring these
methods, making them collaborate or displaying their results become very
efficient and easy to use for all biologists. MPSA can be used as a standalone software or as a local interface to a remote web server like the ~Ptle
Bio-Informatique Lyonnais>>[21.
MPSA provides several protein sequence analysis algorithms: (i)
biologically significant sites, patterns and profiles scans (ProScan,
PattInProt), (ii) homologous protein query (FASTA, BLAST, PSI-BLAST,
etc.) in world databases (SWISS-PROT, TrEMBL, PIP,, non-redundant,
PDB, etc.), (iii) multiple alignment (Clustal W, Multalin), (iv) secondary
structure prediction (C-OR, SOPMA, MLR, PREDATOR, etc.), (v)
physic,o-chemical profile prediction, (...). Using MPSA, the biologist
doesn't worry about algorithm collaboration or data formats. Protein
sequence analysis methods, included in MPSA, can be processed on your
computer, or on a remote computer through Web connection. With these
facilities, the biologist browses databanks dally-updated on remote Web
server, and uses up-to-date algorithms delivered by bio-informatic
laboratories like we do it for PattInProt, SOPMA and more others on our
web server.
MPSA is available for Macintosh, PC, UNIX workstations or
personal computer with Linux system. You can download it and get further
information from its homepage [1].
[1] MPSA: Multiple Protein Sequence Analysis, http://www.ibcp.fr, mpsa
[2] PBIL: Ptle Bio-Informatique Lyonnai s, http://pbil, ibcp._fr

(Mo/1.2/134) Cig30 and Pitx3 genes are arranged in a partially

overlapping tail-to-tail array in the distal region of mouse
Chromosome 19
Abolfazl Asadi*, Petr Tvrdik*, Leslie P. Kozak'~, Edem
Nuglozeh t, Fabienne Parente-~, Jan Nedergaard*, Barbara
Cannon* and Anders Jacobsson*
*The Wenner-Gren Institute, The Arrhenius Laboratories F3, Stockholm
University, S-106 91 Stockhobn, Sweden, t-TheJackson Laboratory, Bar
Harbor, Maine 04609, U.S.A. and ~:lnstabdit~ et altdrations des G~nomes,
UNSA/CNRS UMR 6549, Avenue de Valombrose, F-06107 Ntce, France

The mouse Cig30 gene codes for a 30 kDa membrane glycoprotein,

which appears to have a role in the recruitment of brown adipose tissue. In
order to elucidate the structure of the Cig30 gene, we have isolated a
lambda phage genomic DNA clone containing the entire mouse gene, and
found that Cig30 consists of four exons which are spread over 4 kb of
genomic sequence. Using a FISH assay and interspecific backcross panel
mapping, we have localized the Cig30 locus to the distal region of mouse
Chromosome 19, between the Tlxl and bzsl loci. Sequencing of the
corresponding lambda clone to completion revealed that the insert
contained yet another gene in the opposite orientation. It turned out to be
the newly identified homeobox gene Pitx3. Interestingly, the genes are
very tightly linked, so that the 3' ends of their long mRNA species are
complementary over a short stretch of nucleotldes. Unhke the Pitx3 gene,
Cig30 has no polyadenylation signal at the 3' end of the long mRNA form,

suggesting that polyadenylatlon of those transcripts is governed by other

signals. Our finding provides evidence for bl-directional transcription of a
several hundred base-pair long DNA region as a result of the extremely
tight linkage between Cig30 and Pitx3.

(Mo/1.2/136)

Isolation and characterization of cDNAs involved in

carbon metabolism in Laminaria digitata (phaeophyta) 1.
C. Boyen, P. Moulin, F. Crtpineau, and B. Kloareg
UMR 1931, C.N.R.S. and Laboratoires Go,mar, Observatoire
Oc~anologtque de Roscoff, BP 74, 29682 Roscoff cedex, France.

An expressed sequence tag (EST) approach was used to retrieve

eDNA clones involved in carbon metabolism in Laminaria digitata. Six
partial open reading frames were identified, respectively encoding an c~type carbonic anhydrase (CA), glucose-6-phosphate dehydrogenase
(G6PDH),
6-phosphogluconate
dehydrogenase
(6PGDH),
phosphoglycerate kinase (PGK), glycolate oxidase (GLO) and GDP-4keto-6-D-mannose-3,5-epimerase-4-reductase also known as fucose
synthase (FS). These enzymes were further characterized through
Southern blot analyses, amino acid sequence comparisons and patterns of
expression. In contrast to the other genes, which are expressed in both the
gametophytic and sporophytic generations of L. digitata, the c~-type
carbonic anhydrase messenger RNA was shown by RT-PCR and
Northern blot analyses to be present in the gametophytes only. The
evolutionary relationships of these genes and their interest as molecular
tools for investigating carbon fluxes in brown algae will be discussed.

s202

Abstracts FEBS'99

(Mo/1.2/137) Detection of a novel von HippeI-Lindau (VHL) gene

mutation by F-BESS
J. Brieger, E J. Weidt,, K. Gansen, C. Huber and H J Decker
Johannes Gutenberg Umverstt. 55131 Alamz, Germar(v

The von Hippel-Lindau (VHL) gene has been shown to be a tumor

suppressor gene. Carrier of a germline mutation will be predisposed to a
high variety of benign and malign tumors affecting different organ
systems. The early detection of gene carrier improves significantly the
clinical outcome and the patient's quality of life Direct sequencing
esspecially of large genes might be laborious and time consuming.
Therefore, most laboratories apply single strand conformational
polymorphism (SSCP) analysis as an initial screening technique. Major
disadvantages of this approach are the requirement of specialized
equipment and a limited detection rate of about 70 % To overcome these
problems, we applied the modified technique of fluorescence labelled base
excision sequence scanning (F-BESS)
A young patient without family history of VHL presented with a
hemangioblastoma of the cerebellum as the sole clinical feature not
sufficiant for establishing the clinical diagnosis of VHL. Applying F-BESS,
we detected a frameshift in exon 2 as a de novo germline mutation Direct
sequencing revealed an insertion of C at position 631/632 This is a novel
VHL mutation which results in truncation of the VHL protein. Applying
SSCP on the same DNA, no alteration could be detected Three further
family members were tested negative for the mutation by F-BESS in
accordance to lack of any clinical VHL features. As F-BESS appears to be
a reliable, fast and unexpensive method, we recommend this technique as
an initial screening method.
Keywords BESS, VHL, genetic testing

(Mo/1.2/139)

Cloning and characterization of the rat Tage4 gene :

striking similarities with the human CD155 gene
M.G. Denis, B. Baury, D. Masson, P. Lustenberger
Laboratoire de Biochimie M~dicale, Facultd de Mddecme, Nantes. Fram ~

The Tage4 gene (Tumor-associated glycoprotein E4) is expressed al

the surface of rat colon carcinoma cell lines, as determined by immunofluorescence analysis, in chemically induced rat colon tumors, as determined
by immunohistochemistry [1], and in Min mouse intestinal adenomas, as
determined by in situ hybridization [2]. In contrast, a barely detectable level
was found on normal adult rat colon and lung, and no expression could be
detected on the other normal rat and mouse tissues tested. The Tage4 cDN
has been isolated and sequenced [3]. A search of tile nucleic acid databa.,,:'
using the Basic Local Alignment Search Tool (BLAST) revealed a significw '
homology (47.2%) to human poliovirus receptor, CD155, a member of the
immunoglobulin supergene family [1,3]. The Tage4 gene has been mapped to
rat chromosome 1q22 and mouse 7A2-B I, regions that are homologous to the
long arm of human chromosome 19, where is located the CD155 gene.
In order to gain insight into the molecular mechanims involved in its
overexpression in cancer cells, we isolated and characterized the rat Tage4
gene. A rat PAC DNA library (RPCI-31, http://resource.rzpd.de), was
screened using the rat Tage4 coding sequence as a probe. Eight clones were
isolated. One was selected and further characterized (clone RPCIP712N22604Q2). The rat Tage4 gene spans - i 5 kb. Amplifications were performed using'
oligonucleotides derived from the eDNA sequence. This allowed us to identif?
7 introns ranging in size from 588 bp to -3,300 bp. All splice donor/acceptant
sites conform the GT/AG rule. One belongs to class 0, 5 to class I and 1 n~
class 2. The exons range in size from 35 bp to 354 bp. We compared these
data with the structure of the human CD155 gene. The introns were found tc
be located at identical positions on both genes, and belong to the same classes.
A PCR-based genomic DNA walking technique was used to isolate the 5"
flanking region of the Tage4 gene. An -1 kb fragment has recently been
amplified and cloned. The presence of a putative promoter involved in the
overexpression of this gene in rat cancer cells is being analyzed.
[1] Denis, Int. J. Oncol. 12 (1998) 997.
[2] Chad6neau et al., Int. J. Cancer 68 (1996) 817.
[3J Chad6neau et al., J. Biol. Chem. 269 (1994) 15601.

(Mo11.2/138) Characterisation of the STK11 gene locus in patients with

sporadic colorectai cancer and colon cancer cell lines
A Brieger, J Trojan, J Raedle, S Zeuzem
Department o f Internal Medtcine, Untversity o f Franlqfi~rt,
Germany

Background' A gene mutated m Peutz-Jeghers syndrome (STKll at

chromosome 19p13.3) has been identified recently (Nat. Genet. 1998, 18:
38-4; Nature 1998, 391: 184-7). Due to the cancer susceptibility in this
syndrome and the frequent allelic loss of the STKI 1 locus in hamartomas
of these patients it has been suggested that the STKI 1 gene may act as a
tumor suppressor The aim of this study was to investigate whether the
S T K l l gene is affected in sporadic colorectal cancer Patients and
Methods: Paraffin-embedded tumor specimen from 38 patients with
sporadic colorectal adenocarcinoma were analyzed for loss of
heterozygosity (LOH) at the microsatellite loci D19S886 and D19S878
on 19p13 3 by fluorescent PCR amplification and subsequent automated
fragment analysis In tumors exhibiting LOH the STK11 gene was PCR
amplified and directly sequenced. In addition, the DNA of four human
colon cell lines (CaCo 2, HT 29, SW 948 and Colo 320) was isolated,
and the STK11 gene sequenced. Results: PCR amplification of both
microsatellite loci was successful in 36 patients, whereas in two patients
only one locus could be amplified. LOH at the D19S886 locus, located
telomeric to the STK 11 gene, was observed in one patient (2.6%) In
four tumors (10.5%) allelic loss at D19S878, flanking the STK 11 gene
centromeric, was observed Three out of five cases with LOH at
D19S886 or D19S878 were classified as stage IV tt~mvrs, two as stage I11
tumors. Four &these tumors were either located in the rectum or sigmoid
colon. Somatic mutations of the STK11 gene couldn't be detected neither
m these five tumors nor in the four colon cell lines. Conclusions In
sporadic colorectal cancer allelic loss at the STK 11 locus is a rare event.
In our series LOH at the STK1 l locus was not associated with somatic
mutations of the S T K l l gene, but with advanced tumor stage and left
sided location.

(Mo/1.2/140)

The genome comparison of bacteriophage T4 and the

phylogenetically distant PseudoT-even phage, RB49.
C. Desplats, C. Dez and H. M. Krisch
LMGM, 118 rte de Narbonne, 31062 Toulouse cedex

A PCR analysis of a collection of _60 bacteriophage having T4-type

morphology indicated that four of them were much more phylogenetically
distant from T4 than all the others. DNA hybridization revealed that only one
contiguous segment of about 10% of the genome could hybridize to T4 DNA
under stringent conditions. Nucleotide sequencing of this region showed that it
contained the genes coding for homologues of the major virion structural
components (capsid, collar, and contractile tail proteins). Since their genome
sequences had significantly diverged from the T-evens, these four phage were
reclassified as a new subgroup of the T4-type phage, the pseudoT-evens.
The construction of a library containing cloned DNA of one of these
pseudoT-evens, the bacteriophage RB49, permitted us to sequence about 40 %
of this genome. These random genome sequences had, in general, insufficient
nucleotide sequence homology to be detected as homologous to T4 in a
BLAST analysis using the NCBI database. However, when translated, 70% of
them had a significant homology to T4 encoded proteins (in general varying
between 30 and 70% identity). Thus, many of the homologues of the essential
genes of T4 can be identified in RB49. Nevertheless, 30% of the RB49
sequences had no homology with any entry in the database. We have also
established that the overall organization of the RB49 genome is very similar to
that of T4, although there are differences. For example the T4 genes involved
in the synthesis of the modified base HMC and the glucosylation of DNA
containing it are absent from the segment of the RB49 genome that has its
replication genes. We have identified a putative RB49 early promoter
consensus sequence which differs sigmficantly from that of T4; however, the
RB49 consensus late promoter sequence is identical to T4. For the moment, the
existance of T4-1ike middle mode promoter sequences in RB49 is dubious.
Taken together these results suggest that RB49 and T4 originated from a
common ancestor but that they have now diverged considerably. The pseudoTeven RB49 apparently evolved in genetic isolation from the T-evens, perhaps
because it has occupied a different ecological niche. The further study of the
structure and function of the genome bacteriophage RB49 and its comparison
with T4 should provide fundamental insights about the mechanism of viral
genome evolution.

Abstracts FEBS'99

(Mo/1.2/141)

s203

Isolationof xylanasegenes froma ruminalprotozoan,

(Mo/1.2/142)

Polyplastron multivesiculatum.
b ~INRA de Theix, St Genes Champanelle, France
Rowett Research Institute, Aberdeen, Scotland, UK

In the rumen, plant cell wall polysaccharides are degraded by a

community of anaerobic micro-organisms including protozoa which can
represent up to 50% of the biomass. While the ruminal bacteria and
fungi have been well described for their contribution to the breakdown
of plant cell wall material, the role of the protozoa in this process has
remained uncertain.
To address this issue, a cDNA library was constructed from cells of the
ruminal protozoan Polyplastron
multivesiculatum. This library was
screened for plaques expressing xylanase activity and two positive
clones, XynA and XynB, were further analysed. Sequencing of the
inserts revealed an open reading frame encoding a family 11 xylanase
for XynA and a family 10 xylanase for XynB which are the only known
xylanase families. The coding sequences showed the presence of typical
signal peptides and are followed by a polyadenylation signal and a poly
A ~ tail, supporting the eukaryotic origin of the sequences. Furthermore
the cloned DNA are highly AT-rich, and codon usage highly biased in a
manner typical of protozoa but different from cellulolytic rumen
bacteria and fungi. Phylogenetic analysis show that XynA and XynB
are closer to Gram positive bacterial xylanases than to fungal ones,
suggesting horizontal transfer of these genes. The enzymatic properties
of the two protozoal xylanases expressed in E. coli were alsoc
compared.
This is one of the first reports of the isolation from a ruminal protozoan
of genes sequences encoding enzymes involved in plant fibre
degradation. This work supports the view that these organisms play a
potentially significant role in ruminal fibre breakdown.

(M0/1.2/143)

Chromosomal localization of the human

poly(A)-binding proteins (PABP) genes.
C.F~ral a, A. Pawlak a, M.G Matt~i b, and G.Guella~n a
* Unitb INSERM 99, Hrpital H. Mondor, 94010 Crbteil, France.
bUnit~ 1NSERM 406, Facult~ de mbdecme, 13885 Marseille, France.

The poly(a) binding protein (PABP) regulates eukaryotic mRNA

stability and translation by binding to their poly(a) tail. In human, this
highly conserved protein family displays four isoforms: PABP1,
inducible PABP (iPABP) and two isoforms, PABP3 and PABP4 that
we recently isolated and characterized from human testes [1]. By in
situ hybridization, and using the eDNA coding for PABP3 as probe,
we localized PABP specific sequences on human chromosome at five
different loci: 2q21-q22, 13qll-q12, 12q13.3-q15, 8q22, and 3q24q25. These results are similar to the loci identified recently by an other
group [2], except the location on chromosome 2. In order to assign
precisely each PABP genes to these loci, we designed specific
oligonucleotide primers to amplify a short fragment of each known
PABP gene. The PCR reactions were realized on genomic DNA from a
human-rodent monoehromosomal somatic cell hybrids panel. A
specific oligonucleotide probe was used to reveal each amplification
product. PCR amplification identified specific products on the
following chromosomes. 8 (PABPI), 1 (iPABP), 13 (PABP3), and 15
(PABP4). These results revealed that two loci (iPABP, chromosome
1 ; PABP4, chromosome 15) were not initially detected using in situ
hybridization. In addition other loci (2q21-q22, 12q13.3-qlS, and
3q24-q25) might be indicative of other related PABP genes not yet
identified. This study definitively established that two functional
PABP are localized on 8q22 (PABP1) and the testis specific isoform
(PABP3) on 13ql l-q12.
[1] Feral et al., submitted for publication (1999)
[2] Morris et al., Genomics, 15, (1993) 209

l~lueonaff study au~s ~eaelrate speaes ofdan~osomes with

the majorimptintedgene dusters defines common a~cesWal
relationships.
C. Diatloff-Zko

E. Devillard~'b,J. Newboldb, K. Scottb,

J.P. )ouany', J. Wallaceb, H. Flintb, E. Foranoa

~lSERMU383 ~

Na:l~-~rts M~ad~ 149,ruedeSAr~ 75743P~i~ Franae

Genomic imprinting is an epigenetic phenomenon by which one parental

allelehas partial or total loss of expression. Most of the known imprinted
genes have been identified in man and mouse, the majority of which 16 out
of 28 are grouped into two chromosome regions located in man to l 1p15.5
and 15ql 1-q13 and in mouse to chromosome 7. It has been suggested that
this clustered organization is related to a regional structure and spreading of
the epigenutype, chromatin packaging, regulatory process or function of
genes in these regions. To understand the origin of this clustered
organization a comparative mapping of conserved chromosome segments
was performed in six vertebrate species from man to zebrafish. The question
addressed was: is it possible by using an evolutionary approach to shed light
on the chromosome organization evolution of imprinted genes.
The human and mouse chromosome with the major imprinted domains were
dissected into conserved segments and aligned. To be used as a tool for
comparing the chromosomes, the segments were designated and then
assimilated to blocks which were subsequently used like <<Lego ,. The
organization of the blocks forming the chromosomes in six vertebrate species
from different evolutionary lineages is species specific. Most of the
conserved blocks could also be assigned to chicken and zebrafish
chromosomes. A model for a consensus ancestor could be proposed. It is
suggested that the regions with the clusters of imprinted genes in man and
mouse are located on chromosomes that have a common ancestral origin. The
possibility that imprinting exists in other vertebrate species than mammals
has to be considered.
The analysis could be used for predicting the location of new imprinted
regions. One central question raised is whether the parental expression
pattern of imprinted genes was established before or after the structure
became set. Evolutionary studies of imprinting may help to elucidate the
link between structural organization and function.

(Mo/1.2/144)

Presence of a putative gene within the partially deleted

D4ZA locus of a patient with FSHD
J. Gabri~ls, M.-C. Beckers, D. Collen, A. Belayew
CMVB, Universtty of Leuven, 3000 Leuven, Belgium

Facioscapulohumeral muscular dystrophy (FSHD) is linked to the

polymorphic D4Z4 locus on chromosome 4q35. In non-affected individuals,
this locus comprizes 10-100 tandem copies of members of the 3.3 kb
dispersed repeat family. Deletions leaving 1-8 such repeats have been
associated with FSHD, fur which no candidate gene has been identified [ 1].
We have determined the complete nucleotide sequence of a 13.5 kb E c o R I
genomic fragment comprizing the only two 3.3 kb elements left in the
affected D4Z4 locus of a patient with FSHD. Sequence analyses
demonstrated that the two 3.3 kb repeats were identical. They contain a
putative promoter that was not previously detected, with a TACAA instead of
a TATAA box, and a GC box. Transient expression of a luciferase reporter
gene fused to 182 bp of this promoter demonstrated strong activity in
transfected human rhabdomyosarcoma TE671 cells. In addition, these 3.3 kb
repeats include an open reading frame (ORF) starting 149 bp downstream
from the TACAA box and encoding a 391 residue protein with two
homeudomains (DUX4) [2]. In vitro transcription/translation of the ORF in a
rabbit reticulocyte lysate yielded two 35S Cys/35S Met labeled products with
apparent molecular weights of 41 and 70 kDa on SDS-PAGE, corresponding
to DUX4 monomer and dimer, respectively.
In conclusion, we propose that each of the 3.3 kb element in the partially
deleted D4Z4 locus could include a D U X 4 gene encoding a double
homeodomain protein.
This study was performed in cooperation with A. De Vriese, J. Hewitt, S.
van der Maarel, R. Frants and G. Padberg.
[1] Wijmenga et al., Nature Genet, 2, (1992) 26.
[2] Ding et al., Hum Mol Genet, 7, (1998) 1681.

s204

Abstracts FEBS'99

(Mo/1.2/145)

Montecarlo Simulation Applied To Mahalanobis

Gene Distances
S. Garcia-Vallv6, A. Rojas, J. Palau and A. Romeu

(Mo/1.2/146)

Rovira i Virgih University. Department of BiochemtstO' and

Biotechnolog). E-43005 Tarragona. Spain.

DNA Sequence of the Flagellar Gene Cluster from th e

Thermophilic Bacillus PS3 :Switch, Ring, Fook, and
Biosynthetic Proteins of the Flagellar Motor
M. lshizuka, K. Ogawa, Y. Takahashi, M. Onda
K Tsujimoto, and M. Shinozaki
Department of .4pphed ChennsttT, Chuo Universal To~. o, Japan

Elucidation of the complete genomic DNA sequence of prokaryotic organisms

is now growing up very fast. It is convenient to develop new statistical
computer methods, in order to determine which genes exist within the whole
bacteria/chromosome that significatively deviates from the averaged codon
usage of the own organism. As a measure of the distance between the codon
usage of a gene and the mean of the genomic codon usage of an organism, the
Mahalanobis distance was calculated using the method described by Chou and
Zhang (1995) [1]. Thus each gene corresponds to a vector or a point in the 61D space where its coordinates are the relative frequency of uses of the 61
codons, the stop codons are not included, and each organism corresponds to a
vector or point the coordinates of which are the mean values. The Mahalanobis
distance takes into account the coupling effect among different codon
frequencies since covariance matrix is involved in this computer approach. A
higher value of the distance represents more differences from the mean values.
In our study, we have chosen three bacteria the genomic sequence of which
has been elucidated: Bacillus subtdis which ~s a common soil and water
saprofite; Escherichia coli which is present in the human gut; Mvcobacterium
tuberculosis which causes the human diseases of tuberculosis and lupus. The
Mahalanobis distance for each gene to the organism mean value was calculated
in the three bacteria. The number of repetitions for a given Mahalanobis
distance interval was graphically represented against the increasing values of
these Mahalanobis distances. In order to establish an upper limit that enables
to exclude outliers, the Monte Carlo procedure was used. This consists in the
following: from the mean and c~values for the codon usage for each organism,
a random sample of 10000 sequences were generated, whose Mahalanobis
distances followed a normal distribution. In the case of the three bacterial
organisms, the skewness and kurtos~s values were near to 0 for the random
generated samples, meanwhile were significant higher for the real
distributions. In the case of B. subtilis and E. cob, the Mahalanobis distance
was calculated by the procedure of eigenvalues and eigenvectors, whereas in
the case of M. tuberculosis the procedure of the inverse matrix had to be used
in order to obtain a normal distributmn. Genes with a Mahalanobis distance
higher than 2c~ from the mean value of the Monte Carlo simulation wcre
considered candidates to be extraneous genes of the respective organisms. The
calculated upper limiting values were 35.1 for B. subtilis, 34.4 for E. coli and
25.3 for M. tuberculosis.
1. Chou, K.C. and Zhang, C.T. Crit. Rev. Biochem. Mol. Biol. 30. (1995)

(Mo/1.2/147)

IDENTIFICATION OF DIFFERENTIALLY EXPRESSED mRNAs

AFTER CHRONICADMINISTRATIONOF FLUOXETINETO RATS
A. Jagerschmidt, D. Fage, P. Moser and J.M. Culouscou
Genomic Biology and CN$ Research Departments, Synthelabo Recherche,
Rueil-Malmaison, France.

Depression reflects a complex process in which alterations in the

expression of the genome are supposed to play an important role. In the
present study we investigated changes that occur at the transcription level
in brain of rats treated with the antidepressant drug fluoxetine. Since
maximum antidepressant effect requires several weeks of treatment, we
have studied the effect of a 21 day treatment with fluoxetine (10mg/kg
ip). in order to isolate and identify both up- and down-regulated gene
transcripts, we have constructed and characterized two subtracted cDNA
libraries from rat frontal cortex. Randomly chosen individual clones
from each library were arrayed onto nylon membranes and hybridized
with complex probes generated by RT-PCR from frontal cortex mRNAs
of treated and untreated rats. Candidate clones, identified by their
differential hybridization profiles were sequenced and searches for
homology with genes in sequence databases were performed. Validation
of a selected number of candidate clones was undertaken using semiquantitative RT-PCR (i) on frontal cortex of chronically treated vs
untreated rats, in order to confirm their differential expression, (ii) on
frontal cortex of acutely treated vs untreated rats, in order to demonstrate
that their differential expression was specific to a chronic but not an
acute treatment with fluoxetine (iii) on several brain regions of treated
vs untreated rats to see if the differential expression of the selected genes
was localized in specific brain areas. In conclusion, the subtractive
cloning methodology we used allows the identification of candidate
factors that might provide new information on adaptative changes in the
CNS following chronic antidepressant treatment.

The bacterial flagellum, a rotary device for motility, is composed of three

structurally defined parts, the filament, the hook, and the basal body. The
filament composed of flagellin is the long helical structure. We have already
reported the molecular cloning, sequence and over-expression of thermostable
flagellin as the fusion protein with maltose binding protein [1}.
In order to analyze thermo-stability and construction mechanism of PS3
flagellar motor, the flagellar motor gene cluster (major part offla/che operon)
was isolated from the PS3 genomic library using the two synthetic
oligonucleotides targeted against the conserved region of fld gene product
among several micro organisms, and the complete sequences of 16 genes and
the partial sequences of 2 genes were determined.
The order of their reading frames (size in base pairs of the genes) was. -l/tiE(part of Y-downstream region) - fltF(1568) - fltG(lO17) - fltH(654) tiff(1252) -flU(447) -tifF(474) - fliK(1879) - ylxG(432) - flgE(1065) - fltL(429)
-firM(1002) -.fl~Y(1002) - cheY(360) -.fllZ(666) -fltP(672) -fl~Q(270) -fliR(part
of 5'-upstream region) -//-. The deduced amino acid sequences of the switch
(FliG, FILM, and FIiY/N), MS ring (FIiG), fook (FIgE), chemotaxis (CheY),
flagellar biosynthetic (FliZ, FliP, and FliQ) proteins, etc. were compared with
those of the correspondmg proteins from another microorganisms (BS:Bacdlus
subtilis, EC:Escherwhia cob, ST'Salmonella typhimurium,
TP:Treponema
pallidum, and AA:Aqutfex aeolicus). N-terminal region of FliY(both PS3 and
BS) is absent from FliN(both EC and ST). High conservation of hydropathy
pattern on Flip and C-terminal region of FhY/N raises the possibility of little
variation in protein structure, and the observed substitutions in the
thermophilic subunits seem to increase their propensities to form secondary
structures and extemal polarities to form terttary structures.

(Mo/1.2/148)

Low Copy Number Target Amplification

by Adaptive Hot Start PCR
P. Kainz, A. Schmiedlechner, HB. Strack
Institute of Chemistry and Biochemistry, Untversityof
Salzburg, Hellbrunnerstrasse34, A-5020 Salzburg. Austria

Especially in the amplification of low copy number targets in a background of non-specific nucleic acids "hot start" polymerase chain reaction
(PCR) has been shown to eliminate undesirable amplification products [ 1]
Hot start PCR has been implemented so far by mechanically separating a
reagent, by blocking the polymerase with an antibody, by using modified
forms ofDNA polymerases that are inactive at room temperature or by
using single-stranded DNA aptamers that were shown to inhibit the
activity of various enzymes at temperatures below 40C [2].
We describe a new method for producing hot start conditions: The DNA
polymerases from Thermus aquaticus and Thermusflavas were found to
bind efficiently to short double-stranded DNA fragments without sequence
specificity. This property is exploited by immobilizing the polymerase to
such fragments before its addition to the PCR mixture. The temperature at
which the polymerase molecules are detached upon starting the reaction is
adaptable to the conditions required via the melting temperature of the
DNA fragments. Furthermore - in contrast to existing methods - the
protection against mispriming at a lower than the optimal annealing
temperature is not restricted to the reaction setup, but persists throughout
the PCR reaction.
[I] Chu, Q. et al., Nucleic Acids Res., 20, (1992) 1717.
[2] Lin, Y. et at., J.MolBiol., 271, (1997) 100.

Abstracts FEBS'99

(Mo/1.2/149)

Dynamic o f h o b o transposition in Drosophila transgenic lines

[.adev~'e V ~. Aulard S b Bi~mont C., Chaminade N ~'. P~riquet G ~
Lemeunier F ~ " " 1.13M,I( ~, I "mvervit(" de Poitwr~', l~mtser~ e(. \ R N

s205

(Mo/1.2/150)

J Mates a, C.Ptrez a, L.Olalla a, M.Blanca b, F.Sanchez-Jimenez a

Dpt.Molecular Biology &BIochemtstry, UnivJdtlaga, 29071 M~daga, Spare,
bUnit of Allergy. HospJtal Carlos Haya. 29010 Mddaga, ,Spain

L P G.E., G!f}'rette CCNR.X, L.B.G.RP, I:niverstt~ Lyon 1. ['dleurbanne

J LR.B L, I .htversite t+an~.oisRabelais, 7ours - Prance

Transgenic lines with complete hobo element were obtained from

Hikone line (line without TEs such P, I or hobo). These transgenic lines were
studied along generations to determine the evolution of the structure of the
element (complete or deleted), the number of insertion sites, their localization
on the polytene chromosomes and the possible appearance of new
rearrangements. In these lines, transposition was estimated by the level of
hobo insertions and excisions at different generations.
During the 80 first generations, we demonstrate that hobo transposable
element invades all chromosomes in euchromatin. Some chromosomal
regions have few element inserts although others present a high number of
hobo insertions.The hobo distribution varies between larvae, within a line at a
given generation. This implies excisions and insertions at random.
Nevertheless, some insertions hotspots have been found. They correspond to
identical insertion sites present in most of the transgenic lines and maintained
over generations. Five are located on chromosome 2 and six on chromosome
3. The number of insertions remain low (1 to 10) and the hybridization
signals were unique at each insertion site, except for one line (about 20 at
G80) Then, in that line, at five different loci located on both autosomes,
double, and even triple, hybridization signals were detected and maintained
over the following generations.
These results could be explained by two transposition mechanisms.
Until G80, the increasing number of hobo insertions may be the result of a
random transposition, with excision and insertion in all loci. Then, after G80,
in one line, a regulation of the transposition may appear, without excision in
some loci.

Histamine has been fundamentally associated to the allergic processes and

the anaphylactic reaction. In organisms, L-histidine decarboxylase [HDC
(EC 4.1.1.22)] is the regulatory key enzyme for histamine biosynthesis
Structure of the 24 kb in length human HDC gene was recently identified,
containing 12 exons [1]. The mechanism(s) for the conversion of the
primary translation product (74 kDa) to the 53 kDa form with full activity
are still not well established. It has showed that the level of HDC mRNA
and its activity increases under various stimulations, underlying the great
importance of the inducible expression of HDC
In the promoter sequence has been located several cls-acting elements
important in the HDC gene transcription regulation [2]. In addition, it has
been described aberrant mRNAs of I-IDC in mammalian cells, generated
by alternative splicing and transplicing in different points of the sequence
[3]. This fact shows that HDC gene can present altered expression
products. If as a result of the codification and/or the splicing out of the
gene, truncated or mutated version of the protein were appeared,
enzymatic in vivo activities could be very affected. Thus, the possibility of
the presence of natural more stable HDC mutants could be related with
histamine-dependent pathologies, as ulcer, neurological problems and
allergy.
The goal of our study was to investigate alterations in the HDC promoter
of allergic patients. Genomic DNA from whole blood of patients suffering
from allergy was extracted. Using PCR and agarose eleetrophoresis we
isolated a fragment of 1997-bp, containing the promoter HDC sequence
Multiple restriction analysis and polyacrylamide electrophoresis were
performed to identify putative mutants. Clones were sequenced and some
mutations were found next to the box ACTGG, the consensus site for the
regulatory leader-binding protein-I
[1]
[2]
[3]

(Mo/1.2/151)

Structure and expression of the variant MCH genes.

J.L. Nahon ~, A. Viale, A. Courseaux, F. Richard, C. Ortola,
P. Vernier, F. Presse and B. Dutrillaux
OlPMC/CNRS, Sophia-Antipolis, 06560 Valbonne, France

The initial genetic events associated with the emergence of new functions
during Primate evolution remain largely debated. The so-called "potogenes"
may represent a "reservoir" of modular elements which may be reactivated and
led to a functional phenotype completely different from this of the original
founder gene [1]. P M C H L 1 and P M C H L 2 are two copies, respectively on
human chromosome 5p14 and 5q13, of the variant melanin-concentrating
hormone (MCH) gene, a 5' end truncated version of the P M C H gene mapped
on chromosome 12q23. Both variant MCH genes emerged recently during
Primate evolution by a combinaUon of translocation, duplication and truncation
events occurring sequentially in the Hominoidea lineage [2]. The questions of
the regulation and putative functions of these genes in Primates are central
issues we examined here. We established first the structure and fine
chromosomal mapping of the variant MCH genes on human chromosome 5
and revealed several point mutations and the presence of CA/I'A repeats that
allowed to discriminate between both copies. Using a combination of RACEPCR, RT-PCR and sequencing analysis we provide strong evidences for the
expression of P M C H L 1 but not P M C H L 2 gene in the human fetal, new-born
or adult brain. Sense potentially coding RNAs as well as non-coding antisense
RNAs were identified and they displayed a region-specific expression in the
developing human brain. Strikingly, sense unspliced RNAs of the P M C H L 1
gene comprised a novel open reading frame and they were found actually
translated in a coupled transcription/translation assay, after transfection of
tagged-variant protein vectors in COS cells and in the fetal and adult human
brains. Therefore, the variant MCH genes provide a unique example of a de
novo creation of a gene family in the Primate lineage with a differential
expression in the human brain, one gene copy encoding putative novel
proteins.
[1] Brosius and Gould, Proc. Natl. Acad. Sci. USA 89 (1992) 10706.
[2] Viale et al. Mol. Biol. Evol. 15 (1998) 196.

Alterations in the 5'-flanking region of the human HDC

gene in allergic patients

K. Yatsunami, et al., J. Biol. Chem., 269, (1994) 1554.

Z Zhang et al., J. BioL Chem 271, (1996) 14188.
R. Mamune-Sato et al, Eur. J. Biochem. 209, (1992) 533.

(Mo11.2/152)

Metalioprotein genes from the sulphate reducing

bacterium Desulfovibrio gigas
Solange Oliveira ~'c , G. Silva a , M. Broco ~, M. Agostinho~ , (3
Flores a,b, M. Jo~o Godinho and C. Rodrigues-Pousada a.b

"ITQB- UNL Apartado 127, 2780 Oetras, Portugal, o IGC, Apartado 14, 2780 Oeiras,
Portugal, ' Universi~ of Evora, 7000 Evora, Portugal
Desulfovibrio gigas is a sulphate reducing bacterium that is involved in

processes such as the sulphur cycle, biocorrosion, biomineralization and

metal metabolism. D. gigas genome is about 1.6 Mb, from which we
sequenced several metalloprotein genes:
the rubredoxin oxygen
oxidoreductase (ROO) (encoded in the same polycistronic unit as the
rubredoxin [1], both thought to participate in the electron transfer chain that
couples NADH oxidation with oxygen reduction to water); the neelaredoxin
(a blue monomeric protein of 15 kDa with two non-heme irons, encoded in a
polycistronic unit which contains two additional ORFs homologous to
chemotaxis proteins (methyl-accepting chemotaxis and CheW proteins)[2]);
the molybdenum aldehyde oxido-reductase (MOP) (that shows a high degree
of sequence identity with the mammahan xantine-oxidase [3] and is cotranscribed with a putative nitroreductase and other iron-sulphur proteins);
flavoredoxin and h m c (high molecular weight cytochrome). The genetic
organization of the sequenced metalloprotein operons helped us to understand
the biochemical function of the proteins from the identified genes. We also
started the analysis of Desulfovibrio gigas genome, namely the orgamzation
of coding regions and regulatory sequences, codon usage frequencies, as well
as sequence comparisons with close species like D. vulgaris and D.
desulfuricans in order to establish phylogenetic relationships.
[1] Gomes, C. et al., 1997, J. Biol. Chem., 272, 22502
[2] Silva, G. e t a l , 1999, Eur. J. Biochem. 259, 235
[3] Romao, M.J. et al, 1995, Science, 270:1170
This work was supported by PRAXIS XXI (PRAXIS/PCNA/P/BIO/32/96).
G.Silva has a PRAXIS XXI grant (BD9016/96).

s206

(Mo/1.2/153)

Abstracts FEBS'99

Identification and characterization of a human

testis specific poly(A)-binding protein (PABP).
C. F6ral, A. Pawlak, G. Guella~n.

(Mo/1.2/154) Sinescan: a program for the identification of tRNAderived retroposons in genomic sequences
R. Percudani, C. Mariano, S. Ottonello
Istituto di Scienze Biochimiche. Umversitgt dt Parma, 43100 Parma

Unitd INSERM 99, H6pital Henri Mondor, 94010 Crdteil, France

Poly(A)-binding protein (PABP) regulate the stability and the

translation of mRNAs by binding to their polyA tail. In human three
PABP isoforms have been characterized : PABP1 which is ubiquitously
expressed, PABP2 which results from an alternative splicing of the
PABPI transcript and an inducible form (iPABP). During the
characterization of a large population of human testis "Expressed
Sequence Tag" (EST)[1], we identified two new PABP transcripts,
named PABP3 and PABP4. The analysis of PABP3 mRNA revealed
91.2% identical nucleotide residues with PABP1 mRNA corresponding
to 92.5% identity at the protein level. The main difference resided in the
5'untranslated region of PABP3in which no polyA stretch was detected
as compared to PABP1 (6 polyA streches between a.a. 70 and 130).
Northern blot analysis, using specific oligonucleotides as probes,
revealed two PABP3 mRNAs (2.1 and 2.5 Kb) in the testis and no
messenger in the 16 other human tissues tested. By m silu hybridization
on human testis sections, these mKNAs were localized in round
spermatids. The protein corresponding to PABP3 mRNA, produced in
a m vitro transcription/translation system, exhibited a strong affinity for
poly(A)-Sepharose. This result demonstrates that PABP3 encodes for a
fully functional protein specific for human testis. The analysis of
PABP4 nucleic sequence revealed 87% identical nucleotide residues
with PABP1, but with a frameshift after codon 56. This PABP4
corresponds to an expressed pseudogene. Using polymerase chain
reaction on human genomic DNA, we demonstrate that PABP3 as well
as PABP4 are encoded by intronless genes. In this work, we
demonstrate the presence of a new functionnal PABP isoform
specifically expressed from a retroposon in human testis.

(Mo/1.2/155)

Comparative genomic analysis of the

interferon/interleukin-lO receptor gene cluster.
J. Reboul, K. Gardiner, D Monneron, G. Uz6, G.Lutfalla
lnsfltut de Gdn~flque Mol~culatre,~WRS, Montpether, France.

lnterferons and interleukin-10 are involved in key aspects of the

host defence mechanisms. Human chromosome 21 harbours the
interferon/interleukin-10 receptor gene cluster linked to the GART gene.
This cluster includes both components of the interferon alpha/beta
receptor (IFNAR1 and IFNAR2) and the second components of the
interferon gamma receptor (IFNGR2) and of the IL-10 receptor
(IL10R2). We report here the complete gene content of this GARTcytokine receptor gene cluster in human and the use of comparative
genomic analysis to identify chicken IFNAR1, IFNAR2 and IL 10R2.
We show that the large scale structure of this locus is conserved in
human and chicken but not in the pufferfish Fugu rubripes. This
establishes that the receptor components of these host defense
mechanisms were fixed in an ancestor of the amniotes. The
extraordinary diversification of the interferon ligand family during the
evolution of birds and mammals has therefore oceured in the context of
a fixed receptor structure.

New computer tools are needed for the analysis of the complex
genomes of multicellular organisms. Eukaryotic cells are habitats of a
wide variety of mobile elements that are preserved in DNA repetitive
sequences. Among them, tRNA-derived short interspersed elements
(SINEs) are especially common in higher eukaryotes These elements
have a composite structure with a 5' region homologous to a tRNA, a
tRNA-unrelated 3'-region, and flanking A/T-rich regions and direct
repeats. They transpose through an RNA-mediated mechanism and are
thought to play a pivotal role in genome evolution.
The identification of SINEs in DNA sequences presently relies
on the search of homology with known families described by
consensus sequences or Markov models. Such a strategy, however, is
too conservative and does not allow the detection of elements
belonging to as yet uncharacterized SINEs families. We have thus
exploited RNA polymerase internal promoters and the flanking
hallmarks of the retroposition event as shared motifs for the
identification of new elements in large genomic sequences. Cluster
analysis of all positive instances is utilized to obwate the high false
positive rate (ca. 10 per Mb) resulting from this procedure. In this
way, true positives are grouped in distinct families of sequence
homology while false positives are recognized and discarded as
orphan sequences. Our results indicate that the program is capable to
identify and correctly predict the boundaries of retroposons belonging
to novel SINE families.

(Mo/1.2/156)

Evolution and conservation of the key catabolic

enzyme of aromatic amino acids, HGO
S.R. Sehmidt GPC-AG Martinsried Germany

In the catabolic pathway of aromatic amino acids several sevcrc

inherited metabolic diseases are linked to mutatioM in the
corresponding enzymes. One defect is well known undcr the
term Alkaptonuria. The enzyme which catalyses this only truly
irreversible step, the breakage of the aromatic ring, the
homogentisate 1,2- dioxygenase (HGO, EC: 1.13.11.15), has
been cloned and sequenced from several organisms like human,
mouse, aspergillus, but also from caenorhabditis, drosophila
and arabidospis. Recently for the first time HGO was also
identified in prokaryotic organisms like pseudomonas and
bordetella.
In this presentation the genomic structure of the HGO gene in
eukaryotie organisms will be summarised and compared. The
alignment of all so far completely known 8 HGO amino acid
sequences plus 4 other partially sequenced cDNAs reveals
domains of extremely high conservation which are shared by all
organisms.
All so far described human mutations affect conserved amino
acid residues. The positions of several important amino acids
like Tryptophane (W) and Histidine (H) which are probaby
involved in the binding of the Fe z* cofactor and Proliue are more
than 80% conserved in all organisms. Clearly some areas with
higher variability can be identified.
This alignment data is also used to derive a evolutionary tree
which clearly indicates, that the bacterial HGOs are closer
related to plant HGOs as expected when comparing other
proteins from these species.

Abstracts FEBS'99

(Mo/1.2/157)

Positional cloning of the o c gene: construction of a high

resolution physical map of the candidate region
JC. Scimeca, C. Trojani, It. Parrinello, and G.F. Carle
LEGM, UMR6549CNRS/UNSA, Facultd de M~decine, Nice, France

Bone remodeling relies on a dynamic balance between synthesis and

resorption of the bone matrix. These mechanisms are controlled
respectively by osteoblasts, derived from a common precursor of
ehondroblasts and adipocytes, and by osteoclasts, a multinucleated cell
from hematopoietic origin. This fine equilibrium must be maintained, not
only during development, but throughout the life of every individual to
assure natural bone remodeling as well as post-trattmatic repair. Should this
equilibrium be affected, various pathologies will develop such as
osteoporosis, due to hormonal disfunction, or osteopetrosis, when bone
resorption is impaired.
The mouse osteosclerotic mutant (oc/oc) displays a lack of
resorption of the osteodast cells leading to an osteopetrotic phenotype with
a general increase of bone density. We have undertaken a high resolution
genetic map of this mutation using polymorphic markers in an interspecific
intercross with Mus spretus. Based on a dozen of recombinant individuals,
we were able to restrict the candidate region to the pericentmmeric zone of
mouse chromosome 19 (MMU 19), which forms a syntenic group conserved
through evolution with band q 13 of human chromosome 11 (HSA 11q 13).
Starting from three microsatellites (one displaying a perfect
cosegregation with the disease phenotype, and the other two defining
respectively the centromeric and telomeric recombination boundaries), a
number of ESTs conserved between man and mouse, as well as anonymous
markers, we generated a physical map of this area and constructed Bacterial
Artificial Chromosomes (BAC) contigs over 1.5-2 Mb. In order to increase
the markers density, several approaches were undertaken: (1) sequencing
of the BAC ends, (2) a eDNA selection using the BACs and bone specific
libraries, (3) shotgun subcloning of the BACs, and (4) homology searches
in databases between HSA 11ql 3 conserved markers and mouse sequences.
Thus, in the candidate region, we have been able to isolate 15 BACs
defining an interval close to I Mb and containing 40 markers.

(Mo/1.2/159)

A new phagemid vector for positive selection of recombinants based on a conditionally lethal barnase gene
S. Yazynin, H. Lange, T.Mokros, S.Deyev and H. Lemke
Biochemtsches Institut, Universitdtzu Kiel, Klel, Germany.

Recently we described a plasmid vector (pMT440) which utilized the toxic

effect of the bacterial fibonuclease barnase from Bacillus amylohquefaciens
for positive selection for cloned inserts [1]. In the presence of the coexpressed barnase inhibitor barstar, pMT440 may be carried in any E.coli
harboring the lacP gene. The vector is lethal, however, when the tac promoter in front of the barnase gene is induced by IPTG. For lacla-negative E.coli
strains, pMT440 is lethal without induction. The full multirestriction-site
polylinker (MRS) of pUC 19 was inserted by site-specific mutagenesis into
the barnase gene at a site which does not alter the lethal effects of barnase.
Hence, this pMT440 contained the bamase-barstar cassette, the entire
pUC19 MRS under control of the tac promotor and a vector fragment conraining the ori and the ampicillin resistance gene of the pUC19 plasrnid.
Uncut or religated pMT440 does not support growth, whereas bacteria
survive when transformed by plasmids containing inserts in the barnase gene,
since the insertion of a foreign sequence into the centre of the barnase gene
inactivates the enzyme. However, this modified pMT440 had still the
disadvantages that the MRS was not flanked for the annealing of universal
sequencing primers and that it lacked the florigin for obtaining singlestranded DNA. These drawbacks could be overcome by the creation of a
new vector using the barnase as a conditionally lethal gene in connection
with the pBluescript II KS+ phagemid vector. We amplified the barnase gene
with specific primers for the flanking SacI sites and cloned it into pBluescript
II KS+ again using Sacl sites. In this way, the bamase gene was placed
exactly behind the polylinker. Cloning of an insert into the polylinker of that
new vector causes a frame shill and inactivation of the barnase gene.
Absence of the leader peptide sequence in the barrlase gene and barnase
inhibitor barstar could raise intracellular concentration of active toxic
bamase and interfere with the plasmid amplification. A special mutated
barnase gene coding for an enzyme with reduced activity was used to solve
this problem. This new vector pBa-7 was successfully applied for cloning
and sequencing of PCR-amplified fragments coding for the variable regions
ofimmunoglobulin genes or coding for a fragment of the reprolysin MDC9.
[1] Yazynin et al., Gene 169. 131-132 (1996).

s207

(Mo/1.2/158)

Identification of differential gene expression in murine

thymocytes after a leukemogenic irradiation
M. Verlaet, C. Duyckaerts*, G. Denis, C. Humblet, F.E. Sluse*,
J. Boniver and M.P. Defresne. Laboratory of Pathologtcal Anatomy,
*Laboratory of Bioenergettcs, Universityof Liege. Belgium.

In C57BL/Ka mice, a leukemogemc irradiation induces the

development of thymic lymphomas which are preceeded by the appearance
of thymus-dependent potentially neoplastic cells (preleukemic cells). To
identify and characterize the genes responsible for their preneoplastic
properties, we have used the suppression subtractive hybridization (SSH)
method. SSH is used to selectively amplify target cDNA fragments present
m the preleukemic cDNA but absent (or present at lower levels) in control
eDNA. Nine eDNA fragments were found corresponding to known
mRNAs: the mouse laminin receptor precursor; the profilin; the polyA
binding protein; the mouse high mobility group protein I; the topoisomerase
I; the clusterin; the mouse cytochrome c oxydase subunit 1; the rat
prostatein C3 subunit gene; and the mouse msulinoma mRNA, rig. Four
cDNAs fragments demonstrated significant matches with ESTs and
potentially represent cDNAs from novel preleukemic thymus-specific
genes. Differential expression of three of these eDNA fragments has been
confirmed by Northern blot" the laminin receptor precursor, the PABP and
the profilin. The western Nots for the lammin receptor precursor were also
performed and the results indicated a surexpression of the laminin receptor
of 67 kDa and his precursor of 37 kDA in preleukemic thymuses as compare
to the control thymuses during the preleukemic period. The profilin has
been shown to play a role in different actin-dependent cellular processes.
The PABP play a role m mRNA stability and in translation imtiatmn. The
cytochrome c oxydase activity was analysed by enzymatic tests during the
preleukemic period and compared to oxygen consumptton. The activity first
was increased in preleukemic thymuses 30 days after the last irraditmn, was
decreased to control values 45 and 60 days after the last irradiation, then
reincreased 75 days after the last irradiation as compare to control thymuses.
Similar evolution of cellular respiration was observed. These enzymattc
variations were not observed in thymuses of bone-marrow grafted mice.
Additional experiments are under progress to elucidate the relation between
these observations and the mechanisms ofcancerogenesis.

(Mo/1.2/160)

Deletion at 11q23.1 is frequent in mantle cell

l y m p h o m a and chronic lymphocytic leukemia /
small lymphocytic l y m p h o m a
Y. Zhu 1, O. Monni 1, K. Francilla 2, E. Elonen 2,
H. Joensuu 2, S. Knuutila L2
~Department of Medical Genetics, University of Helsinki;
2Laboratory of Medical Genetics and Department of Pathology
internal medicine and Oncology, Helsinki University Central
Hospital, Helsinki, Finland
ln,roduction:
It has been shown that deletion at I 1@3 is ~mc of
the mo,t l~cquent structural chromosome aberrations in mantle cell
lymphoma (MCL) and chronic lymphocytic leukemla (CLL). The
frequencies of this aberration have been found to be a-s high as 49% in
MCL and 30% in CLL. The purpose of our study was to investigate if
this aberration is common among other lymphoma types.
Methods:
From November 1997 to November 1998, we studted
113 samples of lymphoma / suspicious lymphoma that were sent to the
Laboratory of Medical Genetics of the Helsinki University Central
Hospital. Mononuelear cells of these samples were studied by
fluc,re~erlce in situ hybridization (FISH) using the yeast artificial
chmm,~,,ome (YAC) clone 755bl 1 mapped to 1Iq23.1. The region
represented by this YAC has previously been shown to be the minimal
common region of the 1lq deletion.
Results:
Fourteen of the 113 samples exhibited deletions at
11q23.1. Among them, 7 (out of 38, 18%) were CLLs / small cell
lymphomas (SCL), 4 (out of 16. 25%) were large B-cell lymphomas. 3
(out of 6, 50%) were MCLs. Among the 4 large B-ceil lymphomas with
11q23.1 deletion, two had been diagnosed as CLL earlier.
Conclusion:
In accordance with previous studies, deletion of
I 1c~2"~ 1 was frequent among MCL and CLL t SCL, but present only
occaslOllally in other types of lymphomas.

s208

Abstracts FEBS'99

5.2 RNA editing and other epigenetic modifications

(Mo/5.2/161)

Transfer RNA modification influences the virulence of

(Mo/5.2/162)

Shigellaflexneri by affecting the expression of the virF gene

J. M. B. Durand and G. g. Bjrrk

Department of Mierobzology, Ume&University, Umed,Sweden

Virulence genes of Shigellaflexneri, which are activated at 37 C

but not at 30 C, depend for their full expression of the presence
of the modified nucleosides queuosine (Q34) and 2-methylthioN6-isopentenyladenosine(ms2i6A37) in the tRNA[1,2]. These
modified nucleosides are present in the wobble position (position
34) and adjacent and Yof the anticodon (position 37),
respectively, in a subset of tRNAs. The synthesis of them depends
on the products of the tgt and miaA genes, respectively. We have
shown that the intracellular concentration of the virulence related
transcriptional regulator VirF is reduced in the absence of any of
these modified nucleosides. Moreover, this reduction of the
intracellular concentration of VirF is correlated with a
proportional reduction in the expression of virulence genes.
Overproduction of VirF in the tgt and the miaA mutants
suppressed the tRNA modification deficient induced avirulent
phenotype, suggesting that that the primary target for this
avirulent phenotype is a poor translation of virF mRNA caused by
the lack of these modified nucleosides. These results further
strengthen the suggestion that the concentration of VirF is the
critical factor in the virulence of Shigella.

Roles of RNase E and polyadenylation in

degradation of therpsO mRNA of E. coli:
E. Hajnsdorf and Ph. Regnier
UPR 9073 CNRS, IBPC, 13 rue P. et M. Curie, 75005 Pads, France

The rpsO monocistronic messenger encoding ribosomal protein $15

is degraded by 2 pathways. The first one is initiated by an RNase E
cleavage 10 nucleotides downstream of the coding sequence, the
truncated mRNA is then degraded exonucleolytically from its 3'
terminus mainly by polynucleotide phosphorylase (PNPase). RNase E
also cleaves the messenger at the beginning of the coding sequence
of rpsO. The second pathway allows, in the absence of RNase E,
PNPase and of RNase II, the degradation of the transcript when it is
polyadenylated by poly(A)polymerase I,
These two pathways overlap. Fragments which are generated by
RNase E cleavage(s) are also degraded by the polyadenylation
dependent pathway which is redundant with the exonucleolytic
degradation, and takes place independently of polyadenylation. Our
results show that endonucleolytic cleavage at the beginning of the
coding sequence activates the poly(A) dependent degradation of the
downstream RNA.
The role of polyadenylation is to stimulate the degradation of RNA by
providing an unstructured 3' end to structured RNA allowing the attack
of PNPase, RNase II and also an unknown nuclease.

[1] Durand, J. M. B. et al., J. BacterioL, 176, (1994) 4627.

[2] Durand, J. M. B. et aL, J. Bacteriol., 179, (1997) 577%

(Mo/5.2/163)

The yeast TRMI gene, coding for tRNA(m=2G=6)methylase,

and its homologous genes in some higher eukaryotes
J. Liu and K B. Str~by
Department ofMtcrobtology, Ume~universtty, S-901 87 Ume3. Sweden.

Yeast tRNA molecules with Guanosine at position 26 in the bend between the
D-stem and the anticodon stem is dimethylated by the enzyme
tRNA(m22G26)dimethyltransferase which is coded for by the single nuclear
TRM1 gene. The enzyme modifies nuclear as well as mitochondrial coded
tRNAs and is present in both organdies. Thus the enzyme has both a
mitochondrial and a nuclear targeting signal [1 ].
In Caenorhabditis elegans DNA we found homologies to Saccharomyces
cerevisiae TRM1 and the gene Ce Trml was cloned [2] and shown to code for
an enzyme that in vitro modifies position G26 of mutant SctRNAs that
specifically lack the G26 modification. It also modifies G26 in E. coil tRNAs
(which never has this modification) both in vitro and m vivo. When comparing
the Ce and the Sc wild type and mutant TRM1/Trml genes we find that they
share 39% identity in their amino acid sequence, that two positions in the Cterminal half is of crucial importance for activity, that the Ce C-terminal is
slightly extended compared to the one in the yeast protein and, remarkably, that
the Ce Trml gene seems to lack a sequence that could correspond to a
mitochondrial targeting signal in the modifying enzyme
The tRNA(m22G26)dimethyhransferase and/or its product m22G is known to
exist in many higher eukaryotes. In the data bank for human genomic DNA, a
sequences can be found that after elimination of introns could correspond to a
TRM1 gene, but then with a C-terminal region exceeding other Trml proteins
with about 100 amino acids. Comparisons between our clones of human cDNA
and of published genomic DNA point to several alternative C-terminal peptide
sequences, some of them with lengths more closely to the Ce enzyme. Data
from our cloning and characterisation of human and plant ScTRM1 homologues
and their gene products will be presented.
[1] Rose, A. M. et al.,Mol. Cell. Biol. 12, (1992) 5652; [2] Liu, J. et al., Gene,
226, (1999) 73.

(Mo/5.2/164)

A tRNA modification gene, miaE, affects the ironmetabolism in Salmonella typhimurium.

H. K. Lundgren, R. Leipaviene and G. R. Bj6rk
Ume~tUmversitydepartmentof Microbiology, UmeLSweden

The tRNA modification gene miaE in S. typhimurium codes for the

modification enzyme MiaE which is making the hydroxylation step in the
formation of the modification ms2io6A at position 37 on most tRNA:s
reading UNN codons[l]. The formation of ms2iorA requires among other
cofactors oxygen and iron[l]. A miaE2507::MudJ mutant, which is a null
mutant, is unable to grow on succinate, malate and fumarate and it grows
poorly on acetate and citrate[2]. The uptake of these compounds and the
enzymatic activities of the enzymes in the citric acid cycle as well as the
function of the respiratory chain are unaffected[2]. A miaE2507::MudJ
mutant is also affected in the iron metabolism of the cell. The mutant
lacks siderophores outside the cell and addition of siderophores restores
growth on succinate. The outer membrane profile of a miaE2507::MudJ
mutant three hours after a shift from a glucose minimal medium to
succinate minimal medium indicates that the proteins for siderophore
excretion and uptake are not siginificantly downregulated which is
consistent with the supplementation experiments made with siderophores.
The amount of active Fur (the iron uptake regulator that fimctions as a
repressor) seems to be increased in a minE mutant. Suppressors to the
succinate- phenotype have been isolated and at least two classes exists.
Those that both restores growth on succinate and excretion of
siderophores and those that only restores the growth on succinate. A
putative hypothesis where the miaE2507::MudJ mutation is unable to
produce siderophores through a poor translation of the major siderophore
Enterochelin is presented which might explain the inability of the mutant
to grow on suecinate, malate and fumarate.
[1] Persson B.C and G. R. Bj6rk. J. Bacteriol. 175, (1993) p7776.
[2] Persson B.C et at. J. Bacteriol. 180, (1998) p3144.

Abstracts FEBS'99

(Mo/5.2/165)

qJ residues in the human and S. cerevisiae spliceosomal UsnR~As

tRNAs and U2 snRNA share a common synthase. S. Massenett , 1

s209

(Mo/5.2/166)

AnsrnantI, Y. Motorin2, J. Chen3, J. R. Patton3, H. Grosjean- and C

A. Morina, C. Simona, F. Subrab & H. Grosjeana

B r a n l a n t I / U~4R 7567 CNRS-LHP 54506 l'~mdoenvre-lca ,\ancv cedex I-~,mce 2 LEBS

CNRS. 91198 GlJ-~ur-)'vette France 3 Untver.llr~ of Mede~me Svuth Carolina USA

Depending on intron sequences, maturation of nuclear pre-mRNAs in

vertebrates requires the assembly of complexes containing either the U 1,
U2, U4, U5 and U6 or the Ull, UI2, U4atac, U5 and U6atac snRNA
series ] 1]. Assembly of active spliceosomes with the U 1, U2, U4, U5 and
U6 snRNAs implies the formation of several heterologous RNA-RNA
interactions and the UsnRNA segments involved are highly posttranscriptionally modified in vertebrates. They contain several gt
residues. This suggests an important role of UsnRNA modifications in
spliceosome assembly and function. For a better understanding of this
role, we identified the ~t residues of the human atac and the S. cerevisiae
UsnRNAs. Interestingly, only a limited number of ~gresidues were found
and positions of modifications are similar in the two RNA series [2].
Next, we tested whether the yeast ~g synthases characterized so far
(Puslp, Pus2p, Pus3p, Pus4p and Cbf5p) can catalyze ~ formation in
UsnRNAs. Analysis of UsnRNAs from strains with dismpted V synthase
genes showed that only Pus I p is involved in this process. One of the U2
snRNA ~g residue is not formed in the pusl strain. We verified that the
purified Puslp enzyme catalyzes formation of this residue, in in vitro
produced S. cerevisiae U2 snRNA [2]. This is the first identification of a
gt synthase that modifies UsnRNAs, and this enzyme has the peculiarity
to act both on U2 snRNA and on tRNAs. Sequence and structure
requirements for V formation in U2 snRNA by the Puslp enzyme are
studied. Specific formation by the yeast Puslp enzyme of a V residue in
in vitro transcript corresponding to human U2 snRNA was only obtained
when Sm proteins were associated with the U2 in vitro transcript. This is
due to an unfavourable internal secondary structure in the human U2
transcript, that is disrupted by protein association. The results will be
discussed in terms of UsnRNP biogenesis and of Puslp enzyme
specificity. Disruption of genes of other putative tg synthases from S.
cerevisiae is underway to try to identify the enzymes responsible for the
formation of the other q residues that we identified in the S. cerevisiae
UsnRNAs.
[1] Tam and Steitz, TIBS, 1997, 22, 122
[2] Massenet et al., Mol. Cell. Biol., 1999, 19, 3
(Mo/5.2/167)

Presence of pscadouridine in the anticndnn alters the

genetic code in echinoderm mitochondria
K. Tomtta ", T. Ueda. and K. Watanabe
~bm,er,wty o] tokyo, lbkyo 113, ,htpcm, alBMP ('NRS, Stra.~bourg. /.ram

HIV-1 expression changes the human cell capacity

to modify posttranscriptionally human tRNALy s3.
aLEBS, UPR 9063 CNRS, 91198 Gif-sur-Yvene, France.
bLPPMB, UMR 1772 CNRS, IGR, 94805 Villejuif, France.

HIV-1 retrovirus uses a specific tRNA isoacceptor (tRNALys3) to prime

reverse transcription. The post-transcriptional modifications of the natural
tRNA are required for its specific and efficient selection as a primer. The
modified nucleotides are involved in the initiation of reverse transcription
and the transition step between initiation and elongation. Moreover, they
also play a role in the fidelity and efficacy of plus strand viral DNA transfer.
In the present work, we analyzed the biosynthesis pathways of these
modified nucleotides and we studied the activity of the enzymes which
catalyze their formation in the human tRNALys3 (mcm5s2UUU anticodon).
To this aim, radiolabelled tRNALys3, lacking all modifications, were
obtained by in vitro transcription. Using either microinjection or
electroporation methods, this tRNA substrates were introduced in different
types of eukaryotie cells in culture: Xenopus leavis oocytes, human HeLa
cells and a CEM derived human T-cell line (A3.01). At the end of cell
incubations, total cellular RNAs were phenol-extracted. Full-length tRNAs
were purified by PAGE and completely digested with nuclease P1 or RNase
T2. The modified nucleotides were then analyzed by 2D-TLC. In order to
study the effects of HIV expression, we used the following cell models:
HeLa ceUs transfected with an infectious molecular clone (pNLA-3 derived
from AIDS retroviruses), and chronically infected ACH-2 cells (A3.01derived clone) which were treated with the tumor necrosis factor a to induce
HIV-1 expression. The production of HIV-1 virions was monitored using
ELISA of viral core antigen p24 in cell culture medium.
In all the ceils tested, we identified the formation of nine differents
modified nucleotides (m2G, V, D, m7G, mSC, mlA, Tm, mcm5s2U,
ms2t6A) located at fifteen positions in human tRNALys3. Intermediate
metabolic forms were also identified for the hypermedified nucleotides (Urn
and T for Tm54; cm5U and rncm5U for mcm5s2U34; t6A for ms2t6A37).
Moreover, the results indicate that the capacity of human cells to
posttranscriptionally modify tRNALys3 transcripts was altered
simultaneously with the expression of HIV-1 and especially with the
production of virions: the formation of hypermodified nucleotides (Tin,
mcmSs2U and ms2t6A) was no more detected whereas the intermediate
forms accumulated Wansiently (Urn, cm5U, mcm5U, t6A); some other
modified nucieotides were formed less efficiently (mlA) or were not
significantly changed (v, D).
(Mo/5.2/168)

Catalytic amino acid Cys324 of tRNA modifying enzyme

T r m A is not essential for viability of bacterium E.coli

J.Urbonavi6ius and G.R.Bj6rk

Dept. of Mtcrobiology, Umet) umversity, Umed, Sweden

It has been inferred from DNA sequence analyses that in

echinoderm mitochondria not only the usual aspara~ne codons AAU
and AAC, but also the usual lysine codon AAA are translated as
asparagme by a single mitochondrial (mt) tRNA A~nwith the antlcodon
GUU. Nucleotlde sequencmg of starfish mt tRNA A'n revealed that the
anticodon is GqJu. U35 at the antlcodon second position being
modified to pseudouridme (~). In contrast, mt tRNA t'',
corresponding to another ly sine codon AAG, has the anticodon CUU.
nat tRNAs possessing antlcodons closely related to that of tRNA '8"n,
but responsible for decoding only two codons e a c h - tRNA ff'',
tRNA A'p and tRNA l'r - were found to possess unmodified U35 in all
cases, suggesting the importance of~35 for decoding the three codons
Therefore, the decoding capabilities of two sy nthetlc E. cob tRNA ''~"
variants with the anticodon G~U or GUU were examined using an t/.
coh m vttro translation system. Both tRNAs could translate not only
AAC and AAU with similar efficiency, but also AAA with an
efficiency that was about two-fold higher in the case of tRNAA~GtPU
than tRNAAt~GUU. These findmN imply that qJ35 of echinoderm rnt
tRNA '\'n actually serves to decode the unusual asparagine codon AAA,
resulting in alteration of the genetic code in echinoderm mitochondria
[1].

The trmA gene, encoding tRNA modifying enzyme

tRNA(mSU54) methyltransferase, is essential for viability of
bacterium E.coli, whereas mSU54 tRNA modification is not [1].
In vivo, the TrmA protein is present in three forms: as a native
polypeptide of 42-kDa and as two covalent TrmA-RNA
complexes. The tRNA bound to TrmA polypeptide is mainly
present in the 62-kDa complex, whereas 3 '-end of 16S rRNA is
present in the 54-kDa complex [2]. In vitro, amino acid Cys324
was shown to be a catalytic nucleofile of TrmA protein during
the methylation reaction [3].
Using Western blot technique we demonstrated that trmA4
and trmA6 mutants had significantly reduced amount of both 42kDa and 62-kDa forms of TrmA, whereas trmA5 and trmAlO
mutants had reduced amount of only the 62-kDa TrmA-RNA
complex. However, the amount of 54-kDa TrmA form was
similar to wt amount in all the trmA mutants analyzed.
Sequencing of trmA mutants demonstrated that none of them is
mutated in Cys324. We moved Cys324Ala trmA allele to the
chromosome of E.coli and the resulting mutant lacks covalent
TrmA-tRNA complex. Our results demonstrate that the
TrmACys324-tRNA covalent binding of TrmA to tRNA is not
essential for viability of E.coli and suggests that the essential
function of TrmA protein is formation of covalent TrmA-16S
rRNA complex.

[1 ] Tomita, K.et al., (1999) Nucletc Act& Res , in press

[1] B.C. Persson et al., Proc. Natl. Acad. Sci. USA, 89, (1992)
3995.
[2] C. Gustafsson et al., J. Biol. Chem. 268, (1993) 1326.
[3] J.T. Kealey et al., Biochemistry 30, (1991) 9724.

s210

Abstracts FEBS'99

6.2 Molecular recognition in transcription: the machinery

(Mo/6.2/170)

Characterization of DNA/human NDPK-B complex

F. Agou, S. Raveh & M. V6ron

(Mo/6.2/171)

Dip. Btochtm. e Biotec. Med.,bDip. Biol e Pat. Mol. e Cell.,

Universita' "Federico 1I", vta S. Panslni, 5, 80131, Napoli, Italy

Unitd de Rdgulatlon En~matique des Acttvitds Cellulaires, CNRS

URA 1773,Institut Pasteur. 25 rue du Dr. Rozo: 75724 Paris cedex 15

Nucleoside diphosphate kinases (NDP kinases) are ubiquitous

enzymes which are essential for the maintenance of the cellular nucleoside
triphosphate pool. Prokaryotic NDP kinases are tetramers while eukaryotic
counterparts are hexamers. Another distinctive difference between
eukaryotic NDP kinase and its prokaryotic counterpart is the ability of the
former to activate gene transcription [l]. This property is independant of its
catalytic activity, and is possibly related to the role of the protein in
carcinogenesis as well as in development.
Human NDP kinase B (NDPK-B), the most basic human isoform.
specifically recognizes the DNA matrix. Using filter binding assay and
intrinsic fluorescence, we analyzed the binding of several oligonucleotides
mimicking the promoter region of the c-myc oncogene including variants in
sequence, structure, and length of both strands. We show that NDPK-B
binds specifically to single-stranded oligonucleotides of various sequences,
indicating that the recognition of DNA is a function of the structural
conformation of DNA rather than of its specific sequence [2].
A distinct, albeit, small difference in affinity has nevertheless been
observed when binding studies were performed with a guanine rich strand.
We show that this is due to the presence o f a guanosine at 3' terminus of the
sequence. Furthermore, aptamers with a guanine at 3' terminus are good
competitive inhibitors in the synthesis reaction of nucleoside triphosphates.
Inversely, the nucleosides di- or triphosphates ir~hibit the oligonucleotide
binding with inhibition effects which depends on the base (G>A=T>C) [2].
Taken together, these results show that the active site acts as binding
template for the anchorage of the oligonucleotides and that conunun
determinants of the protein are required for the specific recognition of
nucleotides [3] and for the binding to oligonucleotides.
[1] Postel et al., Science, 261, (1993) 478. [2] Agou et al., (1999) submitted. [3] Schneider
et al., d Biol. Chem, 273, (1998) I I491.
(Mo/6.2/172)

INTERACTIONS BETWEEN NF-KB AND PUTATIVE E-BOX

BINDING PROTEIN(S) ON CYSTIC FIBROSIS TRANSMEMBRANE REGULATOR PROMOTER. F. Brouillard,
T. Leclerc, M. Bali, J. Lipecka, T. Torossi, M Legros,
A. Edelman. L'~'SERM U467. CHU Necker. Parts, France

The level of expression of the cfir gene, responsible for cystic fibrosis, is low
and appears to be, at least in part. dictated by the genomic sequences 5"
upstream of the translation start (ts) of c[?r. This promoter has the features of
a housekeeping gene. Moreover, about 1000 bp upstream of ts is a possible
enhancer region containing putative binding sites for API, SPI and NF-kB, but
the role they play in oqr transcription has not yet been considered. Since NFkB is a mediator of inflarmnation and inflammation is closely hnked to the
pathogenesis of cystic fibrosis, we investigated the possible involvement of
NF-kB in cT?rregulation. Computer analysis of the q[tr promoter revealed a
putative NF-KB element between posnions - 1103 to - 1093.
In a first series of experiments, we searched for the possible binding of NF-kB
on this sequence by performing electrophoret~c mobility shift and supershift
experiments using nuclear extracts from human colon carcinoma T84 cells,
which express a high level of @r, and from HeLa cells, and using the -1111/1090 region of the q[tr promoter as probe. When NF-kB was activated by
treating the cell with 10ng/ml of TNFm specific binding of NF-kB (RelA/p50)
to the probe was obseved. When the probe was enlarged by 14 bp (-l 111/1076), one additmnal DNA/protein complex was detected. To determine the
DNA sequences involved in the formation of this complex, we performed
methylation interference studies. The analysis revealed that the DNA binding
sequence for this protein(s) overlapped with the sequence of NF-kB and
contained the consensus binding site CANNTG (E-box) for basic-Helix-LoopHelix transcription factors. Electrophoretic mobility shift compctition
experiments supported this conclusion.
In a second series of experiments, the functional relevance of the -1111/1076 region was assessed in HeLa cells by using luc reporter gent
experiments. In basal and TNFc~-stimulated cells, a 2.5-fold activation of l l l l/-1076SV40-luc expression hiciferase was observed, suggesting that NF-kB
binding to DNA is not functional. Mutation of the E box dramatically reduced
the - 1111/- 1076SV40-luc expression in basal conditions and led to an increase
of hic activity in TNFc~-stimulated cells, suggesting that when the E-box
binding protein cannot form a complex with DNA, the NF-kB binding to DNA
shmulates transcription activity. Altogether, the results suggest that in the
region -1111-1076 of the cfir promoter, complex DNA/putative b-HLH
protein(s) in the vicinity of c/~r ~f3 element prevent NF-I,zB protcins binding.
These .proteins might be important for the regulation of CFTR gene
transcription.
Supported by INSERM, AFLM and CNRS.

Human aldolase C gene is positively regulated by cAMP

P. Buono ~, A. Alfieri a, S. Cassano b and F. Salvatore

The aldolase C gene is the most abundant glycolytic aldolase

isoenzyme in adult brain. We previously identified two regions in
the promoter that are responsible for the transcription: the proximal
region, of the basal transcription, and the distal region that binds the
transcriptional factor NGFI-B. The binding of this factor upactivates the transcription of the gene in human cell lines [I].
Recently, we demonstrated that the cAMP analog 8-Br-cAMP
induces an increase of human aldolase C messenger and protein of
about 4-fold in rat PC12 cells. This increase is time-dependent and
is more evident 4 h after treatment than at a later times. We
identified the promoter region responsive to cAMP stimulation
(distal region D, -201/-184 bp) and demonstrated that the upregulation requires the removal of a regulatory protein that binds to
this region [2]. Preliminary results showed that human aldolase C
messenger increases in PC 12 cells also after long-term stimulation
with 8-Br-cAMP (9 d at lmM), similar results were obtained in
these cells after NGF 50 ng/ml treatment. In the latter case,
messenger increased after 24 h of treatment Long-term stimulation
with 8-Br-cAMP and NGF is reported to induce neuronal
differentiation in PC12 cells [3]. Our future goal is to identify the
regions of the human aldolase C gene promoter involved in the
regulation during the neuronal differentiation.
This work was supported by grants from MURST (cofin '97) and
CNR (target project Biotechnology).
References:
[1] Buono P. et al. (1997) Biochem. J., 323, 245
[2] Buono P. et al. (1999) submitted
[3] Yao H. et al. (1998) J. Biol. Chem., 273, 8240

(Mo/6.2/173)

p21WAF1/CIPI

A role for Spl and p300 in the induction of

during NGF-mediated neuronal differentiation of PCI2
cells
D. Carlisi a, N. Billon a, M. B. Datto b, X. F. Wang b and
B. B. Rudkin a

"Differentiation and Cell Cycle Group, Laboratoire de Biologic

Moldculaire et Cellulaire, UMR 49 CNRS/ Ecole Normale Supdrteure de
Lyon, 46. Allde dTtalie, 69364 Lyon Cedex 07, France.
bDepartment of pharmacology, Duke University Medical Center,
Durham, North Carolina 27710, USA.

Addition of nerve growth factor (NGF) to PC12 cells promotes

neuronal differentiation while inhibiting cell proliferation. In order to
understand how NGF exerts its antimitogenic effect during
differentiation, we have studied the mechanism by which this factor
activates the promoter of the CDK inhibitor p21. The minimal region of
the p21 promoter required for the NGF-induction was mapped to a
contiguous stretch of 10 bp located 83 bases upstream of the transcription
initiation site. This GC-rich region was shown to interact specifically
with the transcription factor Spl and the related protein Sp3, in either
exponentially growing or NGF-treated PCI2 cells. The transcriptional
coactivator p300 was also found in complexes associated with the NGFresponsive region, and this association was reinforced by the addition of
NGF. Moreover, transcriptional activity of the C-terminal domain of
p300 was specifically induced by NGF m a Gal4-fusion assay. These
data indicate that induction of p2[ during neuronal differentiation may
involve regulation of p300 activity by NGF. Furthemore, they suggest
that p300 and Spl cooperate in actxvating p21 transcription during the
withdrawal of neuronal precursors from the cell cycle . This hypothesis
is suported by experiments showing that p300 and Spl form complexes
in PC12 cells.

Abstracts FEBS'99

(Mo/6.2/174)

Regulations of the Escherichia coligapA gene transcription

B. Charpentier, B. Thouvenot, C. Branlant

s211

(Mo/6.2/175)

Maturation des ARN et EmTmologie Molbculaire,

UMR CNRS 7567, Universitd H. Poincard, Facultd des Scwnces,
BP 239, 54506 Vandoeuvre-les-Nancy, Cedex. France

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key

enzyme for glucose metabolism, catalysing the reversible oxidation of Dglyceraldehyde-3-phosphate into 1,3-diphosphoglycerate. In most bacteria,
the GAPDH-encoding gene ( g a p ) and the pgk gene encoding
phosphoglycerate kinase (PGK) are located in tandem on the chromosome. E.
coli is unusual in this repect : a gapB gene is present upstream of pgk
(forming a gapB-pgk cluster) but no GAPDH activity was measured from this
gene. Instead, gapB is expressed at very low level and the protein displays a
non phosphorylating erythrose 4-phosphate dehydrogenase activity (E4PDH).
A gapA gene, located at a distant site in the chromosome, is the only active
GAPDH-encoding gene.
We showed that gapA is expressed from at least four different
promoters and that these transcription start sites are used differentially during
a batch fermentation [zl]. Three promoters are well identified : PI and P3 are
transcribed by the Eo 0 holoenzyme RNA polymerase, and P2 is transcribed
by the heat shock RNA polymerase Ecy32. In addition, P3 is subjected to the
catabolic repression. Promoter P! is the strongest of the four promoters,
particularly during the exponential growth. Recently, we detected a common
regulation for gapA and gapB-pgk transcriptions. The gapA P1 promoter and
the gapB P0 promoter, producing a gapB-pgk bicistronic mRNA, are
activated in the presence of glucose via the enzyme II~tc (EII61C), encoded by
the ptsG gene [2]. This regulation was first described for the ptsH gene
encoding the histidine-containing phosphocarrier protein Hpr [3].
Our goal is now to define the molecular mechanism of this regulation.
By quantification ofgapA P1 mRNA level in different genetic backgrounds
and growth conditions, we show that gapA P1 utilization is highly dependent
upon the level of DNA supercoiling and interestingly, is activated by protein
FIS. In order to delineate the cis-acting sequences directing the initiation of
gapA PI mRNA, we produced different variants of the PI -10/-35 region.
Analysis of the effect of the mutations show that the activity of gapA P1
depends strongly on an <textended -10 >~sequence.
[I] CharpenUerand Branlant(1994) J. Bacteriol. 176:830-839. I21 Charpentieret al. (1998)
J. Bacteriol. 180:6476-6483.131De Reuseand Danchin(1991)J. Bacteriol. 173:727-733.
(M0/6.2/176)

Study of AP-1 and ATF/CREB family members

dimerization by the LexA-based heterodimer assay
M. Dimitrova, M. Granger-Schnarr
Institut de Biologie Moldculaire et Cellulaire. UPR 9002 du CNRS.
15. rue Descartes, 67084 Strasbourg, France

Protein-protein interactions play a major role in almost all relevant

physiological processes occuring in living organismes, including DNAreplication and transcription, RNA-splicing, protein biosynthesis and signal
transduction. In order to facilitate the study of protein-protein interactions
we have developed a new method (LexA-based heterodimer assay) for
specifically detecting the heterodimerization of two heterologous proteins in
the bacterium Escherichia coil [ 1]. The assay is based on the simultaneous use
of protein fusions with an altered specificity and a wild-type LexA repressor
DNA-binding domain. The hetero-association of two proteins of interest is
specifically measured by the tanscriptional repression of a reporter gene
controlled by a hybrid operator. This method is particularly useful in those
cases where one or both partners are also able to form homodimers, since the
assay is sensitive only to the formation of heterodimers. A typical example of
homo- and heterodimerizing proteins are the AP-1 (activating protein-l) and
ATF (activating transcription factor)/CREB (cyclic AMP responsive element
binding protein) families of transcription factors, which are involved in the
regulation of cell proliferation, transformation and survival. Their members are
characterized by a DNA-binding domain composed of the leucine zipper
(promoting dimerization) and the basic region (promoting specific DNA
binding). With the increasing number of known bZip proteins, the number of
possible combinations of dimers has dramatically increased. In order to
investigate the possible heterodimer formation and interaction specificity, we
have tested the heterodimerization capacity of leucine zipper domains of 14
AP-1 and ATF/CREB family members (c-Jun, JunB, JunD, c-Fos, FosB,
Fral, Fra2, ATF2, ATFa, ATF3, ATF4, B-ATF, CRE-BPa and XBP1) by
using the LexA-based hetemdimer assay. This study allowed us to identify
several particularly strong interactions. Our results can be useful for the
design of suppressor peptides with a dominant negatif effect on the cellular
transformation [2].
[ 1] Dimitrova et al., Mol Gen Genet, 257, (1998) 205
[2] Granger-Schnarr et al., Proc Natl Acad Sci USA, 89, (1992) 4236

TIIIC-independent transcription of a yeast tRNA gene

G. Dieci, R. Percudani, S. Giuliodori, S. Ottonello
lstituto di Sctenze Biochimiche, Universitd di Parma. 43100 Parma, Italy,

Genes transcribed by RNA polymerase 111 exhibit a variety of promoter

structures. 5S rRNA and nearly all tRNA genes are characterized by
intragenic promoters, while promoters associated to U6, 7S and
selenocysteine tRNA genes are all essentially extragenic. In tRNA gene
promoters, intragenic A and B boxes form the binding site for TFIIIC,
which then assembles the multiprotein transcription factor TFII/B. Current
data indicate that TFfflB assembly on yeast tRNA genes is absolutely
TFIIJC-dependent and is not influenced in a sequence-specific manner by
the DNA region preceding the startpoint. Instead, in vitro assembly of a
productive pre-initiation complex on the yeast U6 RNA gene only requires
TFIIIB, which through its TBP component interact with a TATA boxelement at position -30.
We have searched the entire S. cerevisiae tRNA gene database for the
presence of U6-1ike TATA sequences (TATAAATA) upstream of the
transcription start site, and identified several tRNA genes containing this
sequence element around position -30. An in vitro transcription analysis
conducted with a highly purified, reconstituted system revealed that one of
these genes, tDNAneUAU, is efficiently transcribed in the absence of
TF[IIC, while another TATA-containing gene, tDNA~UGG, is not. These
preliminary data indicate that TFIIIB can be correctly assembled on
particular tRNA genes in the absence of TF[IIC, and that a consensus
TATA-box element at position -30 is not by itself sufficient to promote
"IFIIIB assembly. The transcriptional properties of other TATA-containing
tRNA genes as well as the sequence and factor requirements of TFIIICindependent tDNA transcription are currently being investigated.

(Mo/6.2/177)

Molecular Organization of the RNA Polymerase II Initiation Complex : Role of Transcription Factor TF31A.
D. Forget*, A. Rojas*, M.-F. Langefier*, J. Greenblatt~ and
B. Coulombe*, *Biologic, U. de Sherbrooke, Sherbrooke (Qudbec)
J I K 2R1 Canada. # BBDMR, University of Toromo, Toronto, Canada

Wrapping of promoter DNA around RNA polymeras II has recently

emerged as a novel model to describe transcriptional mechanisms (Robert et
a/. (1998) Mol. Cell 2, 341-351). The DNA wrapping model describes
specific roles for the general initiation factors (TBP, TFIIB, TFIIF, TFIIE
and TFKI-I), provides a plausible explanation for prdnitiation complex
isomerization,
suggests mechanisms underlying synergy between
transcriptional activators, and suggests an unforeseen role for TBPassociated factors (TAFs) in transcription. However, the role of transcription
factor TFIIA in transcriptional initiation is not clear. TFIIA can bind to
TBP/TFIID and stabilize binding of this factor to the TATA element of
promoters. Roles for TFIIA in anti-repression and co-activation have also
been reported. In order to clarify the role of TFILK, we have analyzed the
effect of TFIIA addition on the molecular organization and the formation of
the initiation complex. We have shown that, in addition to binding TBP and
TAFs, TFIIA specifically interacts with both TFRE and TFIIF. We have
identified the subunit contacts responsible for each interaction and defined
the corresponding binding domains. We have also obtained evidence that
TFRA modulates both the topological organization of the preinitiation
complex and the pathway of its formation. These results provide information
on the function of TFRA and, when they are interpreted in the context of the
DNA wrapping model, help to address some confusing observation
concerning the role of TFIIA in transcriptional initiation.

s212

(Mo/6.2/178)

Abstracts FEBS'99

Elements implicated in the transcriptional regulation of sea

urchin replacement H3.3 histone gene
L Fuccia, ]~vl.Dentlcea, p. Mancinia, M. Piscopoa, G. La Muraa, F Aniellob,
M. Branno

(Mo/6.2/179)

"Dept Genettcs, General and Molecular Btology, Um~erst O, of ,rvktples

~Zoologtcal Statton Anton Dohrm, ?vaples, Italy

The replication-dependent histone genes, that are devoid of introns, are

transcribed during the S-phase of the cell cycle into non-polyadenylated
mRNAs. In addition to this major class there is a sub-class of genes, called
replacement histone genes, that is constitutively transcribed at low rate. These
genes present introns and are transcribed into polyadenylated mRNAs. The
replacement subtype of the H3 class, the H3.3 histone, is a minor component ot
chromatin in dividing cells but it becomes relatively abundant in nondividing
cells. Using the cDNA isolated in our laboratory we have identified and
characterised the H3.3 histone gene of the P. lividus sea urchin with the
proximal promoter. In order to understand the differential transcriptional
regulation of replication-dependent and replacement histone genes, we studied
the binding of nuclear proteins to elements of the promoter and of the coding
regions of the H3.3 histone gene in comparison with the cell-cycle dependent
H3 late and H3L histone genes [1]. By band shift analyses we showed that
nuclear proteins from P. lividus embryo, mainly at blastula stage, bind to two
CRE/TRE elements located at -356 and -120 relative to the start of transcription
of the H3.3 gene. The human AP1 consensus oligonucleotide was unable to
compete with the formation of the complex. A CRE/TRE element present
within the human H3.3B histone gene has been previously shown to contribute
to the cell-cycle independent expression of the gene [2]. The promoter region ol
the H3L histone gene lacks the two motifs although presents various other
boxes similar to the H3.3 gene. In addition we studied the binding of the nuclear
proteins to the two coding region elements, CRAS t and f2, involved in the
transcription of replication-dependent histone genes in mouse and man. CRAS
elements of P. lividus H3 late and H3L histone genes interacted tightly with
nuclear factors while the putative CRAS elements of H3.3 gene formed a very
weak protein-DNA complex. The reported data support the hypothesis that the
cell-cycle independent expression of the H3.3 histone gene involves multiple
elements within the promoter region but no within the coding region, that mighl
be implicated in the cell-cycle dependent expression of the H3 histone genes.
[1] Fucci L. et al., FEBS Letters 407 (1997) 101
[2] Witt O. et aI., Biochem. J. 329 (1998) 609

(M0/6.2/180)

Characterization of the influenza virus PB1 protein

to binding to vRNA and cRNA.
S. Gonz,'ilez and J. Ortfn.
Centro Nacional de Biotecnologfa ( CSIC). UAM. 28049 Madrid, Spain

The interaction of the PB1 subunit of the influenza virus

polymerase with the vRNA and cRNA templates has been studied in
vitro [1]. The experimental approach included in vitro binding of
labeled model vRNA or cRNA to PB 1 protein immobilized as an
immunoprecipitate, as well as Northwestern analyses. The binding to
model vRNA and cRNA was specific and with vRNA an apparent Kd
of about 2 x 10 -8 M was determined For the vRNA, the interaction
with the isolated 3'-arm of the panhandle was detectable, although the
interaction with the 5'-arrn was prominent and the binding was
optimal with a panhandle analog structure (5'+3' probe). While with
the cRNA, PB 1 protein interacted with both arms of the panhandle
independently and with a similar intensity. When presented with a
panhandle analog mixed probe, PBI was able to retain the T-arm as
efficiently as the 5'-arrn in the vRNA and in the cRNA.
The sequences of the PB1 protein involved in vRNA and
cRNA binding have been studied by in vitro interaction tests using
PB1 deletion mutants. For the vRNA, two separate regions of the PB1
protein sequence proved positive for binding: the N-terminal 83
amino acids and the C-proximal sequences located downstream
position 493. All mutants able to interact with model vRNA were
capable of binding more efficiently the 5'-arm than the 3'-arm of the
panhandle. Taken together, these results suggest that two separate
regions of the PB1 protein constitute a vRNA binding site that
interacts preferentially with the 5'-arm of the panhandle structure.
[1] Gonzfilez, S. et al. (1999) J. Virol., 73,631.

CREB-2 cooperates with Tax to transactivate the

HTLV-I promoter
F Gachon, C Devaux, J M. Mesnard
ln/~c~lons Retrol.Jrales et Slgnalisatton Celhdatre, CRB31-C.\TCS ~ P R
1086, Instltut tle Btologte, 4 B d ttenrt II : 34060.11ontpelher, F r a n c e

The primary function of Tax is to transcriptionally activate the

HTLV-I promoter but Tax is also known to stimulate expression of cellular
genes It has been reported to associate with several transcription factors,
as well as proteins not involved in transcription To better characterize
potential cellular targets of Tax present in infected cells, a yeast twohybrid screen was carried out with a cDNA library constmcted from the
HTLV-I infected MT2 cell hne From this study, we found 158 positive
clones representing 7 different cDNAs We have focused our attention on
the cDNA encoding the transcription factor CREB-2 CREB-2 is an
unconventional member of ATF/CREB family in that it lacks a PKA
phospho~'lation site and has been described to negatively regulate
transcription from the cAMP response element of the human enkephalin
promoter [1] We have demonstrated that CREB-2 colocalizes with Tax in
the nucleus, interacts wzth Tax m vtvo, and cooperates with Tax to enhance
viral transcripuon, and that the basic leucine zipper domain (bZip) of
CREB-2 is sufficient to perform this function [2] Analysis of deletion
mutants Indicates that the 50 amino acid long C-terminal domain of
CREB-2 bZlp is reqmred for interaction with the C-terminal acidic domain
of Tax Moreover, by using immobilized templates containing the 21 bp
repeats & t h e viral promoter, we have found that Tax increases the binding
of CREB-2 to the 21 bp repeats Our results confirm that the activation
of the HTLV-I promoter through Tax and factors of the ATF/CREB family
is PKA-independent, probably by a mechanism occuring with the CREB
Binding Protein [3]
[1] Karpmski B A et al, Proc Natl Acad Sci USA, 89, (1992) 4820
[2] Gachon F et al, J Virol, 72, (1998) 8332
[3] Kwok R P S e t al, Nature, 380, (1996) 642

(Mo/6.2/181)

Evolutionary conservation of primary ~70-type

transcription factors between plants and bacteria.
M. A. Hakimi, I. Privat and S. Lerbs-Mache
Laboraloire de G~netique Molbculaire des Plantes, Universit~ J. Fourier
and CNRS. B P 53 - 38041 Grenoble. France.

We have cloned and sequenced the cDNAs coding for three different
sigma70-type transcription factors from the higher plant Arabidopsis
thaliana. In order to analyse the degree of evolutionary conservation of
components of the transcriptional machinery we analysed whether these
plant sigma factors can complement E. coli r-poD (ts) mutants (rpoD285 and
rpoD800).
We analysed the entire plant sigma factors and also hybrid sigma factors,
being composed of domains originating either from plants or from E. coli.
Our analyses indicate that the N-terminal domains of plant sigma factors
seem to have aquired different functions during evolution. The C-terminal
domain of one out of the three plant factors analysed can fully substitute all
functions of the primary sigma factor of E. eoli indicating strong
evolutionary conservation of promoter structures and promoter/transcription
factor/RNA polymerase interactions.

keywords: sigma factors, plants, procaryotes

Abstracts FEBS'99

(Mo/6.2/182)

s213

Nitric oxide in hibits DNA binding of the transcriptionfactor

cJun by specifically targeted S-glutathionylatioo
P. Klatt, E. Pineda Molina, M. Garcfa de Lacoba, S. Lamas
Centro de InvestigacionesBml6gicas, 1RSIN,CSIC. Vel~izquez 144,
E-28006 Madrid, Spain

Accumulating evidence suggests that the free radical nitric oxide may play a
role in the control of transcription by modulating the DNA binding activity of
transcriptional activators The transcription factor activator protein-I (AP-1),
which is composed by homo- or heterodimers of Jun and Fos proteins, has
been proposed as one potential sensor of nitrosative stress at the level of
transcription. However, the molecular mechanisms underlying inhibition of
AP-1 DNA binding activity by reactive nitrogen species remain to be established Here, we show that in the presence of the physiological sulfhydryl
glutathione, nitric oxide modifies the two cysteine residues contained in the
DNA binding module of cJun in a selective and distinct way While nitric
oxide induced the formation of an intermolecular disulfide bridge between
cysteine residues in the teucine zipper domain of cJun monomers, this same
radical directed the covalent incorporation of stoichiometric amounts of glutathione to a single conserved cysteine residue in the DNA binding site of the
protein We found that covalent dimerization of cJun did not affect its DNA
binding activity, whereas the formation of a mixed disulfide with glutathione
correlated well with the inhibition of the transcription factor We show that
NO-induced S-glutathionylation of cJun is not mediated by NO-induced
oxidation of glutathione to glutathione disulfide but involves the generation of
S-nitrosoglutathione as reactive, thiol-transferring species. We provide evidence that the reaction of S-nitrosoglutathione with cJun to yield the mixed
disulfide does not require homolytic cleavage of the nitrosothiol but rather
involves a direct and reversible transfer of the glutathionyl moiety to the cJun
protein. Molecular modeling of S-glutathionylated cJun further indicates that
the incorporation of glutathione into the basic DNA binding domain of cJun
is facilitated by specific electrostatic interactions In conclusion, our results
support the inhibition of cJun by NO/nitrosothiol-induced S-glutathionylation
as a potential mechanism by which nitrosative stress may be transduced into a
functional response at the level of transcription

(Mo/6.2/lg4)

MOLECULAR C]tARACTERISATION OF THE

RAT PARP GENE PROMOTER
M.A. Laniel a, G. Lansaca,G.G. Poirier ~b. S.L. Gudrin a
~Molecu/ar Endocrinology and ~Health and Enwronment Re,~earch
Untt, CHUL Research 6k~ntre. Ste-Foy, Quebec, Canada G t 1"4G2

Poly(ADP-ribose) polymerase (PARP) is a highly-conserved nuclear

protein found in almost all eukaryotes. This enzyme catalyses the posttranslational poly(ADP-ribosyl)ation of different proteins, including
itself, in response to DNA strand breaks. Although PARP is
constitutively expressed, its expression level varies in different tissues,
and in various physiological situations such as cellular differentiation and
proliferation. Characterisation of the PARP gene and of its promoter is
essential to understand the transcriptional mechanisms underlying PARP
expression. The human, rat and mouse PARP promoter share a similar
structure proper to housekeeping genes. They are GC-rich, have no
functional TATA or CCAAT boxes and contain an initiator sequence.
We have recently shown that transcription factor Spl binds five sites on
the rat PARP proximal promoter and functions to activate PARP
transcription. Furthermore, binding of transcription factor NF1 at two
regions overlapping Spl sites suggests that NF1 might regulate PARP
expression by interfering with Spl binding. We have also demonstrated
the binding of nuclear proteins to the transcription initiation site of the
rat promoter. This region is completely conserved in all three known
mammalian promoters and includes a consensus initiator element.
Analysis of the conserved structure of the PARP promoter reveals that
the transcriptional mechanisms regulating PARP expression are more
complex than previously thought.

(Mo16.2/185)

In vivo characterisation of DNA and protein interactions

of XapR - a LysR type protein

R. Linding Raun, G. Dandanell
Dept. of Biological Chemtstry, lnstttute of Molecular Biology
University of Copenhagen, Solvgade 83H, 1307 Copenhagen, Denmark
E. coil can utihze the purine nucleoside xanthosine as the sole carbon and
energy source. At least five genes (xapAB, xapR, nupC, nupG) are

involved in the uptake and degradation of xanthosine (1,2). Three

nucleoside transporters (XapB, NupC & NupG) are responsible for the
uptake of xanthosine whereas xanthosine phosphorylase (XapA) catalyses
the degradation of xanthosine to xanthine and ribose-l-phosphate.
Ribose-l-phosphate can be metabolised in the pentosephosphate shunt,
and xanthine can enter the nucleotide metabolism. In wild type cells the
induction of xapAB both requires the xapR gene product that encodes a
LysR type transcriptional activator and the presence of the inducer
xanthosine (3). XapR upregulates the xapAB expression by binding to the
xapAB promoter where it probably directly interacts with the
transcription apparatus.
It has not been possible to purify enough XapR to perform in vitro
footprints, therefore the interaction studies made so far has been based on
mutational analysis. A library of promoter/operator mutants have been
constructed by PCR amplifying the promoter region in the presence of
Mn(II) with subsequent cloning upstream of lacZ. By scoring different
lac phenotypes on indicator plates mutants affected in XapR binding has
been isolated and characterised. Using a similar procedure the interaction
between XapR and the RNA polymerase ~x-subunit has been studied.
Revertants in XapR that can complement RpoA mutations can give
specific information about the interactions between XapR and the RNA
polymerase.
Since purification of XapR has been difficult due to formation of
inclusion bodies we are trying to set up an in vivo footprinting system
using Guanine/Adenine-Linker-Mediated PCR(G/A-LMPCR) and DMS
as footprinting reagent. An alignment of the mutant profile and
footprinting profile, indicate hotspots of interaction between XapR, DNA
and the RNA polymerase.
[1] R. S. Buxton et al. Molec. Gen. Genet., 179,331.
121 K. Hammer-Jespersenet al. Molec. Gen. Genet., 179,341.
[31 C. Seeger et al. J. Bacteriol., 177.5506.

s214

(Mo/6.2/186)

Abstracts FEBS'99

About plant protein complexes interacting with both RNA

phi H tef cis-acting elements and rDNA spacer sequences.
A. Manevski,C Bardet, D. Tremousaygueand B Lescure.

(Mo/6.2/187)

L B.M.R P.M.. CNRS-1NRA, BP 27, 31326 Castanet-Tolosan, France

The tef box, a cis-element identified in promoters of several plant genes

encoding components of the translation apparatus, is involved in the
activation of gene expression in cycling cells [1]. This element mediates in
vitro the formation of two protein complexes called C 1 and C2 A tef-like
box is located within the intergenic transcribed spacer of several plant rRNA
genes. This sequence, already described in radish as a protein binding site and
putatively involved in the rDNA transcription regulation process [2], is
sufficient to form C 1 complexes. By using mutated tef boxes, we show that
the tef-dependent activation of transcription is correlated to both C1 and C2
complex formation in a context-dependent manner. In transient expression
experiments the activation of a minimal promoter/GUS gene fusion is
connected to the formation of C2 complexes. In contrast, the ability to form
C1 complexes appears sufficient to activate the expression of the reporter
gene in root meristems of transgenic ArabMopsis. SDS-PAGE analyse of
purified protein fractions containing either the C1 or the C2 activity indicates
a complex heteromerie structure of these potential regulators [3]. Thus, the
t e f b o x seems to be a central element for regulation of gene transcription in
distinct, superimposed developmental programs and could be involved in a
co-regulation of RNA polymerase I and RNA polymerase II transcription
systems.
[1] Regad et al., Mol. Gen. Genet., 248, (1995) 703
[2] Echeverria et aL, Mol. Gen. Genet., 243, (1994) 442
[31 Manevski et al., Mol. Gen. Genet., submitted

CYPIAI autoregnlation mediated by

oxidative repression of NFI
Y. MoreP, N. Mennod b and R. Barouki a
alNSERM U490. 45, rue de~ Saints-POres. 75006 Paris
hLaboratoire de Biotech Moldculatre, UNIL-EPFL, I015 Lausamw

Reactive oxygen species (ROS) have been shown to activate

stress-response genes. Conversely, we have shown that, in HepG2 cells,
eytochrome P450 IAI (CYPIAI) transcription was specifically
decreased by a shift of the cellular redox status to more oxidant conditions
(H202 or TNFc~ treatment, glutathione depletion). The induction of this
xenobiotic metabolising enzyme by aromatic hydrocarbons such as
TCDD (dioxin) is strongly impaired by H202. We showed that this
oxidative repressive effect was mediated by the transcription factor NFI
which transactivating function is particularly sensitive to oxidative stress
[1]. Its DNA-binding is impaired in vivo, likely through the oxidation of
critical cysteines. In addition, its transactivating domain (TAD) is
repressed by micromolar H202 concentrations (that do not alter the
DNA-binding), as assessed with GaI4/TAD fusion proteins. Using
deletions and point mutations, we are currently investigating the amino
acids of the TAD that are targeted by oxidative stress. It appears that a
critical cystcine is required to mediate the H202 repressive effect. To our
knowledge, this is quite a novel observation since oxidative stress
mediated inhibition of transcription factors was usually reported to target
cysteines within the DNA binding domain.
The redox sensitivity of NFI could be involved in a repressive
autoregulatory loop controlling CYP1AI expression. Indeed, H,O_, is
released within the cell during the catalytic cycle of this enzyme which
can inhibit NFI transaetivating function and repress the CYP1A1 gene
promoter. This mechanism could limit CYP1A1 expression and the
subsequent possible deleterious effects such as ROS production or
procarcinogens activation. We are investigating similar regulations with
other CYP isofomls.
[1] Morel Y. et al., J. Biol. Chem. 273 (1998), p. 26969

(Mo/6.2/188)

Interaction of yeast Poll with the general

factor Rrn3p is mediated by A43 subunit.

transcription

G P e y r o c h e 1 , P Mltkereit 2, N Blschler 3, P.Schultz 3. H , T s c h o c h n e r 2,

A.Sentenac t C.Carlesl and M R t v a | .
t SBGM. CEA/Saclay~91919I Glf /Y,ette edex, France, 2 BZ, Neuenhe~merFeld 328, 69120
Heidelberg, Ge~any: [GBMC. BPI63, lllklrcll edex. France

In all eukaryotic cells, large ribosomal RNA precursor is

synthesized in the nucleolus by RNA polymerase I (Pol I). In
Saccharomyces cerevisiae, Pol I is a multimeric complex composed of
14 distinct subunits. The initiation competent subpopulation of Phi I is
complexed with the general transcription factor Rrn3p. The
dissociation of the Pol l-Rrn3p complex may serve as a molecular
switch tbr the down-regulatmn of rRNA synthesis in various
physiological conditions. We investigated whether A43, the only
essential specific subunit of Pol I, is the interaction target of Rrn3p.
We constructed strains harboring mutant rpa43 alleles generated
by random mutagenesis. One of these mutant strains was further
chosen tbr biochemical investigation. Purification of the mutant Pol IRrn3p complex indicated that this complex was almost inactive in an in
vitro specific transcription assay. Western blot analysis showed that
the mutant fraction was devoid of Rrn3p, suggesting the dissociation
of the Pol I-Rrn3p complex upon purification. The stability defect
exhibited by the Pol l-Rrn3p complex purified from the mutant strain
strongly suggests that A43 subunit participates to the interaction of Pol
I with Rrn3p.
This conclusion was strengthened by genetic data showing that
A43 and Rrn3p are functionnaly linked in vzvo. The thermosensitive
phenotypes of strains harboring rpa43 mutant alleles could be partially
suppressed by the overexpression of Rrn3p. Furthermore,
combination of each of these rpa43 alleles with the rm3-8 mutant "allele
resulted in synthetic lethality.
Finally, structural data, obtained by immuno-electronmicroscopy, showed that A43 and Rrn3p colocalize on the surface of a
3D model of the Pol I-Rrn3p complex, supporting the hypothesis of a
direct interaction between A43 and Rrn3p within this complex.
Altogether, our results strongly suggest that A43 subunit is the
interaction target of the general transcription factor Rm3p.

(Mo/6.2/189)

Association of SR and Sm Splicing Factors with the RNA

Polymerase H Hoioenzyme.
F. Robe~*, M. Blanchette#, B. Chabot~ and B. Coulombe*
*Biologie, U. de Sherbrooke, Sherbrooke (Quebec)J1K 2RI Canada.
I Microbiiologie et Infectiologie, CUSE, U. de Sh*,rbrooke, Canada

mRNA formation in mammals is a multistep process including transcription,

capping of the transcript 5' end, polyadenylation the transcript 3' end, and,
for most genes, splicing of the pre-mRNA to produce a mature mRNA.
Classically, each of these steps has been studied independently and specific
systems have been developped for their detailed analysis. More recently,
evidence for the coupling between the various stages of mRNA formation
have accumulated. Although a number of physiological data suggests a
coupling between transcription and splicing, biochemical evidence in support
to this notion is missing. Here, we describe the purification of a human RNA
polymerase H holoenzyme which contains most of the general transcription
initiation factors, core RNA polymerase II and a number of additional
polypeptides including both SR and Sm splicing factors. SR and Sm have
previously been shown to be involved in splicing signals recognition and
exon bridging, two steps essential for exon definition and formation of the
commitment complex. The association of both SR and Sm with the
holoenzyme is resistant to extensive treatment with RNase A, an indication
that protein-protein interactions are sufficient to sustain the interaction.
Interestingly, only a specific subset of both the SR and Sm proteins are part
of our human holoenzyme, suggesting a putative role of transcription per se
in the regulation of the splicing reaction. Functional date addressing the role
of splicing factors in the RNA polymerase H holoenzyme will be presented.
These results, in ctmjunction with structural data from various laboratories
including our own, provide compelling evidence for transcription-coupled
splicing in vitro.

Abstracts FEBS'99

(Mo/6.2/190)

s215

Crystallographic studies of histone fold-containing

proteins from Transcription Factor IID (TFIID)
C. Romier, C. Birck, O. Poch, M. Ruff, I. Davidson, D. Moras

(Mo/6.2/191)

C. Rouanet 1, K. Nomura2, S. Tsuyumu2, and W. Nasser 1

l: LGMM1C, INSA bdt 406, 20 av Einstein, 69621 Villeurbanne, France
2: Faculty of Agriculture, Sh~zuoka University, 836 Ohya, Japan

IGBMC, 1 rue Laurent Fries, B.P. 163, 67404 lllkirch Cedex. France

Transcription initiation in eukaryotes by RNA polymerase II requi~

the assembly of a macromolecular
complex containing the general
transcription factors TFIIA, -liB, -liD, -liE, -IIF and -IIH at the core
promoter. TFIID is involved in specific recognition of the core promoter and
has been shown to interact with many transcription activators. It is composed
of several subunits: the TATA-box Binding Protein (TBP) and the TBPAssociated Factors (TAFs). Recently, some of the TAFs have been found in
other macromolecular complexes involved in transcription: yeast SAGA and
human TFTC and PCAF.
The topology and three dimensional structure of TFIID remain poorly
tmderstood. We have started the structural study of subcomplexes of TFIID.
This work has recently lead to the determination by X-ray crystallography of
the structure of the complex between human TAFI8 and TAF28 [I]. The
crystals obtained belong to space group P212t21 with a=45.4 A, b=48.4 A,
and c=77.6 A. The structure was solved by using exclusively anomalous data
from pCMBS-soaked crystals that showed a decrease of 7 A in the c-~xis
compared to the native crystals. The structure was refined at a resolution of
2.6 A with final R-factor and R-free of 20.5% and 25.7%, respectively.
Although undetected by sequence analysis, both proteins interact
through histone folds. These histone fold motifs cannot be classified within
the four canonical families (H4, H3, H2A or H2B). Further sequence analysis
also revealed that the protein SPT3 - found in TFTC, SAGA and PCAF contains at its N- and C-termini two histone fold motifs which are strongly
homologous to the histone fold motifs of TAF18 and TAF28, respectively.
The fact that human TAF18 and TAF28 and their yeast homologues are not
found within SAGA, TFTC and PCAF argues in favor of an homologous, but
not identical role for SPT3 in these complexes since their subunit composition
is different from the one of TFIID.
[1] Birck et al., Cell, 294, (1998), 239.

(Mo/6.2/192)

ldeatificafionof a novd SFN2 randy member that modulatesandrogen

receptor-depeadmtgema-anscri~L

NaRaule~M~Redz~n,A.-M.Moil~en,J Palvimo,andO,~ J'~lne.

Department of Phystology, lnstttute of Biomedicine University o! Helsmkl
FIN-O0014 Helsmki, Finland

Androgen receptor (AR) belongs to the steroid receptor (SR)

super~amily that are ligand-activated transcription factors. After ligand
binding SRs are transported to nucleus were they bind to cognate response
elements and regulate expression of their target genes. To accomplish gene
regulation, SRs have to interact with a variety of proteins, leading to
complex molecular mechanisms controlling transcriptional activity. Several
studies have suggested that SRs can interact with some members of SNF2
family of proteins. The founding member of this family, SNF2/SWI2,
represents the ATPase subunit of the well-characterized SWI/SNF complex
which facilitates gene regulation in a chromatin environment. The way by
which ATP is used in this complex to remodel chromatin structure is not
well understood. To date, the SNF2 family comprises over 100 members;
only few of them have been biochemically characterized as well as
SNF2/SWI2. Each of the SNF2/SWI2-related proteins possesses a similar
domain (SNF2 domain) that contains consensus motifs needed for binding
and hydrolysis of ATP. Outside the conserved domain exists nonhomologous regions to direct the ATPase activity where needed. In search
for proteins interacting with AR, we have used a yeast two-hybrid system
and found a new SFN2-1ike family member. SRIP (Steroid Receptor
Interacting Protein) interacted reproducibly with the AR DNA-binding
domain (AR-DBD) and with other SR-DBDs both in the yeast and
mammalian cells. The interaction domain on SRIP is located just before the
conserved SNF2 domain. Northern blot analysis identified a ubiquitous 7
kb transcript with a higher expression level during embryogenesis and in
adult mouse brain. Cotransfection experiments demonstrated that SRIP
represses to one fifth the AR-dependent activation of a reporter gene under
the control of the probasin promoter. Immunocytochemistry studies
demonstrated that the protein was always present the nucleus. Presently.
GST pull-down and coimmunoprecipitation experiments are being
performed to confirm that there is a direct interaction between SRIP and
AR. We propose that SRIP utilizes the energy of ATP to modulate ARdependent transcription using molecular mechanisms currently unknown.

Erwinia chrysanthemipelD/pelE expression regulation.

The main virulence factors of the phytopathogenic bacterium Erwinia

chrysanthemi are pectinases which attack pectin, the major constituent of
the plant cell wall. Of these enzymes, the alkaline isoenzyme named PelD in
strain 3937 and PelE in strain EC 16, was described of great relevance for the
virulence on plants. The expression of the corresponding gene is tightly
modulated by various regulators including the KdgR repressor and the
cAMP-CRP activator complex [1]. The use of a lacZ reporter gene revealed
that the E. chrysanthemi 3937pelD expression is also regulated by PecS, an
other repressor of the pectinase synthesis. In vitro DNA-protein interaction
experiments, led on the pelD/pelE wild-type or mutated promoter region,
allowed us to precisely define the sequences involved in the binding of these
three regulators and of the RNA polymerase (RNAP). These studies
revealed an unusual binding of the KdgR repressor and suggested the
presence of an UP element in the corresponding genes. Investigation of the
simultaneous binding of CRP, KdgR, PecS and the RNAP to the regulatory
region of the pelD/E gene showed that: i) CRP and the RNAP bind
cooperatively, ii) PecS partially inhibits the binding of the CRP activator and
of the CRP-RNAP complex, iii) KdgR stabilizes the binding of PecS and
prevents the transcriptional initiation by the RNAP [21. Taken together, our
data suggest that PecS attenuates the pelD/pelE gene expression rather than
acts as a real repressor like KdgR. As a whole, the control of the alkaline
pectate lyase crucial for the pathogenicity of E. chrysanthemi appears
complex and original.
[1] W. Nasser, J. Robert-Baudouy and S. Reverchon. Mol. Microbiol. 26 :
1071-1082 (1997). Antagonist effect of CRP and KdgR in the transcription
control of the Erwinia chrysanthemi pectinolysis genes.
[2] C. Rouanet, K. Nomura, S. Tsuyumu and W. Nasser. Submit for
publication in J. Bact. Regulation of the pelD/pelE virulence gene encoding a
alkaline pectate lyase in Erwinia chrysanthemi: involvement of the basic
transcriptional factor.

(Mo/6.2/193)

Flexible zinc finger requirement for binding of the

transcriptional activator Star to U6 snRNA and
tRNA sec promoters
M. Schaub, A. Krol and P. Carbon
UPR 9002, IBMC/CNRS. 15, rue R. Descartes, 67084 Strasbourg, France

Enhanced transcription of the majority of vertebrate snRNA and

snRNA-type genes, transcribed by RNA polymerases I1 and IlI, requires the
transcriptional activator Staf [1] [2]. Interaction between Staf and its specific
targets in gene promoters is mediated through a DNA binding domain
containing seven tandemly repeated C2-H2 zinc fingers.
Using a binding site selection assay, we have determined that the highest
affinity binding site for Staf consists of a 21 bp consensus sequence. The
Staf site identified in the Xenopus selenocysteine tRNA (tRNAs) promoter
does not contain the 5'-part of the selected consensus sequence, whereas
that found in the human U6 snRNA promoter lacks the T-part.
By using a combination of approaches, we analyzed the interaction
between wild-type and accurately truncated Star zinc finger domains and the
consensus, Xenopus tRNA scc and human U6 snRNA sites. We found that
different sets of zinc fingers are responsible for the high affinity interaction
and sequence specificity on the different sites. Two main conclusions
emerged from our data. The first one clearly indicates that zinc finger 7 does
not establish DNA contacts in Staf-DNA complexes. The second conclusion
concerns zinc finger 1, which is required for the binding to the Xenopus
tRNA see site, but is dispensable in the case of the human U6 snRNA site.
Taking into account the sequence differences between the two sites, these
findings demonstrate that Staf utilizes zinc finger 1 in a rather flexible
manner, illustrating how a protein can interact with DNAs containing targets
of different sequences. The consequences of this binding flexibility at the
transcriptional level will be presented.
[1 ] Schuster C. et al., EMBOJ., 14, (1995) 3777
[2] Schaub M. et al., EMBOJ., 16, (1997) 173

s216

(Mo/6.2/194)

Abstracts FEBS'99

Molecular mechanism of the interaction between

ERct and AP-1
C. Tcyssier, F. Galtier and D. Chalbos

(Mo/6.2/195)

Laboratory of Cell Biology, Howard Hughes Medical Institute, The

Rockefeller University, 1230 York Avenue, New York, NY 10021

hVSERM U146 60 rue de Navacelles. 34090. Moutpelher. France

AP-I are homo or heterodimeric transcription factors, composed of either

two Jun family proteins or one Jun and one Fos family protcins, which have
been implicated in the control of cell proliferation in a large number of cells.
We had prewously shown that, in the human breast cancer cell line MCF7,
estradiol increased the growth factor-induced AP-I response without
affecting c-Fos and c-Jun synthesis and that the estrogen receptor c~ (ERos)
was necessary for this effect. In the present study, we investigated the
molecular mechanism of the ERo~/AP-I cross-talk.
ERct mutant analysis, in transient transfeetion experiments, revealed that the
hormone binding domain and the DNA binding domain of ERo~ were needed
for stimulation of AP-1 activity by estradiol. However, ERa dad not bind to
AP-1 sequence and an ERo~ mutant unable to bind an estrogen responsive
element still regulated AP-I activity suggesting that the effect of ERa was
striggered by some protein-protein interaction.
In fact, a physical interaction between ER and c-Jun was detected by GST
pull-down and Far Western experiments. Under these in vitro conditions,
E R a bound to c-Jun irrespective of it was unoccupied or occupied with
agonist or antagonists. JunB was as efficiently bound to GST-ERct than cJan while JunD only weakly hybridised with the fusion protein and no
member of Fos family (c-Fos, FosB, Fra-I and Fra-2) could directly interact
with ERc~.
We looked for the domains ofc-Jun and ERct which were implicated m the
protein-protein interaction. Wath the use of deletion mutants, we showed that
the carboxy terminus of c-Jun, containing the bZIP domain, was sufficient
for ERot binding and that the minimal part of ERcc necessa~ for the physical
interactmn with c-Jun encompassed amino acids 251-302.
Our data altogether suggest that estradiol stimulation of AP-1 activity is a
&rect effect of activated ER which is likely to result from protein-protein
interaction with Jan proteins.

(Mo/6.2/196)

Interstitial telomeric motifs involved in the control of gene

expression in Arabidopsis bind a protein related to Purer.
D. Tremousaygue, A. Manevsl~, C Bardet, N. Lescure and B. Lescure.
L.B.~.R.P.~L, CNRS-INRA, BP 27, 31326 Castanet-Tolosan France

Telomeres, which are extremities of linear chromosomes, are essential for the
stability and the complete replication of eukaryotic chromosomes. They
consist of simple repeated sequences with a G-rich strand always in the 5' to 3'
orientation towards the chromosome terminus. In addition to telomere repeats
found at the end of chromosomes, more or less degenerated telomere
sequences are often observed at interstitial sites within the genome. These
sequences can be classified arbitrarily into two classes. The first class contains
many tandemly repeat telomeric motifs which are more or less degenerated.
These sequences are usually found in subtelomeric or pericentric regions but
also at distinct internal sites, sometimes clearly as relic of ancestral telomeretelomere fusions A second class is constituted by interstitial telomeric
sequences harbouring only one or few telomere motifs. Apart from the ciliate
Stylonychia lemnae and the plant Arabidopsis thaliana, this category of
sequences has not been extensively searched [1,2]. We have shown that most
of Arabidopsts genes encoding components of the translational apparatus
contains one or several telomeric motifs AAACCCTAA in their 5' region.
These motifs are involved in the activation of gene expression in cycling cells
of root primordia. In addition, south-western screening of an ArabMopsis LZAP II expression library with a double stranded (AAACCCT)4yielded
cloning of a gene encoding a protein related to the conserved Pumt nuclear
protein. The implications of these observations are discussed.
[1] Stoll et al., Nucl. Acids Res., 221, (1993) 1783
[2] Regad etal., J. Mol. Biol., 239, (1994) 163

The Karyopherin Kap122 imports both subunits of

the transcription factor IIA into the nucleus
A. A. Titov and G. Blobel

We discovered a novel nuclear import pathway that is mediated by

the product of the previously identified S. cerevisiae gene PDR6 (pleiotropic
drug resistance). We found that Pdr6p functions as a karyopherin (Kap) for
import into the nucleus. Consistent with previously proposed nomenclature we
propose the name Kap122p for Pdr6p. Kap122p was localized both to the
cytoplasm and the nucleus. As a prominent import substrate of Kap122p we
identified the complex of the large and small subunit (Toalp and Toa2p,
respectively) of the general transcription factor IIA (TFIIA). In wild type cells,
Toalp and Toa2p were located primarily to the nucleus. Consistent with
Kap122p being the principal Kap for import of the Toalp/Toa2p complex, we
found that deletion of KAPI22 results in diffuse cytoplasmic mislocalization of
both Toalp and Toa2p. Deletion of KAP122 is not lethal, although deletion of
TOA1 and TOA2 is. Together these data suggest that although Kap122p is the
major Kap for the import of Toalp/Toa2p into the nucleus, other pathways may
substitute for Kap 122p, albeit with lower efficiency. The Toa I p/Toa2p/Kap 122p
complex isolated from the cytoplasm was dissociated by incubation with
RanGTP.

(Mo/6.2/197)

The two closely-related transcription factors FLI and ERG

display similar binding specificities
P. Sticgler and P. Remy
CNRS. UI'R 9005 u ,tf~;cam~rues Molt;culaire~ de/a Dwt ~ton ('ellulalre et
du Ddveloppement ~, 15. rue Rend De.scartes - 67084 Strasbourg. France

FLI and ERG are tv, o members of the ETS transcription factor titmily. All
the members of this family share a highly conserved region termed the ETS
domain which is about 85 amino acids in size and mediates binding to a DNA
target harboring a central GGA core. Different ETS proteins bind similar, but
not identical, purine-rich sites on the regulatory regions of different genes. FLI
and ERG which belong to the same subset of ETS proteins share a 96 %
sequence identity in tile ETS domain as compared to an overall 67 % identity.
During embryogenesis in Xcnopus. expression of the Xlqli gene occurs
earlier than the expression of the Xl-erg gene and their expression patterns in
the developing embryo partially overlap, indicating redundant functions in
gene transcription regulation. We therefore addressed the question whether the
sequence differences in the ETS domains of XI-Fl,l and XI-ERG proteins may
determine subtle DNA binding specificities.
As a first step to identify target sequences for XI-FLI and XI-ERG. a
cyclic binding site selection technique was developed using a pool of
randomly synthesized oligonucleotides. The alignment of the cloned binding
sequences showed a very similar consensus site for both proteins,
AAACCGGA A/G Y/c. We will discuss here whether differences in the base
pairs that flank the consensus core sequence can modulate the DNA binding
specificity of XI-FLI and X1-ERG. or if accessory cellular protein(s) that bind
nearby in a cooperative fashion are required to achieve specific transcriptional
activation.

Abstracts FEBS'99

(Mo/6.2/198)

Evidence for the presence of a NF- kB signal

transduction system in Dictyostelium discoideum
F. Traincard1, E. Ponte1, J. Pun2, B. Coukell2 and M. Veronl
1 Unit~ de R~gulation enzymatique des Activitds cellulaires,lnstttut
Pasteur, Paris, France ; 2 York University, Toronto, Ontario, Canada

The Rel/NF-rd3 family of transcripion factors and regulators have

been discovered so far only in vertebrates and arthropods where they
mediate responses to many extracellular signals. No counterparts of genes
coding for such proteins have been identified in the Caenorhabditis elegans
genome and no NF-r,B activity was found in Saceharomyces cerevisiae. We
describe here the presence in the lower eukaryote Dictyostelium discoideum,
of proteins reacting with antibodies raised against mammalian Rel/NF-rd3,
InB and IKK components of the NF-nB pathways. Using Gel shift
experiments, we demonstrate the presence in nuclear extracts of developing
Dictyostelium cells of proteins binding to nB consensus oligonucleotides as
well as to a GC-rich r.B-like sequence, lying in the promoter of ebpA, a
developmentally regulated Dictyostelium gene encoding the Ca2+-binding
protein CBP1. Using immunofiuorescenee, we show a specific nuclear
tranlocation of the p65 and p50 homologues of the NF-KB transcription
factors as vegetatively growing cells develop to the slug stage. Taken
together, our results strongly indicate the presence of a complete NF-nB
signal transduction system in Dictyostelium discoideum that could be
involved in the developmental process.

s217

s218

Abstracts FEBS'99

8.1 Protein degradation

(Mo/8.1/199)

Degradation of Hypoxia Inducible Factor-I alpha (HIFlet) depends on the transcriptional activity of H1F-I
E. Berra, D.E. Richard, E. Gothi6, J. Pouyss6gur

(Mo/8.1/200)

Continuous hydrolysis of goat whey in an enzymatic

ultrafiltration reactor: Generation of alpha-lactorphin.
S. Bordenave*, F. Sannier, G. Ricart + and'J.M. Plot.
LGPC, UFR Sciences, Avenue Martllac, 17042 LA ROCHELLE, FRANCE.
*Laboratolre de SpectromOme de Masse. USTL, 59655 VILLENEUVE
D'ASCQ, FRANCE.

'hire de Biochtnlie C%;RS LMR 6543 (Untver~Hb de Ntce) ,arc Ualro.~e.

06108 Nice. France

Bovine whey hydrolysates have been developed and applied to areas such
Hypoxia Inducible Factor 1 is a key mediator of adaptative
responses to hypoxia. Recent evidence has demonstrated the
essential role played by the HIF-Ia subunit in HIF-1
activation. HIF-la is a short lived protein which is present at
very low levels in normoxic cells. Hypoxia rapidly increases
HIF-Ia protein levels by relaxing ubiquitin-proteasome
dependent degradation. In this study, we show that the rate of
HIF-la degradation upon reoxygenation of hypoxic cells
depends on the duration of hypoxic stress. More interestingly,
we demonstrate that degradation of HIF-Ia is suppressed by:
i) inhibiting general transcription with actinomycin D or ii)
specifically blocking HIF-l-dependent transcriptional activity
by the expression of a HIF-la dominant negative mutant. In
keeping with these findings, we postulate that HIF-Ia is
targetted to the proteasome via a HIF-Ia Proteasome
Targetting Factor (HPTF) which expression is directly under
the control of HIF-1 transcriptional activity.

as nutrition and culture media preparation. More recently, bioactive peptides

have been isolated from bovine whey hydrolysate. Goat whey also constitutes a
food processing by-product, nevertheless, information about goat whey
hydrolysate products is scarce. Hydrolysis of goat whey by a physiological
enzyme, pepsin, was carried out in a continuous enzymatic membrane reactor.
The resuhmg permeate contained a pept~de hydrolysate which was resolved by
reversed phase-HPLC. Second order derivative spectroscopy, amino acid
analysis and mass spectrometry revealed the presence of a biologically active
pept~de called alpha-lactorphin. Alpha-lactorphin opioid and ann-hypertensive
effects were evaluated by measuring the inhibition of the guinea pig ileum
(GPI) contractions and the angiotensin-l-converting enzyme (ACE) inhibttory
activity respectively. The GPI-ICs0 value was 300taM and the ACE-ICs0 value
was 7681aM. On the basis of th,s primary observation, it would be of interest to
undertake extensive studies concerning the generation of other b~oact~ve
peptides from this edible protein source.

(Mo/8.1/201)

Nuclear localization of the C2H2 Zinc-finger protein

Msn2p is regulated by stress and protein kinase A
activity!
E. Durchschlag, W. Grmer, J. Wolf, C. Sch/iller, H. guis.
Department of Biochemistry and Molecular Cell Biology at the Universzty
of Vienna; Dr. Bohrgasse 9, A-1030 Vienna, Austria

Changes in environmental conditions like stress or nutrient starvation provoke

a rapid molecular response in unicellular organisms. One mechanism to manage
gene expression is to regulate nucleocytoplasmic transport of transcription
factors. In Saccharomyces cerevisiae, the C2H2 Zn-finger transcription factor
Msn2p plays a crucial role in gene regulation in response to stress (e.g. heatshock, osmotic stress, nutrient starvation) and protein kinase A (cAPK) activity.
Stress and low cAPK activity cause rapid nuclear accumulation of Msn2p,
whereas stress-relieve and high cAPK activity promote nuclear export. We
identified two structural domains, NES and NLS, necessary for Msn2p
nucleocytoplasmic transport, and found both to be regulated by cAPK activity.
Msn5p, a member of the karyopherin-beta family, appears to be the transportin
for Msn2p export. Another aspect that indicates multiple layers of Msn2p
regulation seems to be protein-stability, as the factor is unstable in the nucleus.

(Mo/8.1/202)

Proteasomal activities in different tissues of the rat

L. Farout, M. Harrison*, G. Boissonnet, Y. Briand & M. Briand
Laboratotre de Biochimte, EA 995, UnlverMl~ Blaise Pascal, 63177
Aubt~re Cedex, France, *Department of chemistry and biochemistry.,
University of Oklahoma, USA.

The distribution ofproteasome among different mammalian tissues is not

well documented, Tanaka et al (1) using an enzyme immunoassay method
and Rivett and Sweeney (2) by immanoblot showed large differences in the
rat tissues but the methods did not reflect activities. Other datas compared
only two tissues and were performed on isolated proteasomes.
We have measured on crude extracts the 5 peptidic activities including
Chymotrypsine-like activity (LLVY and AAF substrates), Trypsin-like
activity (LSTR substrate), Peptidyl glutamyl peptide hydrolase activity
(LLE substrate) and the BrAAP and SnAAP activities (GPALG and
GPAGG substrates respectively) as a tool to reflect the real activities of the
proteasome in the cell. The caseinolytic activity was also measured with
z4C-casein. Experiments were made on ten tissues including liver, testicles,
spleen, brain, lung, kidney, heart and three skeletal muscles.
Results show large differences of activities between different tissues
ranging from one to ten times. If we consider the chymotrypsine-like activity
(LLVY) our classification is very closed to that obtain by Tanaka et al,
however this classification is not the same according the activity measured
showing a putative difference in subunlt composition of proteasome
depending on the tissue. In an other hand only few differences were showed
with the caseinolytic activity (the higher activity observed in kidney is only
twice the lower activity in muscle). This result showed that in the
proteinolytic activity, probably all the peptidic activities were concerned
and the lower one is rate limiting, reducing the differences between tissues.
References:
(1) Tanaka et al., J. Biol Chem, 261, (1996) 15197.
(2) Rivett and Sweeney, Biochem. J.,278, (1991) 171.
BrAAP and SnAAP substrateswere generous8it~ from ChristopherCardozo(Mount Sinai
School of Medicine, New York, USA)

Abstracts FEBS'99

(Mo/8.1/203)

Thyroperoxidase is largely retained and degraded in

the ER. Depending on its folding state, its
degradation involves two different s y s t e m s
L. Fayadat, S. Siffroi, P. Niecoli-Sire, J.L. Franc

s219

(Mo/8.1/204)

aLGPC, La Rochelle, VCEBC/CNRS, B/Niort, ~IPMC/CNRS,

Valbonne.

u38 INSERM, Facult~ de M~decine, Marseille, France

Human thyroperoxidase (hTPO) is a type I transmembrane heme containing

glycoprotein that plays a key role in thyroid hormone synthesis, hTPO was
stably expressed in the ClIO cell line and its folding was studied with two
mAbs: mAb47 recognizing a linear epitope accessible only when hTPO is
unfolded and mAbl5 recognizing a conformational epitope. We previously
showed (Fayadat etal, 1998 Endocrinology 139, 4277-4285) that only 15% of
the newly synthesized hTPO molecules were able to acquire a conformation
suitable for recognition by mAbl5 and that only 2% were able to reach the cell
surface.
In the present study, we focused on the degradation of hTPO and on its
interaction with molecular chaperones. The hTPO molecules recognized by
mAb47 and which were not able to acquire a eonformational structure
recognized by the mAbl5 were rapidly degraded (half life: 4h). This
degradation was inhibited by proteasome inhibitors, lactaeystin and MG132. In
contast molecules recognized by mAbl5 and that stayed at the ER level had a
longer half life (11 h). The degradation of these conformational forms was not
inlaibed by lactacystin nor by NH4C1 or chloroquine (lysosomal inhibitors).
Using a panel of protease inhibitors we showed that this degradation was
slowed by leupeptin, aprotinin and ALLN. Thus, depending on its folding
state, the degradation of hTPO at the ER level involves two different systems:
(i) the proteasome, for the unfolded forms and (ii) an unknown system
(including different protease activities) for the partially folded forms.
Moreover, we show that after synthesis hTPO molecules associate with
calnexin (CNX) and calreticulin (CRT). Treatment of cells with
eastanospermine led to a dramatic decrease both in the percentage of hTPO able
to acquire a tfidimentionnal structure suitable for recognition by mAbl5 and in
the half life of the molecules (3h versus 1 lh for control). This degradation was
inhibited by proteasome inhibitors. Thus CNX and/or CRT interaction with
hTPO is required for initial folding of this protein.

Endogenous lysosomal cathepsin proteases processing of

hemoglobin gives rise to bioac~tive ~eptides : hemorphins.
I., Fruitiera, I., Garreau~L A., Lacrolx ~, A., Cupo c, J.M., Piot ~

Biological peptides are released from proteolytic treatment of

hemoglobin. Some of them exhibited an opioid-like activity and were called
hemorphms. Besides its main function as an oxygen carrier, hemoglobin
seemed to be implicate as an in vivo putative source of bioactive peptldes.
Therefore, it was of a great interest to characterize the putative processing
enzymes leading to the release of these bioactive peptides. Concerning the
hemorpbins, preliminary studies indicated that hemoglobin incubated with
mice peritoneal macrophages generated bemorphins. However, the proteolytic
enzymes ~mplicated in the release are yet unknown. The lysosomes constitute
the roam compartment of the endocytlc pathway, so their putative implication
in the generation of hemorphins was studied. Previously a specific antiserum
against the C-terminal part of the hemorphin-7 peptide was developped. In the
present study the quantification and the characterization of the peptide release
was performed using immunological and biochemical approaches. Incubation
of hemoglobin with the lysosomal protease content induced an early
generation of hemorphin-7 related peptldes : the hemorphin-7, the VVhemorphin-7 and the LVV-hemorphin-7 peptldes.
Since a link between increased lysosomal hydrolases activities (such as
cathepsm ones) and many diseases has been reported, this work strongly
suggests that the lysosomal hydrolases could be amplicated in the generation
of biological active peptides such as hemorphms and could have important
implications in the pathophysiology of these diseases.

In conclusion hTPO is a powerful model to study (i) the coordinated and

sequential interaction with molecular chaperones leading to the protein folding
and (ii) degradation of a protein by different systems depending on its folding
state at the ER level. (This work was supported by INSERM U38 and by
Association pour la Recherche sur le Cancer).

(Mo/8.1/205)

E. coli HtrA protease participates

in oxidative stress,
B. Lipifiska, J. Sk6rko-GIonek, D. Zurawa, E. Kuczwara,
M. Wo~niak, Z. Wypych; Univ. of Gdafisk, Medical Univ. of
Gdafisk, Marine Biology Center,Gdafisk, Poland

HtrA(DegP) serine protease, indispensable for cell survival at elevated

temperatures, is a peripheral membrane protein, localized on the
periplasmic side of the inner membrane and the biochemical and
genetical evidence indicate that the physiological role of HtrA is to
degrade denatured proteins formed during heat shock in the cellular
envelope [1,2]. The aim of this study was to find out if the HtrA
protease participates in protection of cell against oxidative stress. We
compared influence of various oxidizing agents on the htrA mutant cells
and the wild type bacteria and found that the htrA mutation did not
increase sensitivity of the cell to hydrogen peroxide or paraquat but
made the cell extremely sensitive to ferrous ions(II), which are known
to enhance oxidation of proteins. Ferrous ions caused higher increase in
the level of protein carbonyl groups in the membrane fraction of the
cell when compared to the periplasm and cytoplasm. The iron-induced
oxidation of membrane proteins was higher in the htrA mutant than in
the wild type cells. Inhibition of the htrA mutant growth by iron could
be alleviated more efficiently by the nitroxide antioxidant locaiising in
the membranes than by the antioxidant acting mainly in the soluble
fraction of a cell. Growth inhibition of htrA mutant was more
pronounced during treatment with cumene hydroperoxide, anchoring in
membranes, than with t-butyl hydroperoxide, that forms a radical
mainly within cytosol. Both ferrous ions and cumene hydroperoxide,
but not hydrogen peroxide, paraquat and t-butyl hydroperoxide, induced
synthesis of HtrA protein. Our results show that HtrA protease
participates in the defense against oxidative shock and support
hypothesis that HtrA participates in degradation of oxidatively damaged
proteins localized in cellular envelope, especially those connected with
the membranes.
1. Lipifiska, B. et al., J. Bacteriol. 172. (1990) 1791.
2. Sk6rko-Glonek. J. et al., J. Biol. Chem. 272.
(1997) 8974.

(Mo/8.1/206)

Translational attenuation of CIpP in the chloroplast of

Chlamydomonas : effect on the degradation of a thylakoid
proteins.
W. Majeran, F-A Wollman and O. Vallon
CNRS/UPR 1261, lnst. BioL Phys-Chtrn., 13, r P. & M Curie. Paris, France

The chloroplast-encoded CIpP protein is essential for cell viability in

Chlamydomonas reinhardtii. We have obtained a 3-4-fold reduction in its

expression level by mutating the translational start codon from AUG to

AUU. The elpP-A UU mutant displays a reduced rate of proteolysis of the
cytochrome b0c complex of the thylakoid membrane, in two conditions
where it is subject to accelerated proteolysis: in nitrogen starvation and
when the Rieske Fe-S protein is absent or mutated. In the latter case, the
Rieske protein itself was stabilized, possibly as a result of decreased
degradation at a pre-translocation step. In contrast, attenuation of CIpP
expression did not rescue the unassembled subunits in a series of mutants
fully defective in cytochrome b6f assembly. Hence, a Clp protease appears
to be involved in the degradation of cytochrome b6f once it is assembled in
the membrane. In ATP-ase CF0-mutants, the attenuation of CIpP also
resulted in a protection of PSII complex during light-stress.
The clpP gene in C. reinhardtii displays a large insertion sequence
IS 1, in reading continuity with the rest of the protein, and found only in this
genus. It has been proposed that IS1 is eliminated by protein splicing.
However 1S 1, bears no sequence similarity to classical inteins. An antibody
raised to the entire reading frame (59 kDa) expressed in E. coli recognizes
mainly a 26 kDa band which we identified as "mature" CIpP. A 59 kDa
"precursor" was also recognized, totaling 5-10% of the immunoreactivity.
Both forms are attenuated in the clpP-AUU mutant and are therefore
products of the same clpP gene. Both forms are highly stable in vivo, when
chloramphenicol is added to block their de novo synthesis. Hence, protein
splicing if it occurs, must take place immediately after translation in a
subpopulation of primary translates.

s220

(Mo/8.1/207)

Abstracts FEBS'99

The 20S proteasome of Frankia

M-N. Pouch,, B.Coumoyerb, and W. Baumeister~

(Mo/8.1/208)

aMax-Planck-lnstttut fitr Biochemie, D-82152 Martmsrted, Germany

bLaboratotre d'Ecologie Midrobienne du Sol, U M.R C N,R.S. 5557
F-69622 Villeurbanne Cedex, France

Frankia is an actinomycete which fixes atmospheric nitrogen in symbiotic

association with the root systems of a variety of nonleguminous plants
denominated actinorhizal plants. Information on Frankia proteases is rather
scarce as it is extremely difficult to grow this organism. We have purified
20S proteasomes from Frankia strain ACNl4a/ts-r. It is composed of one (x
subunit and one B subunit assembled into four rings of seven subunits each.
The enzyme displayed a chymotrypsin-like activity against synthetic
substrates and was sensitive to lactacystin, a specific proteasome-inhibitor.
Analysis of the structural genes and the flanking regions revealed a similar
organization to Rhodococcus, Mycobacterium and Streptomyces and showed
that the /3 subunit is encoded with a 52 amino-acids propeptide which is
cleaved in the course of the assembly. We report also for the first time the in
vitro reconstitution of chimeric proteasomes composed of Frankia and
Rhodococcus subunits, which are correctly assembled and proteolytically
active.

Effects of storage on activity and subunit structure of AMP

deaminase from skeletal muscle of bass.
M. Ranieri-Raggi,D Martini,C. Pret'tib,A. Sabbatini,A. Raggi"
"DIp Scwnze ( -omo e .4mbwnte. bDtp l'atotogm, tmmale. ( )m" lh ~a, ltaha

It is well established that NH3 liberated in fish muscle during severe

exercise or during rigor morris arises solely from deamination of AMP to
IMP, catalysed by AMP deaminase (AMPD). We previously observed
that on storage rabbit muscle AMPD undergoes fragmentation, the 79
kDa subunit being cleaved to a product of approx. 70 kDa which exhibits
hyperbolic kinetics at low K' concentration even in the absence of ADP
[1] Similar alterations of kinetic and structural properties of AMPD are
observed during storage of the enzyme prepared from skeletal muscle of
bass (Dicentrarchus labrax) suggesting that also in fish muscle the
activation of AMP deamination, causing depletion of the adenine
nucleotide pool during rigor mortts, is due to a protease-induced removal
from AMPD of a fragment acting to hold the enzyme in an inactive
conformation An activation offish muscle AMPD similar to that brought
about by storage results from limited proteolysis with trypsin. Both the
native and trypsinized enzymes are strongly inhibited by heparin, a
mucopolysaccharide that interacts with human plasma histidine-prolinerich-glyeoprotein (HPRG) [2] These results together with the
observation that an antibody against human plasma HPRG cross-reacts
with both native and trypsinized AMPD from fish muscle strongly
suggest that an HPRG-like protein is an integral component of the
AMPD complex extending to the fish enzyme the evidence previously
given with rabbit and human skeletal muscle AMPD [3]
[1] Ranieri-Raggi M and Raggi A, Biochem J , 189, (1980), 367.
[2] Lijnen H R. et al., J. Biol. Chem, 258, (1983), 3803
[3] Sabbatini A R M et al. J Histochem Cytochem, 47, (1999) 255

(Mo/8.1/209)

Myofibrillar turnover in rabbit skeletal muscle.

W. Reville a, O. Walsh a, M. Motherway a, M. Zeece b.
"Btoehemistry Department, Untversity College. Cork, Ireland.
t'Department of Food Technology, UniverstO/of Nebraska, USA

Myofibrils in skeletal muscle turnover continuously but many details of the

underlying mechanism remain unknown. There is substantial evidence that
the myofibril surface plays a special role in turnover. A subset of easily
releasable myofilarnents (ERM) is easily shed from the myofibril surface
under relaxing conditions, i.e. 0.1M KC1, 3raM Mg-ATP, 3mM EGTA, pH
7.2. There is considerable evidence that ERM play an important role in the
mechanism of myofibril turnover [1]. If ERM are important intermediates in
myofibril degradation it would be expected that their yields would increase
under conditions of increased myofibrillar catabolism and would decrease
under conditions of depressed catabolism. Previous work in this laboratory
has confirmed this correlation in several atrophic conditions and in
clenbuterol-stimulated muscle hypertrophy [2]. The present study measured
ERM yields during the maturation of rabbits.
Rates of myofibrillar
degradation are known to be higher in immature animals and to decline as the
animal matures. If the hypothesis regarding ERM and turnover is true, it
would be expected that ERM yields would decline as the animal matures. In
the present study ERM yields were measured in the back muscle of rabbits at
3 different ages - 2.5 months old. 5.0 months old and 15.0 months old. ERM
yields declined significantly over this period from a mean of 2.53% total
myofibrillar protein at 2.5 months, through a mean of 2.01% at 5.0 months, to
a mean of 1.14% of total myofibrillar protein at 15 months old. These results
support the hypothesis that ERM play a role in the 'in vivo' mechanism of
myofibrillar degradation.
[1] van der Westhuyzen, D.R., et al., J. Biol. Chem., 256, (1981), 11791.
[2] Murray, B.A., et al., Proc. 23 rd FEBS Mtng., Basel, (1995), P14.8.

(Mo/8.1/210)

NFr.B activity, II~B~ deficiency and chemokinelL-8

overproduction in CF bronchialbglands
0. rabary . S. Esc0tte. D Dusser. D. Hubert. J P C00cn[ . E
Puchelle~,l Jacqu0t~.
"INSERM Unit6.514, 0S1092, R('t ,s,/'H61 a a l C
Broussais. Parts, Fram e

, I'trts. 'It@ira[

We have recently shown a selective upregulation of chemokine IL-8 in cystic

fibrosis (CF) bronchial gland cells in vivo and in vitro [I]. Evidence suggests
that, in airway epithelial cells, activation of the nuclear factor kappa B (NFk B ) whmh regulates the level of mt]ammatory genes transcription, is mainly
controlled by the cytosohc inhibitory factor I~,Bc(. We demonstrate here that
in human CF bronchial tissues, submucosal gland cells exhibit an absence of
inhibitor factor lkBCZ and high levels of chemokine IL-8 expression. These
results are confirmed by in vittw studies using primary cultures of CF
bronchial subnmcosal gland cells in which a lack of cytosolic IkBa. high
levels of activated NF-kB (as shown by EMSA) associated with a
dysregulation of IL-8 production (a 13-fold increase, as assayed by ELISA)
are found when compared to non-CF (control) disease bronchml gland cells.
Interestingly, treatment of CF gland cells with the isoflavone genistein, a well
known tyrosine klnase inhibitor, results in a significant decrease (p<O.001) in
IL-8 production down to those levels of IL-8 by non-CF gland cells. This
decrease in IL-8 production is associated with a 85 % increase of the IkBo~
protein level in CF gland cells. Addition of gcnistem also reverses the effects
of LPS Pseudomonas aerughwsa-induced nuclear translocation of NF-~,B by
increasing cytosolic IkBCt protein in CF gland cells. We also demonstrate that
genistein induces a 75 % inhibition of endogenous IKK-a kinase activity (as
demonstrated by Western blot), known to specifically phosphorylate IkBa
factor, leading to its degradation and translocation of NF-kB to the nucleus.
Taken together, this strong inhibition of constitutively activated N F - k B with
the resulting down regulation of IL-8 production by genistcm in the CF
airway glands highlights the key role of cytosolic [kBOt factor and IKK-~
kinase actiwty in the regulation of pro-lnflammatory cytokines in airways of
CF patients.
[11. Tabary O. et al., Am. J. Pathol.. 153, (1998), 921.
O. Tabary is a fellowship funded by GOEMAR laboratories.
Supported by AFLM (Association Franqaise de Lutte
Mucoviscidose).

contre

la

Abstracts FEBS'99

s221

9.2 Endocytosis
(Mo/9.2/211)

Endocytosis of angiotensin II receptor subtype

AT~ in isolated rat hepatocytes
M.C. Caro, M. Montiel, E. Jimdnez
Department of Bio~'hemi.stry ctJut Molecular Bi~logy.
Fatal O' of Medicine. UniverviO, r~[Mcllaga. 29080Malaga, Spcdn.

Angiotensin 11 (Ang lI) receptors sequestration is a mechanism of receptor

desensitization required for Ang I1 degradation and for recycling process
which returns internalized receptors to the cell surface [ 1,21. Moreover. Ang
ll-induced receptor intemalization is necessary to maintain the production of
certain intracellular signals in some target cells 13,41. In this study we have
investigated the relationships between Ang 11 receptor endocytosis and the
generation of second messengers in rat hepatocytes. The results of the
present study demonstrate that in response to exposure of hepatocytes to
Ang I1 a decrease in surface Ang 11 receptors occurs consistent with a rapid
endocytosis of the receptor-bound hormone complex. Pretreatment of cells
with okadaic acid (OA) did not have any effect on receptor mediated
internalization. In contrast, a marked reduction of the Ang II receptor
endocytosis process occurred after treatment of hepatocytes with
phenylarsine oxide (PAn), indicating that cysteine residues could be
involved in the receptor-mediated endocytosis. Stimulation of cells with Ang
I1 blocked the generation of cyclic adenosine monophosphate (cAMP) which
follows the stimulation of hepatocytes with forskolin. Moreover. Ang 11
increased both inositol 4,5-bisphosphate (1P,) and inositol 1,4,5trisphosphate ([P,) generation, and enhanced intmcel[ular ca[cium
concentration (ICa~'*],). Exposure of cells to PAn did not alter the effect of
Ang 11 on the accumulation of cAMP after forskolin stimulation, indicating
,that endocytosis of the agonist-receptor complex is not involved in adenylate
cyclase inhibition. Conversely, PAn and OA markedly reduced IP. and IP~
synthesis, and the plateau phase of the Ang ll-induced Ca"* mobilization. In
summary, the relationship between Ang 1I induced endocytosis and the
generation of phosphoinositols and increment in ICa2+l, indicates that
sequestration of the Ang 11 receptor is necessary to maintain the production
of these intracellular signals in rat hepatocytes.
111Crozat A eta1. Endocrinology 118, 11986) 2312.
121 Ullian MEetal. J. Clin. Invest. 84, (1989) 840.
131 Kapas Set al. Biochem. Biophys. Res. Commun. 204, (1994l 1292.
[41SchellingJR etal.J. Clin. Invest. 90, (1992) 2472.
Supported by a grant of DGICYT (PB94/1476)

(Mo/9.2/213)

Two novel sec7-domain containing proteins, Arno3 and

Efa6, involved in different stages of membrane trafficking.
M. Franco I, J. Boretto, S. Monier, P. Peters, C. D'SouzaSchorey and P. Chavrier 2. IIPMC CNRS, 660 route des luctoles 06560

(Mo/9.2/212)

Endoeytosis of LDL-Proteoglyeans complexes in

Fibroblasts and Monocytes/Macrophages
R. Coinu, M.A. Meloni, S. Maggio, F. Lumbau, P. Pippia
Dipartlmento di Sclenze Fisiologlche. Biochimiche e Cellalari,
Universifftdl Sassari,via Muroni25. 07100 Sassan,Italy

Accumulation of lipids, notably cholesteryl esters, in the arterial wall

cells is an important early event in atherosclerosis. The association of low
density lipoproteins (LDL) with arterial wall proteoglycans (PGs) appears
partially responsible for the lipid accumulation during atherogenesis. This
interaction leads to lipopmteins entrapment, modification and uptake by
maerophages and smooth muscle cells thereby promoting foam cells
formationvl. However, the "in vivo" mechanism of LDL mediated foam cells
formation is unknown. The aim of this work is to visualize the intraeellular
pathway of fluorescent labeled LDL and LDL-PGs complexes and contribute
to better understand the mechanism of LDL-PGs complexes uptake by human
monocyte derived macrophages and fibroblasts, in order to explain the role,
not yet clarified, of lipoproteins-proteoglycans interaction on foam cells
formation and atherogenesis.
Proteoglycans were isolated from human aortas, purified by
chromatographic technique and conjugated with fluorescein isothiocyanate
(PGsFITC). LDL were separated from normal human donors plasma by
ultracentrifugation and labeled with the fluorescent dye dioctadecyltetramethyl-indocarbocyanine (LDLDiI). The insoluble complexes, obtained
incubating LDLDil and PGs or LDL and PGsFITC, were collected by
centrifugation and solubilized in appropriate culture medium. The complexes
were incubated with rat fibroblasts and human monocyte derived macrophages
at 15-30 min and 1-2-4-6 h, observed by fluorescence microscopy and
spectrofluorometrically quantified in chloroform extracts. Binding specificity
was determined by competition with 20-fold excess of unlabeled LDL.
Our results indicate that: fibroblasts and monocyte derived
maerophages bind, internalize and accumulate LDL and LDL-PGs complexes;
the fluorescent distribution in both cells is consistent with receptor mediated
uptake; native LDL and LDL-PGs complexes seem to be internalized through
the same receptor pathway; the uptake of LDL-PGs complexes is greater than
the native LDL one; PGs are internalized only when they are bound to LDL.
Although our data indicate that the internalization of LDL-PGs complexes by
fibroblasts is higher then LDL alone, further studies are needed to understand
the role of PGs in the intracellular lipid accumulation. To verify whether LDL
bound to arterial PGs can lead to lipid critical accumulation in fibroblasts and if
these cells can be transformed into foam cells further investigations should be
undertaken.

(Mo/9.2/214)

Haemoglobin binding sites on renal brush-border

membranes
J. Gburek, J. Osada, WroclawUmverstty oj Medicine,
Faculty of Pharmacy, Dept Btochemtstry, Wroctaw, Poland

Valbonne, 2CIML, parc de Luminy, 13288 Marseille Cedex 9 France,

ADP-ribosylation factors (ARF) are small GTP-binding proteins that

play a regulatory role in secretory and endocytic pathway of eukaryotic cells.
ARF1 and ARF6, are the best characterized members of the ARF family.
ARF1 plays a role at the Golgi apparatus level where it triggers the
recruitment to the membrane of coat proteins that are "instrumental" for
vesicle budding. ARF6 which localises to the cell periphery, appears to be
involved in receptor-mediated endocytic trafficking and in actin cytoskeleton
rearrangement. Activation of ARF protein is catalyzed by a guanine
nucleotide exchange factor (GEF). ARNO was the first mammalian ARFGEF discovered. It contains a Sec7 domain which have been shown to be
responsible for nucleotide exchange activity in vitro on ARF 1.
Here, we describe a novel member of the ARNO family, that we
referred as ARNO3. We show that ARNO3 is more active as GEF in vitro on
ARF1 than on ARF6. Its overexpression in mammalian cells results in both a
redistribution of Golgi resident proteins and an inhibition of protein transport
across the Golgi. In contrast, distribution of endocytic markers is not affected.
We conclude that ARNO3 by regulating ARF1 activation, controls
morphology and function of the Golgi apparatus.
By screening for new Sec7 domain containing proteins, we have cloned
and caracterized a new ARF-GEF. In contrast to ARNO family, this protein
we referred as EFA6 (for Exchange Factor for ARF6) is more active in vitro
on ARF6 than on ARF1. EFA6 localizes to a dense matrix on the cytoplasmic
face of plasma membrane invaginations, induced by its expression. EFA6
regulates endosomal membrane recycling and promotes the redistribution of
the transferrin-receptor to plasma membrane Furthemore, expression of
EFA6 induces actin-based membrane ruffles that are inhibited by coexpression of dominant-inhibitory mutant forms of ARF6 or Racl. Our
results demonstrate that by catalyzing nucleotide exchange on ARF6 at the
plasma membrane and by regulating Racl activation, EFA6 coordinates
membrane dynamics at the cell periphery with cytoskeletal rearrangements.

In several haemolytic diseases the level of haemoglobin appearing

in the circulation exceeds the binding capacity of haptoglobin, therefore
free haemoglobin passes the glomerular filter. Prolonged exposure of renal
tubules to haemoglobin markedly reduces renal function and eventually
leads to the acute renal failure called pigment nephropathy. Intracellular
haemoglobin toxicity is one of the most important pathomechanisms
involved in the development of the disease [ I ]. However, the process in
which haemoglobin is taken up by kidney tubular epithelium, has not been
characterized so far.
In the present study we used isolated renal brush border
membranes of the rat and radioiodinated rat and human haemoglobin.
Our experiments showed that both human and rat haemoglobins
can be specifically bound by renal brush border membranes. Fall in
binding activity after preincubation of membrane vesicles with peptidases
suggested proteinacous nature of the binding sites. Analysis of binding
data revealed one class of low affinity (Kd 7.7 p.M), high capacity
(Bmaxl1.2 p.g/mg protein) binding sites. In competition experiments the
sites were relatively selective for haemoglobin. Albumin did not compete
with hemoglobin at any tested concentrations. Cationic molecules i.e.
cytochrom C and lysine exhibited some competition, while strong
competition of myoglobin, was observed. The binding was greatly affected
by EGTA indicating Ca reqmrement for the mteracUon. There was
considerable rise in binding in pH 5.4. The binding protein has been
partially purified and characterized.
Existence of binding sites for haemoglobin in renal brush border
membranes strongly suggests that uptake of the protein by tubular epithelia
occurs via adsorptive endocytosis. Increased binding of haemoglobin to
membranes under acidic conditions may explain exacerbation of
haemoglobinuric acute renal failure in aciduric states.

2 +

[1] Zager R. A., Kidney Int., 49, 1996, 314.

s222

(Mo/9.2/215)

Abstracts FEBS'99

Expression and fate of Nerve Growth Factor receptors TrkA

and p75 NTR
J r r r m e Jullien*, Jos6 Luis Urdiales*, and Brian B. Rudkin*.
* UMR 5665, 46 All6e d'Ialie, 69364 Lyon codex 07, FRANCE.

Previous work has described the cell cycle phase-specific action of nerve
growth factor (NGF). In the pheochromocytoma cell line PC12, NGF ellicits
an anti-mitogenic effect, i.e. inhibition of the G1 to S transition, and provokes a
differentiation response in the GI phase, while a survival effect is stimulated
elsewhere in the cell cycle. The proto-oncogene c-fos is also induced in the
G1 phase of the cell cycle. These observations suggest that the infrastructure
necessary for signal transduction is different depending on the cell cycle
phase. Since signalling starts at the receptors, we have undertaken studies of
the expression and fate of the receptors for NGF, TrkA and p75NTR, in the
PC12 model. The results of experiments designed to evaluate the synthesis,
maturation and turnover of the receptors in the absence and presence of NGF
are presented.
This work was supported by grants from the "Association pour la
Recherche Contre le Cancer", the "Ligue National de Recherche Contre le
Cancer", the Rh6ne-Alpes Region, the Ministry of Research and Technology
and the European Union Biomed program.

(Mo/9.2/217)

Discovery of a novel receptor for

secretory phospholipases A2 in porcine brain
I. Kri~aj, N. Vu~emilo, A. t%pi~, F. Guben~ek
Department of Biochemzstr) and Molecular Bzology.
Jo~.ef Stefan Institute, 1000 Ljubljana, Slovenia

A specific phospholipase A2 receptor from porcine cerebral cortex has been

characterized (Kd = 145 nM, B.l,x - 0.4 pmol/mg membrane protein), using
a radioiodinated derivative of ammodytoxin C (AtxC), a snake venom
neurotoxic phospholipase A2 [1]. After the receptor was solubilized in a
ligand-binding form, it was approximately 14,000-fold enriched by
chromatography on Wheat germ Lectin-Sepharose and AtxC-Affi-Gel 10.
The receptor is a single-chain glycoprotein with an apparent molecular
mass of 180 kDa and binds toxic and non-toxic phospholipases A2 of either
type 1 or II. It also recognizes mannosylated BSA. In its molecular mass
and pharmacological profile the AtxC receptor resembles the M-type
receptor for secretory phospholipases A2 from rabbit skeletal muscle [2,3]
(a C-type multilectin, homologous to macrophage mannose receptor) yet by
relative abundance in brain and antigenicity these two receptors are
completely different. An additional AtxC receptor of approximately 200
kDa discovered in porcine liver, was, however, recognized by anti rabbit
M-type phospholipase A2 receptor antibodies. There are, therefore, two
immunologically distinct secretory phospholipase A2 receptors of about
200 kDa in the same species. While the liver receptor is related to the Mtype secretory phospholipase A2 receptors, the brain AtxC receptor is not
and belongs to a novel group of secretory phospholipase A2 receptors.
[1] Kri2aj, I. et al., Biochim. Biophys. Acta, 999, (1989) 198.
[2] Lambeau, G. et al., J. Biol. Chem., 265, (1990) 9526.
[3] Lambeau, G. et al., J. Biol. Chem., 269, (1994) 1575.

(MoD.2/216)

Rab22, a small GTPase involved in endocytosis

M. Kauppi a, A. Vieira a, H. Stenmark b, and V. M. Olkkonen a
"Dept Biochemisto:, National Public" Health Institute. Helsinki, Finland.
ADept Biochemisti3; The Norwegian Radium Hospital, Oslo, Norway

Rab22 is a small GTPase related in sequence to the well characterized

regulator of early endocytosis, rab5 (53% amino acid identity). The rab22
mRNA displays ubiquitous expression in mammalian tissues, suggesting that
the encoded protein has an important housekeeping function. In the present
study we have analyzed the localization and function of rab22 in Baby
Hamster Kidney (BHK) cells using Semliki Forest virus-mediated
overexpression of the protein (cloned from MDCK cells) and its mutant forms
defective in GTP binding (S19N) or hydrolysis (Q64L). Further, we have
produced and affinity purified rabbit antibodies against rab22 to analyze its
endogenous expression pattern at the protein level. Expression of wild-type
rab22 or the Q64L mutant in BHK cells resulted in formation of abnormally
large endosomal structures which were positive for EEA 1 and negative for the
late endosomal/lysosomal marker LAMP, thus representing early endosomal
elements. In contrast, the S 19N mutant protein appeared largely cytosolic and
caused fragmentation of the EEAl-positive early endosomes. The wt and
Q64L proteins enhanced the endocytic uptake of fluid-phase HRP. These
effects are similar to those of overexpressed rab5 and its corresponding
mutant forms, suggesting that in BHK cells the function of rab22 is related to
that of rab5. Analysis of dog tissues by Western blotting and the highly
sensitive ECL-plus detection system with the affinity-purified rab22 antibody
revealed the presence of the protein in brain, testis, and kidney, and possibly
in lung and spleen. This distribution may imply predominant function of the
protein in neurons and polarized epithelial cells.

(Mo/9.2/218)

Infuence of lipid formulations of amphotericin b on

immunostimulating effect of maerophages
M. Larabi, P. Legrand, M. Appel, M. Lepoivre, G. Barratt.
Laboratoire de Phvswo-Chimie-Pharmacotechnie-Biopharmacie
UMR
CNRS 8612, Facult~ de Pharmacte, Chatenay-Malabry Cedex, Prance.

To compare the activity of amphotericin B (AmB) and AmB

associated with different lipid carriers on uptake in relationship with
the production of effectors.
Different commercially available AmB formulations were used:
liposomes AmBisomes and Ampholiposomes and complexes
Abelcet, Amphocil and a laboratory formulation prepared by a
solvant displacement method. These formulations were incubated with
routine peritoneal macrophages infected or not by Leishmania
Donovani in presence of a costimulator (INFy). After this activation
phase, the effectors nitric oxide (NO) and tumor necrosis factor alpha
(TNFcQ, and AmB associated with macrophages were measured and
NFKB induction was determined.
We observed that AmB was able to induce both TNFct and NO
production by macrophages in a dose-dependent manner only in the
presence of INFy. AmB associated with lipid carriers stimulated
significantly less NO and TNFct production in the presence of INFy
than free AmB, this reduction was not clearly related to the amounts
of AraB associated with macrophages. The NFKB induction activity
of AmB was dose and carrier-dependent and relatied to the amounts
of AmB associated with infected macrophages This results had be
confirmed by Western blot.
These results seem to provide evidence of the direct action of AmB
1/against Leishmania donovani within macrophages at low doses of
AmB rather than macrophage-mediated effects 2/ indirecte action by
ativation of macrophage effector at subtoxic dose. These results must
be confirmed in macrophages infected by yeast or parasite in vivo.

Abstracts FEBS'99

(Mo/9.2J219)

Cloning of Rabip4, an effector of the small GTPase Rab4.

Caracterisation of its functions in endocytosis.
M Marl, M Cormont, S Marl, Y Lc Marchand-Brustel

s223

(Mo/9.2/220)

Rabef4: An effeetor protein of the small GTPase Rab4

I. Met6n, M. Cormont, S. Mari and Y. Le Marchand-Brustel
INSERM E 99-11 and U145. Facult~ de M~decme. Nice. France

I N S E R M E 99-11 a n d U145. Faculty; de mddecine. Nice. France.

Rab4 is a small GTPase of the Rab family involved in the regulation of

endocytosis at the level of sorting endosomes. It plays a role in the recycling
of Glut 4 in adipocytes and in the receptor-mediated antigen processing in B
cells. To better understand the role of Rab4 in endocytosis, we decided to
characterize its effectors. A eDNA library was screened in the yeast two
hybrid system using Rab4 without GTPase activity as a bait. The sequence
coding for the protein corresponding to one of the isolated clones was
obtained combining dBEST interrogation. RACE-PCR and RT-PCR
approaches. The protein, named Rabip4 (for Rab4 interacting protein),
possesses 600 aa and presents 40 % homology with a Rap2-binding protein
in its N-terminus while its C-terminus is 40 % similar to EEA1, an effector
of Rab5. The protein possesses a FYVE finger and two putative coiled-coil
domains. Using the yeast two hybrid system, the protein was shown to be
an effector of Rab4 since its interacts only with Rab4-GTP The domain of
interaction of 10 aa with Rab4 '~as localized at the end of the second coiledcoil domain of Rabip4. Rabip4 was fused with the green fluorescent protein
GFP and overexpressed in CHO cells. By confocal immunofluorescent
microscopy, overexpressed Rab4 gave a punctate labeling, corresponding to
endosomes. In cells overexpressing GFP-Rabip4 alone, punctate structures
of a bigger size were labeled. When GFP-Rabip4 and Rab4 (Wild type or
GYP-hound) were co-expressed, the two markers were predominantly
colocalized, and an enlargment of the vesicular structures was observed.
There was no substantial colabeling of Rabip4 with GFP-Rab5, GFP-Rab7
and GFP-LAMP, markers of early endosomcs, late endosomes and lysosomes, respectively. However, there was a partial, but significant, colabeling
of GFP-Rabip4 with overexpressed transferrin receptors that disappeared
from the plasma membranes. In CHO cells stably expressing GFP-Rabip4.
no major modifications of the fluid phase-endocytosis were observed. These
results strongly suggest that Rabip4 is an cffector of Rab4 and that it might
participate in the enlargement of a Rab4 positive endosomal compartment
that seems not to be involved in the route to lyzosomes.
Supported by INSERM APEX 97-01 and JDH 198302.

(Mo/9.2/221)

Endosome-like compartments at an active synapse

O. Shupliakov a, J. Crum b, M. Ellismanb, L. Brodin a
a Karolinska Institute, Dept. Neuroscience. 171 77 Stockholm. Sweden,
b NCMIR, UCSD School ofMedwine. La Jolla, CA 92093-0608. USA

Clathrin-dependent endocytosis appears to be essential for the recycling of

synaptic vesicles. It is still an open question, however, whether it revolves a
single or two sequential membrane budding steps. The classical model [1]
proposed that synaptic vesicles were retrieved from the plasma membrane in a
clathrin-mediated budding reaction. The "primary" coated vesicles were then
thought to fuse and form endosomes from which synaptic vesicles were
generated in a second budding reaction. In an alternative model, synapnc
vesicles were proposed to be generated directly by uncoating of primaLw clathrin
coated vesicles [3, 4]. According to this model, the endosome-like structures that
appear in nerve terminals after intense stimulation would primarily represent
deep invaginations of the plasmalemma. Both hypotheses have received support
from in vitro studies [e.g. 2, 4]. To elucidate the nature of the membrane
structures appearing at active zones after stimulation, we have performed a 3-D
morphological analysis of this area in stimulated synapses.
Experiments were performed on giant synapses established by reticulospinat
axons in lamprey, which have been extensively used to study vesicle recycling
mechanisms. The synapses were stimulated at 0.2 and 5 Hz, respectively, fixed
in glutaraldehyde and embedded for EM analysis. In some of the experiments a
fusion protein containing the SH3 domain of amphiphysin was rejected into the
axons prior to stimulation, to perturb synaptic vesicle budding from the plasma
membrane compartment [5]. 0.5gin thick sections were examined at 400kV and
electron tomographic reconstructions were produced of endosome rich regions.
Serial thin section analysis was performed on similar material.
The results showed that endosome-like structures at stimulated synapses
represent either (1) membrane invaginations connected to the plasma membrane,
(2) cisterua of smooth endoplasmic reticulum. Free endosomes were also
encountered, but they were not associated with vesicle clusters. After injection of
the SH3 domain of amphiphysin the plasma membrane invaginations were
covered with constricted clathrin coated pits, whereas no budding structures
similar in size ti synaptic vesicles were observed on other structures. The results
provide morphological support to the hypothesis that recycling of synapnc
vesicles involves only a single budding event.
[1]
[2]
[3]
[4]
[5]

Heuser J.E., Cell Biol. Intern. Reports, 13, (1989) 1063.

Faundez et al., Cell 93, (1998) 423-32.
Cremona, O. and De Camilli, P. Curt. Opin. Neurobiol., 7, (1997)323.
Schmidt, et al., J. Cell Biol. 137, (1997) 445-58.
Shupliakov et al., Science 276, (1997) 259.

Small GTPases of the Rab family are known to play an essential role in
the regulation of vesicular transport and membrane traffic. Rab proteins
cycle between an active (GYP-bound) and an inactive (GDP-bound) form.
The GTP/GDP exchange and then the state of activation of Rab proteins
are determined by a number of factors. Among Rab proteins, Rab4 is
involved in the recycling of membrane material back to the plasma
membrane from endosomal compartments. Specifically, Rab4 plays a role
in insulin-mediated translocation of glucose transporters in adipocytes and
on the pathway of receptor-mediated antigen processing in B cells. We
used the yeast two-hybrid system to search for proteins interacting with a
constitutively active form of Rab4 (GYP-bound). The complete protein
sequence corresponding to a positive clone was obtained using RACEPCR and mouse fat cell eDNA library screening approaches. The presence
in the polypeptide sequence of three SH3 domains, two proline-rich
regions and a coiled-coil domain suggests a possible role in protein-protein
interactions. Using the yeast two-hybrid system, this protein was found
to interact specifically with GYP-bound active forms of Rab4, as Rab4
Q67L. even when they lack the geranylgeranylation site and thus do not
anchor to membranes (Rab4 Q67I.ACT and Rab4 Q67LA10CT). No
interaction was observed with wild type Rab4 or with mutated forms not
able to bind GYP (Rab4 $22N and Rab4 N121I). This protein was likely
to be a Rab4 effector, and was named Rabef4. Confocal fluorescent
microscopy showed the apparition of a fine punctate staining pattern in
CHO cells transfected with either Rab4 or Rabef4. Following
cotransfection, both proteins exhibited large colocalization and the stained
structures were enlarged compared to those observed with any of the two
proteins alone, suggesting a rearrangement of the subcellular compartments
containing both Rab4 and Rabef4 in the cotransfected cells.
Supported by APEX 97-01 (INSERM) and JDFI 198302

(M0/9.2/222)

Stomatin is present in endosomes and in detergentinsoluble membrane domains.

L. Snyers, E. Umlauf, R. Prohaska.
lnstitute of Biochemistry, Dr. Bohrgasse, 9/3, 1030-Vienna, Austria.

The 31 kDa integral membrane protein stomatin has a monotopic

topology and a cytofacial orientation. The expression of stomatin can
be up-regulated in the human amniotic cell line UAC after treatment
with interleukin-6 and dexamethasone. Using this cell line, we have
shown previously that stomatin is located in plasma membrane
protruding structures and coloealizes with cortical actin. It is also
present in juxta-nuclear vesicles. In addition, stomatin forms high
order homo-oligomers, suggesting that this protein has a structural
function in the cortical morphogenesis of the cells. Here we show that
stomatin colocalizes with the GPI-anchored proteins PLAP and M F R a
in UAC cells. This observation enabled us to demonstrate two
different aspects of stomatin. First, using antibody internalisation
assays, we show that the peri-centrosomal vesicles containing
stomatin correspond to a subset of endosomes and are labeled with the
Iate endosome/lysosomal marker LAMP-2. Secondly, we found that
stomatin is partially present in lipid rafts and co-patches with PLAP
on the plasma membrane, after cross-linking of PLAP by antibodies.
These data indicate that stomatin and GPI-anchored proteins are
linked through lipid rafts and undergo the same sorting events. We
propose a model in which one function of stomatin is to concentrate
raft-associated proteins in microvillar structures.

s224

Abstracts FEBS'99

14.2 ABC transporters

(Mo/14.2/223)

INHIBITION OF PKC AND TYR K DECREASES MRP

FUNCTION
Z. Benderra, H. Morjani, M. Manfait

(Mo/14.2/224)

Unit~ M~DIAN, 1FR 53, UFR de Pharmacie, Universit~de Reims

Champagne-drdenne, 51 rue Cognacq Jay, 51096 Reims, France.

Mukidrug-resistance (MDR) is a major limitation to chemotherapeutic

treatment of cancer. MDR, conferred by overexpression of P-glycoprotein
(Pgp) and/or the multidrug-resistance associated protein (MRP), is
characterised by a decreased cellular drug accumulation due to an enhanced
drug effiux. In the present study we have examined phosphorylation
proprieties of Pgp and MRP. Both proteins are probably phosphorylated by
protein kinase C (PKC) and/or tyrosine kinase (Tyr K).
The efficacy of genestein (GNST), CGP57148B (CGP) (inhibitors of Tyr K)
and GF109203X (GFX) (inhibitor of PKC) on the sensitivity and nuclear
accumulation of daunorubicine (DNR) was examined using K562 cells lines
overexpressing MRP (KH30) and Pgp (KH300). Nuclear accumulation of
DNR was carried out using microspectrofluorometry confocal laser. We show
that nuclear accumulation and cytotoxicity of DNR were increased in KH30
ceils in presence of GNST, CGP and GFX. CGP and GFX have a moderate
effect on modulation o f MDR in KH300 cells. GNST was unable to increase
the DNR nuclear accumulation and sensitivity of these cells.
These findings suggested that MRP protein is highly phosphorylated by tyr K
and PKC. In KH300 cells, CGP and GFX probably interact with Pgp directly.
This result- does not support the concept of a major contribution of tyr K and
PKC to a Pgp-associated MDR. Finally, GNST, GFX and CGP might be a
useful for discrimination between Pgp and MRP phenotypes,

(Mo/14.2/225)

Diagnostic motifs and search for novel members of the

mouse ABCl-like gene family
C. Broccardo, M.-F. Luciani, B. Jordan, and G Chimini.
CIML, case 906, 13288 Marseille cedex 9, France.

Several subclasses can be defined in the family of the ATP Binding Cassette
(ABC) transporters on the basis of a number of structural criteria, e.g. the
number of subunits required to assemble a functional molecule, the topology
and number of the transmemhrane spanners and the presence of extra domains.
In this context, ABC1 defines a structural subclass since it possesses a putative
regulatory domain reminiscent of the R domain of CFTR, but split here into
two halves by a stretch of highly hydrophobic amino acids. This peculiarity is
shared by three recently identified mammalian ABC transporters (ABC2, ABC3
and ABCR) and by the Caenorhabditis elegans gene ced-7. It is worth noting,
that none of the 29 ABC transporters present in yeast shows these features.
We report here the identification and characterization of an additional ABC1like gene, ABC4, which is located on mouse chromosome 10B4-CI. This gene
shows an exon-intron organization perfectly mirroring that of ABC1, ABC2
and ABCR and encodes a transporter more than 50 % identical to the other
members of the subfamily.
A comparative analysis of the expression patterns of the ABC l-like transporters
shows that, in spite of an ubiquitous low level transcription, they have distinct
and non-redundant preferential areas of expression. For instance, out of a panel
of adult mouse tissues, ABC1 is preferentially expressed the pregnant uterus,
ABC2 in the brain, ABC3 in the lung, ABCR in the retina and ABC4 in the
spleen.
A comparative analysis of these highly related sequences allowed to highlight
specific motifs conserved throughout the ABCl-like family and absent from
other mammalian ABC transporters. In addition when extended to the domain
structure this analysis evidenced an extremely high conservation of discrete
transmembrane spanners, possibly correlated to a basic functional constraint as
opposed to the very low conservation for instance of the regulatory domain,
likely thus to reflect specific functional diversification.
The presence of highly conserved diagnostic motifs at fixed positions together
with the extreme conservation of the exon-intron organization allowed us to set
up a PCR based- extensive search for novel genes belonging to the ABCl-like
family. Preliminary results indicate that at least three other putative novel
members are yet to be identified.

Use of a cell culture system (Caco-2 cells) to

investigate the intestinal transport of xenobiotics
V. Bergera, Y. Larondelleb, A. Trou~t c and Y.-J. Schneidera
Laboratoires de abioch#me cellulaire, de biochimie de la nutrition,
de ' bwlogie cellulaire, Universit~catholique de Louvam,
Pl. Louis Pasteur, I, B-1348 Louvain-la-Neuve, Belgium

Caco-2 cells, derived from a human colon adenocarcinoma, spontaneously

differentiate into small intestine absorptive cells. Cultivated onto microporous
membranes within bicameral inserts, they express carrier systems for amino
acids, peptides, bile acids, vitamins and the P-glycoprotein (an effhix pump)
at their apical side. RT-PCR indicates that Caco-2 cells express the mRNAs
for the two MRP (Multidrug Resistance-Associated Protein) isoforms, MRP 1
and MRP2 (cMOAT) (unpublished results).
We used this in vitro model to study the intestinal transport of the mycotoxin
ochratoxin A (OTA). OTA contamines animal feed (oat, barley and rye) and
human food (wheat, rice, coffee, beer, pig meat) and is nephrotoxic for several
animal species including humans. OTA is also immunosupppressive,
genotoxic, teratogenic and carcinogenic. This mycotoxin is a weak organic acid
with a pKa value of 7.1. A decrease of pH from 7.4 to 6.0
(microenvironmental pH of the small intestine) increases the fraction of
uncharged (non-dissociated) molecules from 33% to 93%.
At pH 7.4 at both sides of the Caco-2 cells, O T A is transported selectively in
the basolateral (BL) to apical (AP) direction. Acidification of the donor
compartment to pH 6.0 (pH of the acceptor compartment being maintained at
7.4) increases intracellular accumulation and transepithelial transport. In
conditions similar to the in vivo situation (AP pH 6.0, BL pH 7.4), AP to BL
transport is higher than BL to AP transport, indicating that OTA is absorbed.
Metabolic inhibitors decrease BL to AP transport and increase AP to BL
transport and accumulation from BL side.
Verapamil, an inhibitor of P-glycoprotein, does not affect the transport of
OTA across Caco-2 cell monolayers. In contrast, indomethacin, an inhibitor
of MRP, decreases BL to AP transport at pH 7.4 and increases AP to BL
transport and intracelhilar accumulation from both AP and BL sides at pH
6.0. These results suggest that the transepithelial transport of OTA across
Caco-2 cell monolayers is mediated by MRP.
The results illustrate that the Caco-2 cell culture system provides an
interesting model of the intestinal mucosa to investigate the interaction with
xenobiotics.

(Mo/14.2/226)

Investigating the expression and function of a putative

ABC transporter in peHwinlde.
M.Cannieux a, M. Rideau b, S. Hamdi b
Institute of Biological Sciences, Aberyst'~h, Wales, UK. SY23 3DA
b Biomolceuleset Biotechnolo~iesVe~,etales.37200 Tours, France

ABC (ATP-Binding Cassette) have been proven competent in active

transmembranous movement of numerous chemically unrelated substrates,
both in proearyotie and eucaryotic cells. In mammals, their importance is
focused by the diseases associated with their malfunction, like cystic fibrosis
or multidrug resistance of cancerous cells [ 1].
An increasing number of ABC transporters have also been identified
in higher plants, but their function remains obscure, with exception to the
Arabidopsis MRPs working as glutathion-conjugate ATPases [2].
We have isolated a partial eDNA (CrABC1) from a Catharamhus
roseus cDNA library that shows highest similarity with TUR2, a PDRS-like
ABC transporter previously characterized in the water lens Spirodella
polyrrhiza [3]. Unlike the stress related TUR2, CrABC1 fails to show a
regulation by abscisic acid. Its expression in periwinkle cell suspensions is
strongly up-regulated by trans-zeatin; high expression levels have also been
shown to be correlated with indolie alkaloid culture conditions (suppression
of 2,4-D, addition of trans-zeatin or methyl jasmonate).
The yeast PDR5 transporter is involved in pleiotropic resistance by
pumping various hydrophobie drugs, including cycloheximide, out of the
cells We could show no enhanced expression of CrABCI in a
cycloheximide resistant periwinkle cell line, indicating a probably
completely different function for the transporter in higher plants

[1] Higgins et al., Annu. Rev. Cell Biol. 8, (1992) 67.

[2] R e a e t al., Annu. Rev. Plant Physiol. Plmlt Mol. Biol. 49, (1998) 727.
[3] Smart etal., J. Biol. Chem. 269, (1996) 19351.

Abstracts FEBS'99

(Mo/14.2/227)

P-glyeoprotein-catalyzed doxorubicin transport into

DNA-loaded proteoliposomes
M. Chambon and O. Viratelle

s225

(Mo/14.2/228)

Mechanisms of CFTR regulation by xanthines.

V.Chappel,y.Mettey2,J.M.Vierfond2,F.Becq3,M.Golal &B.Verrier1
I uPRg024 CNRS, Marseille, France, 2,3 UMR6558, Poitiers, France.

Centre de Recherche Paul Pascal/CNRS, 33600 Pessae, Prance

P-gtycoprotein (P-gp), a member of the ATP-Binding Cassette transporter

family, is involved in multidrug resistance phenotype (mdr) by
transporting drugs as doxorubicin (Dox) out of the cells. A powerful in
vitro system to study
this transport would be DNA-loaded
proteoliposomes, as they combine the possibilities to control all the
relevant parameters and to take advantage of Dox fluorescence and its
quenching by DNA. The characterization of this system, obtained by
reconstitution of P-gp after solubilization by octylglucoside of plasma
membranes, has shown that the freeze/thaw procedure successfully
entrapped DNA in all proteoliposomes, and that P-gp was still active as
determined by its verapamil-stimulated ATPase activity. The passive
diffusion of Dox was quantified through its fluorescence decrease upon
time. Surprisingly, in the presence of ATP/Mg 2. which is required Ibr Pgp activity, diffusion rate was slower than in its absence. ATP without
Mg> induced a similar inhibitory effect. Understanding this inhibition was
a crucial point to define conditions allowing transport measurements. We
have studied the interaction of Dox and ATP, in the presence and in the
absence of Mg z+, by (i) Dox fluorescence, (ii) Dox partition between
octanol and buffer, (iii) Dox passive diffusion into DNA-Ioaded liposomes.
Results clearly demonstrated that Dox exhibited complex interactions with
ATP, and that, in our conditions. Mg > had no influence on these 3
parameters. It was necessary to confirm that it was also the case with
proteoliposomes. For such a study, P-gp-devoid proteoliposomes had to
be used. They were obtained from sensitive ceils in which no P-gp was
expressed. The same ATP inhibitory effect was observed with and without
Mg 2+, whatever be the ATP concentration. Therefore, P-gp-catalyzed Dox
transport could be determined by the comparison of fluorescence decreases
in the presence of ATP with and without Mg 2-, as this cation was
absolutely required for P-gp activity. The internalization rate of Dox into
DNA-loaded P-gp-containing proteoliposomes in the presence of
ATP/Mg 2+ was faster than in the presence of ATP alone. This effect was
reversed by addition of verapamil, an alternative substrate of P-gp. In
conclusion, DNA-loaded proteoliposomes allow the quantitative study of
P-gp-cata[yzed transport rate.

(Mo/14.2/229)

Flavonoids as potent bifunctional modulators of cell

multidrug resistance
G. Conseil ~, J.-M. P.4erez-Victoria-, F. Gamarro 2, D. Barron ~,
E. Balzi, A. Goffeau4, A. Di Pietrot
~IBCP, CNRS, l<yon and 3UCBL, Villeurbanne - FRANCE
ZlPB, CSIC, Granada - SPAIN - ~UCL, Louvain-la-Neuve - BELGIUM

Multidrug resistance (MDR) is a cell phenotype found from human to lower

eukaryotes as related to overexpression in plasma membranes of ABC (ATPbinding cassette) multidrug transporters.
Flavonoids am natural products found in plants, some of which being 'already
known for their anticancer properties. Their effects have been studied here at
different levels of multidrug transporter functioning :
1) Direct binding to a purified target, by using the recombinant Cterminal nucleotide-binding domain from mouse P-glycoprotein, in order to
establish structure-activity relationships. Hydroxyl groups at positions 3 and 5
of the flavonoid, in addition to the ketone group at position 4, enhance the
binding-affinity, most likely by mimicking the adenine moiety of ATP (as
demonstrated for other proteins). Additional hydrophobic substituents
markedly inct~ease the binding affinity, probably at the steroid-interacting
region [1].
2) Inhibition of ATP hydrolysis and of quenching of fluorescent
Rhodamine 6G. The experiments have been can'ied out on membranes
containing the overexpressed ABC muhid,'ug transporter PDR5 from
Saccharomyces cerevisiae. Flavonoids inhibit both activities, suggesting a
bifunctional interaction at the two vicinal sites of the transporter.
3) Inhibition of daunomycin cellular efflux and reversion of MDR cell
phenotype. Flavonoids produce accumulation of daunomycin in Leishmania
rropica protozoan parasite cells overexpressing the ltpgp multidrug transporter,
as monitored by flow cytometry. They also chemosensitize the parasite growth
to the presence of the drug. Their efficiency is directly convlated to their
binding affinity towards a purified recombinant target. [2]
The experiments lead to the conclusion that flavonoids behave, in all cases, as
potent bifunctional modulators of celt multidrug resistance.
[1] Conseil et al. (1998) Proc. Natl. Acad. Sci. USA, 95, 9831-9836.
[2] Perez-Victoria et al. (1999) Biochemisu'y, 38, 1736-1743.

Cystic fibrosis (CF), the most common lethal genetic disease is

characterized by alterations in transepithelial salt transport. The defective
apical conductance of chloride results from either mistargeting or abnormal
function of the cAMP-regulated CF transmembrane conductance regulator
(CFTR) channel, a member of the ATP binding cassette (ABC)
transporters superfamily.
Using cell-attached patch clamp and iodide efflux techniques we
previously described the molecular features that enable xanthine
derivatives (IBMX, DPMX) to activate CFTR, stably expressed in CHO
cells (BQ1) [1].
We demonstrate here that P2u receptor signaling pathway constitutively
regulates CFTR activation. 1) CFTR activation by forskolin or xanthines
was inhibited by extracellular ATP 2) Reactive Blue-2 lowered ATP
induced inhibition and acted together with IBMX or DPMX to enhance
CFTR activity. 3) P2u receptor activation induced intracellular cAMP
elevation through a calcium dependent signallingpathway.
We used combined application of inactive and active 3,7-dimethyl-1-alkyl
xanthine derivatives to detect CFTR inhibitors. Among the inactive
derivatives, 3,7-dimethyl-l-(2-propenyl) xanthine was found 1) able to
prevent CFTR activation by xanthines or forskolin, and 2) to inhibit both
P2u receptor mediated calcium elevation and cAMP production. 3)
Substitution at C 1-position of 2-propenyl to -isobutyl or-oxiranyl methyl
resulted in CFTR activator or fully inactive compound respectively.
These results indicate that active, inactive or antagonist forms of xanthine
derivatives can be obtained by merely manipulating the nature of the alkyl
substitution at Cl-position on the xanthine skeleton. Moreover, these
results suggest a complex regulation of CFTR activity by second
messenger pathways. CFTR activation involved both P2u receptor
inhibition and intracellular cAMP increase. We suggest that the effect of
xanthine derivatives on CFTR activity primarily depends on their
inhibitory potency of P2u receptor.

(Mo/14.2/230)

Phylogenetic analyses of ABC transporters

in complete micoroorganism genomes
E. Dassa
UPMTG, Institut Pasteur, F75724 Paris Cedex 15, Prance

ATP-binding cassette (ABC) systems, also called Traffic ATPases, are

found in eukaryotes and prokaryotes and almost all participate in the
transport of a wide variety of molecules. ABC systems are characterized by
a highly conserved ATPase module called here the ABC module, involved
in coupling transport to ATP hydrolysis. We have used the sequence of
one of the first representatives of bacterial ABC transporters, the MalK
protein, to collect 250 closely related sequences from a non redundant
protein sequences database. The sequences collected by this objective
method are all known or putative ABC transporters. Having eliminated
short proteins sequences and duplicates, the 197 remaining sequences were
subjected to a phylogenetic analysis based on a mutational similarity
matrix. An unmoted tree for these modules was found to display two major
branches, one grouping all collected uptake systems and the other all
collected export systems. This remarkable disposition strongly suggests
that the divergence between these two functionally different types of ABC
systems occurred once in the history of these systems and probably belbre
the differentiation of prokaryotes and eukaryotes.
We have now analyzed the ABC transporters in 18 completely sequenced
microorganisms. We have identified the different components of such
transporters and proposed that they cluster in 43 groups of orthologous
sequences. By this analysis, we could predict the substrate specificity of
hypothetical transporters. In prokaryotes components of ABC transporters
form in general operons, with two notable exceptions in the genomes of
Synechocystis and o f A q u i f e x aeolicus. Our results are consistent with the
idea that all components of ABC transporters evolve with very tittle
shuffling [1, 2]. It appears also that Archaea lack a particular subfamily of
ABC transporter otherwise found in all organisms, whose most known
representative is the P-glycoprotein.
[1] Saurin W. et al., Protein Sci, 3, (1994) 325
[2] Kuan G. et al., Res Microbiol, 146, (1995) 271

s226

(Mo/14.2/231)

Abstracts FEBS'99

C h a r a c t e r i z a t i o n o f a Sinorhizobium meliloti A B C - h i s t i d i n e
t r a n s p o r t e r also i n v o l v e d in b e t a i n e s a n d p r o l i n e u p t a k e

(Mo/14.2/232)

S.Ellouk-Achard, CMartin, H.Dutertre-Catella,

M.Thevenin and J.-MWarnet.

L. Dupont, E. Boncompagni, T. Mignot, J.C. Trinchant, D. Le

Rudulier

Laboratoire de Toxicologie, Facult~ de Pharmacie. F-75270 Parts Cedex 06

Biologie vFgdtale et Microbiologie, UNSA , E SA6169, 06]08 Nice, France

In the rhizosphere or during the symbiosis with its host plant

alfalfa, Sinorhizobium meliloti is submitted to osmotic fluctuations
which affect root colonization and nodulation. To overcome growth
i n h i b i t i o n due to osmotic stress, S. m e l i l o t i accumulates
osmoprotectants such as glycine betaine (GB) by de novo synthesis
from choline or choline-O-sulfate or by uptake from the
environment.
In S. meliloti, a high-affinity GB transport activity, strongly
stimulated by elevated osmolarity, and the presence of a periplasmic
GB binding protein suggest that an osmoregulated ABC transporter
analogous to the Escherichia coli ProU system might exist in this
bacterium. Amino-acid sequence comparisons of the ATPases
involved in GB uptake systems allowed to identify strongly
conserved boxes : degenerated primers were designed and used to
amplify a PCR fragment of the expected size on S. meliloti total
DNA. This fragment was used as a probe to isolate a S. meliloti
p r o U - l i k e locus from a S. ineliloti genomic library.
The sequence analysis of this locus, called hisT, identified
three genes (hisTx, hisTw, h i s T v ) whose products share strong
homology with proteins of betaines uptake systems from E. coli
(ProU) and B. subtilis (OpuA and OpuC). Downstream hisT, a gene
encoding an e n z y m e involved in the first step of histidine
catabolism (hutH) was found.
Chromosomal h i s T mutants were constructed to determine
the substrate specificity of this transport system and its contribution
in GB uptake. HisT is an ABC-transporter involved in histidine,
proline, dimethylproline uptake at high affinity and GB uptake at
low affinity. Construction of a h i s T x - l a c Z fusion showed that HisT
does not play a role in S. meliloti osmoregulation since its
expression was not induced by salt but rather by histidine .The
presence of a gene involved in histidine catabolism downstream
h i s T suggests that this transporter has a role in nitrogen assimilation
and not ni osmoregulation.

(Mo/14.2/233)

P-I~lycoprotein
pharmacology

i m p o r t a n c e a n d r o l e in t h e c e l l u l a r
of anthracyclines
in C a C o 2 cells.

L Englebert~ C. Remacle b A. Trouet b and Y.-J. Schneider ~

' de abtochtmte
. . .' cellulatre,
de bbtologte
.
. celhdatre,
.
Laboratotres
UniversitF cathohque de Lota,ain,
PI. Louts Pasteur, I, B-1348 Louvmn-la-Neuve, Belgium.

Multidrug resistance in tumor cells reduce the toxicity but also the
pharmacological activity of different antineoplastic drugs (anthracyclines,
vinca alcalo'/ds, ...) by an energy-dependent active effiux mechanism mediated
by the mdrl gene product, the P-glycoprotein (P-gp). We investigated the role
and importance of P-gp in the cellular pharmacology of anthracyclines by
using monolayers of CaCo 2 cells, deriving from a human colon
adenocarcinoma. Grown and differentiated on microporous substrates, these
cells express P-gp at their brush-border membrane (apical pole).
CaCo 2 cells transport daunorubicin (DNR), doxorubicin (DOX), idarubicin
(IDA) and various amino acid derivatives preferentially from the BL pole than
from the AP pole. DNR, IDA and various amino acid derivatives are more
accumulated than DOX. Cell fractionation and laser scanning confocal
microscopy indicate that the subcellular distribution of these compounds is
very different. DNR DOX and IDA accumulate in the nuclei whereas the
amino acid derivatives, which have a lower affinity for DNA, show a vesicular
and cytoplasmic localization. In the presence of verapamil, a P-gp inhibitor,
the net transport of the anthracyclines from the BL pole to the AP pole is
inhibited and the cellular accumulation of these compounds from both poles is
increased. In contrast, verapamil unaffects the accumulation of IDA. HPLC
separation and fluorescence detection indicate that DOX is not metabolized in
CaCo 2 cells whereas the other anthracyclines are biotransformed into the "o1" form, which is produced by cytoplasmic aldo-keto reductases. Part of
these metabolites is expulsed preferentially to the AP pole of the cells. This
transport is inhibited in the presence of verapamil, which suggests that the
metabolites are also substrates of the P-gp.
These results indicate that CaCo 2 cells provide an interesting tool to
investigate transport, accumulation and biotransformation of anthracyclines in
polarized epithelia and confirm that P-gp plays a crucial role in the
bioavailability of drugs in gastrointestinal tract.

Co-inhibition of liver C Y P 3 A and P-glycoprotein by

Cyelosportn A and Cyclosporin G in Wistar rats

Cyclosporin A (CsA) is widely used as an immunosuppressive drug to

prevent graft rejection after transplantation. It is established that therapeutic
and toxic effects of this drug are dependent on inhibition of calcmeurin, a
calcium- and calmodulin-dependent phosphatase. However inhibition of
calcineurin requires CsA binding to its protein cofactor, cyclophihn, which
can act as a reservoir for the drug in the cell and may inhibit cellular efflux
of CsA.
Otherwise, P-glycoprotein is known to increase the rate of CsA efflux
(MDR-like activity). The disparity in P-glycoprotein expressmn within the
different tissues (high level in the liver or the small intestine versus low level
in the kidney) results in such a situation that CsA dosage required for
therapeutic use largely exceeds toxic dose.
Moreover, in the liver, CsA is metabolized by cytochrome P450 3A
(CYP3A), producing less toxic metabolites, and, at the same time, is
subjected to the drug efflux activity of P-glycoprotein. The proportion of
native CsA leaving the liver via the P-glycoprotein pump may exert its
toxicity on the kidney, where P-glycoprotein exhibits a low expression.
This study was investigated to test the hypothesis that P-glycoprotein and
CYP3A are important determinants of CsA cellular specificity with regard to
both therapeutic and toxic effects. Liver subcellular fractions were obtained
from non-induced or Rifampin-mduced Wistar rots. Rifampin treatment
(400 mg/kg/d for 4 days, then 200 mg.'kg/d for the next 4 days per os) was
applied m order to reduce CYP3A. CsA and CsG, a promising analog, were
given i.p (50 mg/kg/d for the last 4 days of Rifampin treatment). CYP3A
and P-glycoprotein were assessed by SDS-PAGE according to the method
of Laemmli. Erythromycine N-demethylase activity was determined m
order to assess the SDS-PAGE results obtained for CYP3A The results
showed a weak inhibition for CYP3A and erythromycme N-demethylase
activity with CsA and CsG in non-reduced rats, and a stronger inhibition in
Rifampin-induced rats (CYP3A: about 30% for CsA and 20% for CsG;
erythromycine N-demethylase activity' 25% for CsA and 15% for CsG). In
the same way, P-glycoprotein, increased by Rifampin treatment, exhibited
an inhibition after CsA or CsG treatment. Our results showed that both Pglycoprotem and CYP3A are regulated by CsA m the rat liver
We can conclude that the co-inhibition of CYP3A and P-glycoprotein by
CsA and CsG in the liver may have important implications concermng the
bioavailabihty of these drugs, particularly with regard to their
nephrotoxlcity.

(Mo/14.2/234)

M D R m o d u l a t i o n by N - b e n z y l p i p e r a z i n e - f l a v o n o i d s .
R o l e o f the p r o t o n a t e d form.

J. Fert6 a, G. Lewin b, M. Schwallerc

a Btophystque des Protdmes Membranaires, DSV/DBCM/SBPM
CEA Saclay, 91191 Gij:su~-Yvette, France

Many modulators of P-glycoproteln-mediated multldrug resistance are basic

hpophilic compounds. Recently, we described a new famdy of such MDR
modulators, the N-benzylpiperazine-flavonoids (BPF) [1]. The structureactivity relationship study suggested that the protonated form of these weak
bases is the pharmacologically active species. Here we present further support
for this hypothesis, by investigating the interaction of BPFs displaying
varying MDR modulating potency with SDS, an anionic surfactant. This was
achieved by following the micelhzation process of SDS in the presence of
relevant BPF concentrations (5-20 gM), using merocyanine 540 as a
fluorescence probe. At physiological pH 7.4, where both protonated and
neutral forms coexist, all derivatives affect SDS micellization equally. By
contrast, at pH 5, where the cationic species is predominant, a marked
interaction with the surfactant occurs, well below the critical micellar
concentration of SDS alone. More importantly, the magmtude of this eftect
was correlated with MDR modulating activity on cultured cells
As a whole, this correlation supports the idea that, at physiological pH, the
protonated ti3rm of BPFs is involved in the modulation of MDR. Since a
permanently charged BPF derivative is inactive on MDR cells, it is likely that
the target(s) of this family of MDR modulators can be accessed only from the
cytoplasmic sxde, and that the neutral form is mandatory for crossiug the
plasma membrane. Finally, these results underscore the importance of the
strong amphiphilic character conferred by the cationic charge. This would be
consistent with a preferential adsorption of BPFs at the interface of the tuner
leaflet of the plasma membrane, where they could exert their MDR
modulating activity, enher through interactmn with membrane lipid
components [21, or through direct interaction with P-glycoprotein at the lipidprotein interface.
[1] Fert6 et ah, .L Meal Chem., 42 (1999) 478.
[2] Wadkins and Houghton, Biochim. Biophys. Acta, 1153 (1993) 225
b LaborarotJ e de Phalmacognosie, Facultl de Pharmacte, 92290 Chdtenay MalabJ3,
c Transports lontques Membranaires, Ihliversitd Paris 7, 75005 Paris

Abstracts FEBS'99

(Mo/14.2/235)

A role for ABC proteins in plant ion channels control

C Forestier. N Lconhardt. E Marin and A Va~asseur
CE.q Cadarache, DEI3,I, LE/~IS, BPI - 13108. Nt Paul-lez-Duran~e.

s227

(Mo/14.2/236)

France cforestter~a cea fr

The ABC super-famdy is probably the largest family of proteins that medmte ATPdependent transfer of solutes In plants, only a few ABC proteins have been recently
idenufied. The funcUonal charactenzatton obtained for some of them suggests their
involvement in the cellular detoxification of xenobiotics or in the regulation of cell
elongation If the vast majority of ABC proteins in animal cells are active
transporters, it has emerged that another fundamental role of these proteins Is their
involvement in so far as ion channels [1] Our group is mterested by the
identification of plant ABC proteins and by interactions that could occurred wtth
plant ion channels (http://xs'~vw-dsv.cea ff/gtso/gtso_us htm).
Stomata, located in the epidermal leaf are formed by a couple of guard cells and
control gas exchange between the plant and the atmosphere Incubauon of epidermal
strips in the presence of glibenclamlde induced stomatal opening in darkness
Conversely. stomata previously open in light, are closed by potassmm channel
openers. Apphcation of the whole-cell patch-clamp technique on guard cell
protoplasts demonstrates that glibenclamide inhibits the outxsard-potassium channel
In northern blots, a 7 kb transcript was revealed when total RNA from guard cells
were hybridized w~th an EST tdenttfied as an encoding sequence homologue to SUR
Thin confirms that a transcript encoding a sulfonylurea-receptor-like protein is
present in guard cells and that this protein is able to control, directly or not, the
actwlty of the plasma membrane outward potasstum channel [21 Using the same
EST, we cloned AtMRP2, a cDNA encoding a putative ABC transporter from
Arabidops~s [3] but different from the SUR expected AtMRP2 ~s now charactertzed
as a transporter involved m the detoxification of xenobiotics and we are currentl3
investigating its role m the control of ion channels using Xcnopus heterologous
expression Moreover. we observed that glibenclamlde strongly affect anion
channels and the stomatal closure induced by the phytohormone ABA Elements
concerning a more detailed Identification of this receptor, tts interaction with ton
channels and with the ABA-slgnaling cascade will be presented at the meeting
[1] Fliggins CF (1995) Cell. 82:693
[2] Leonhardt Net aI., (1997) Proc Natl. Acad. Scl USA, 94 14156
[3] Mann E et al. (1998) Btochim Blophys Acta. 1369 7

(Mo/14.2/237)

Subcellular localization of the mammalianABC transporterABCI

Hamon Y ". Lucmni M F ", Freyssinet J M ~, Marguet D ", Chimini G"
"( 'ettD'~"d'llplmlt~ologle de Ih*rwdh~-Lumtm

( "a~e 906, 132~4,~-Ahw~ell/e e e r i e r 09, ]'ronce

~hl~tltllt d'[lem~ltologle el d']tm~ntnohJgw, 4 ~te htr~thlege~ 6~O,'%Slra~homg /,~,#/t e

ABCI is a mammalian ATP Binding Cassette (ABC) transporter required by

macrophages during the engulfment of cell corpses generated by apoptosls [1] To
further analyze the function and the subcellular localization of this transporter, v,e
generated stable transfectants in HeLa-tet offcelts [2] An ABCI/Green Fluorescent
Protein (GFP/chimera construct has been generated by overlap extension-PCR This
plasmid encodes a fusion protein between ABC1 and GFP as an "in frame" Cterminal tailpiece The tetracycline inducible system was chosen since it allows the
conditional expression of ABCI/GFP In all the analyzed transfectants. ABC1/GFP
is addressed to the plasma membrane, where it displays an heterogeneous
distribuuon, concentrating in discrete spots and at cell-to-ceil contacts In addition,
ABCI is present on intracellular vesicles, identified as lysosomes by
immunofluorescence colocalization with known markers of this compartment From
a funcUonal point of view, ABC1 expressing HeLa Cells show an increased abihty
to expose pbosphatidylserine (PS) on the outer leaflet of the plasma membrane after
Ca2~ ionophore treatment, as assessed by annexin-V binding This evidences a role
of ABCI in the control of phospbolipid equdibrium across the membrane bilayer,
which is consistent with its specific functional requirement during engulfment [3] It
is well knov,n, in tact, that the membrane dtstribution of PS is widely modified
during apoptosis On the other hand, it is not known bow PS recognluon by the
phagocyte takes place nor whether a specific PS receptor exists Our results suggest
that the requirement for ABC] during the engulfment of PS exposing cells, ts related
to its abihty to promote movement of PS across the membrane
[1] Luclani. MF etaI,(I996) EMBO, 15,226
[2] Gossen, M et al ,(1992)Proc Natl Acad Sc~ USA 89, 5547
[3] Moynault et al 1998, Biochemical Society transactions 26. 629

Effects of hydrophobic cyclic peptides on the

P-glycoprotein ATPase activity
A. Garrigues, S. Orlowski, M. Delaforge, M. Garrigos
D@artement de Biologie Cellulaire et Mol~culalre, DSV/ CEA
Saclay, 91191 Gif/Yvette Cedex, France

P-glycoprotein (P-gp) is a plasma membrane ATPase which is able to

expel various anticancer drugs out of tumor ceils, such a mechanism being in
part responsible for the multidrug resistance phenotype (MDR). Moreover, Pgp is also able to transport a large variety of structurally unrelated
amphiphilic molecules among which some hydrophobic cyclic peptides like
cyclosporin A, a well-known MDR-reversing agent. Likewise, pristinamycin
IA, a cyclic peptide antibiotic, was shown to be transported by epithelial cells
expressing P-gp [1]. In order to investigate the coupling between substrate
transport and ATP hydrolysis by P-gp, we first focused our attention on the
influence of cyclosporin A and pristinamycin IA on the ATPase activity of Pgp. These enzymatic experiments were performed on P-gp-containing
membrane vesicles prepared from highly resistant MDR cells. Our results
show that cyclosporin A and pristinamycin IA both inhibit the basal P-gp
ATPase activity, above 0.1 tiM and 10 p.M, respectively. Likewise, both
molecules inhibit the verapamil-stimulated P-gp ATPase activity, by an
apparent competitive mechanism. Assuming a 1:1 competition stoichiometry
between verapamil and each of these two molecules, the inhibition constants
were evaluated as 0.02 tiM and 5 tiM for cyclosporin A and pristinamycin IA,
respectively. Despite the very different inhibiting concentration ranges for
cyclosporin A and pristinamycin IA, these data are consistent with a specific
interaction between pristinamycin In and P-gp.
It has been shown that pristinamycin Ig displays a synergistic
bactericid action when associated with pristinamycin IIA, another
hydrophobic cyclic peptide. We were thus prompted to investigate whether a
synergistic effect could also occur with respect ~o P-gp. Using our enzymatic
method, it was found that, similarly to pristinamycin In, the concentration of
pristinamycin IIA which inhibits P-gp-mediated MgATP hydrolysis was
above 10 laM, whereas 50 tiM was necessary to inhibit the verapamilstimulated P-gp ATPase activity. However, synergistic effects in the presence
of both pristinamycin [g and pristinamycin IIA on P-gp-mediated MgATP
hydrolysis could not be detected.
[ 1] Phung-Ba et al., Eur J Pharmacol, 288, (1995) 187.

(Mo/14.2/238)

Protein Kinase C Inhibitors in Multidrug Resistance

J. Hofmann, M. Rybczynska, M. Spitaler, H. Grunicke
Institut of Medical Chennstry and Biochemistry,
University of Innsbruck, Fritz-Pregl-Str. 3, A-6020 lnnsbruck. Austria

There are many reports showing an involvement of protein

kinase C (PKC) in mdrl-mediated multidrug resistance (MDR). We found
that in mdrl-expressing CCRF-ACTD400, VCR1000, ADR5000, KB-8-5,
KB-C1, Hela-MDR1 (transfected with a m d r l gene) and their matched
sensitive cells, the expression o f m d r l is not associated with the expression
o f a certain PKC isoenzyme. The PKC inhibitors CGP 41251, ilmofosine
and dexniguldipine-HC1 do not reduce the expression o f m d r l - m R N A . The
PKC inhibitor GF 109203X inhibits PKC in the nanomolare range and
reverses multidrug resistance at approximately 10 p.M, whereas
dexniguldipine-HC1 inhibits PKC in the range of 100 ~tM and reverses
multidrug resistance at 0.1 tiM. The specific PKC inhibitor CGP 41251
reverses mdrl-mediated resistance in medium to a similar extent as CGP
42700, a derivative which does not inhibit PKC. In serum CGP 42700 is a
more potent modulator than CGP 41251, because the latter is inactivated
by binding to cx-acidic glycoprotein. M d r l - and MRP-expressing cells are
also resistant to the phospholipid analogues ilmofosine, miltefosine and
D21266. Miltefosine is used for topical treatment o f breast cancers, The
expression of these genes may be responsible for treatment failures.
Ilmofosine and miltefosine interact with the regulatory domain o f PKC.
They do not reverse multidrug resistance and seem not to be transported by
the mdrl or the mdr2 gene products, indicating that not direct interaction
o f the m d r l - or MRP gene products, but an indirect mechanism linked to
the expression o f these genes is causing resistance to phospholipid
analogues. One conceivable indirect mechanisms seems to be resistance to
apoptosis in m d r l - or MRP-expressing cells. However, miltefosine induces
apoptosis to a similar extent in mdrl-transfected HeLa cells than in
sensitive non-transfected cells. In conclusion, (i) reversal o f multidrug
resistance by PKC inhibitors is performed by direct interaction with Pglycoprotein and not via PKC. (ii) Expression o f m d r l or MRP leads to
resistance to phosphotipid analogues which may be the reason for
resistance to miltefosine treatment o f breast cancers. (iii) P-glycoprotein
seems not to have any influence on induction o f apoptosis.

s228

(Mo/14.2/239)

Abstracts FEBS'99

Physiological and molecular characterisation of a fructose

transport system from Sinorhizobium meliloti
A. Lambert, M. Osterfis, MC. Poggi, K. Mandon, D. LeRudalier

(Mo/14.2/240)

Laboratoire de Biochimie, Ecole Polytechnique, 91128 Palaiseau, France

Biologie vdg6tale et Microbiologie, CNRS ESA 6169, UNSA,

Parc Vahose 06108 Nice. France.

Only few sugar transport systems have been characterised at the

molecular level in the Rhizobiaceae although these bacteria can use a large
variety of sugars as energy source. We have investigated the transport of
fructose in Sinorhizobium meliloti, an agronomically important symbiotic
bacterium.
The strain Rm 5000, a [rifampicin% Pho']-derivative of the wild
type RCR2011, showed a high-affinity uptake activity (Km = 6t.tM) with a
Vmax of 32nmol/min/mg proteins. This transport activity was repressed by
growing the cells in the presence of succinate. Mannose and ribose were
strong competitors for fructose uptake, with respectively 91 and 86%
inhibition when present in 50-fold excess. Using 14C-fructose and
antibodies against the fructose-binding protein from Agrobacterium
radiobacter, a 35kDa periplasmic protein was detected by radiolabelling
and Western blot. The fructose-binding activity of this protein was also
abolished in the presence of excess mannose or ribose.
In order to identify the genes encoding this fructose uptake system,
a random mutagenesis with a TnphoA transposon was conducted on
Rm5000 and a mutant [rifampicin,, Pho , Neon] unable to transport
lYuctose was isolated. The TnphoA mutation did not affect the symbiotic
phenotype of S. meliloti [Nod+ and Fix+].
The fructose transport activity was restored by complementation
with the plasmid pMRFRU which contains a 4.9kb genomic fragment
from the parental strain. DNA sequence analysis of the cloned locus
allowed the identification of four open reading frames. Three genes
showed significant identity (25-70%) with the ABC-ribose transport
system from Radiobacter capsulatus : frcB, the first gene encodes the
periplasmic fructose-binding protein, frcC, interrupted by TnphoA,
encodes the permease protein, andfrcA, the ATP-binding protein. The last
open reading frame, upstream o f f r c B C A is transcribed divergently and
showed low but significant identity (10-16%) with repressors of xylose
transport from various bacteria.
(M0/14.2/241)

Effect of Mg 2+ on l9Cd extrusion via the P type Cd 2+ATPase in Staphylococcus aureus

A Maim, Z Tynecka, Z Szcze~niak,

Inactivation and phenotypic analysis of the yeast ORF

YLL015w encoding an ABC protein
M. Lazard, O. Gueldry, P. Plateau, S. Blanquet

Most of the ATP-binding cassette (ABC) proteins are ATP-driven

transporters that mediate the selective transport of a wide range of substrates
across biological membranes. The significance of this family is exemplified by
the numerous human diseases that are associated with defects in ABC protein
functions. Therefore, understanding the physiological roles of these proteins is
of major importance. Because it allows easy genetic manipulations, the yeast
Saccharomyces cerevisiae is a powerful model organism for such studies. The
determination of the DNA sequence of the entire Saccharomyces cerevisiae
genome has made possible an inventory and classification of all the ABC
protein sequences from this organism [ i,2].
One of the yeast ABC proteins subfamily deduced from genome analysis
contains members which exhibit striking similarities with the clinically
important MRP (multidrug resistance-associated protein) and CFTR (cystic
fibrosis transmembrane regulator) proteins. Out of the six members composing
this subfamily, three only benefit from biochemical or physiological studies.
Among those, the best characterized one is the product of YCF1 [3]. In order
to understand the role of additional yeast ABC proteins, we have undertaken a
functional study of the product of the ORF YLL015, the closest match to
YCF1. We disrupted YLL015 by single-step genetic replacement in a wildtype background or in a YCF1 null-mutant. Both the single and double
disruptants are viable indicating that YLL015 is non-essential. The effects of
various drugs on the growth of wild-type, or single and double disruptants of
YCF1 and YLL015, were compared. Surprisingly, the results indicate that
disruptions of each of the two genes produce opposite phenotypes. Whereas
deletion of YCF1 lead to increased sensitivity to drugs such as 1-chloro-2,4dinitrobenzene (CDNB) or diamide, deletion of YLL015 confered increased
resistance to the same compounds.
1. Decottignies A. and Goffeau A. Nature Genetics 15, (1997) 137.
2. Taglicht D. and Michaelis S. Methods Enzymol. 292, (1998) 130.
3. Li Z. et al. J. Biol. Chem. 271, (1996) 6509.

(Mo/14.2/242)

Reversion of the multidrug phenotype of cancers

E. Marthinet, L.G. Baggetto
LB.C.P. CNRS, 7 Passage du Vercors, 69367 Lyon Cedex 07

Department of Pharmaceutical Mlcrobtolog~. 3iedtcal AcadamL

Lubartowska 85. 20-123 Lubhn. Poland

Cadmium resistance in Staphylococcus aureus is mediated via the plasmidcoded, energy-dependent Cd 2+ efflux system identified as a P type Cd2+ATPase This membrane-bound system extruds Cd2+ from the cytoplasm of the
resistant cells immediately after its entry via the Mn2+ transport system [ 1,2]
According to our preliminary data, the cadmium effiux system may require not
only ATP needed for conformational changes but also electrochemical proton
gradient (A~H+) essential for Cd2+/nH+ exchange during Cd2+ extrusion
In this work we assayed the effect of 5 mM Mg 2+ on l9Cd efflux in the
resting cells of cadmium-resistant S. auretts 1781OR. Cells were preloaded with
10 ~tM lgCd under special experimental conditions (1 mM phosphate buffer,
pH 7 plus nigericin) Steady-state or net efflux of 109Cd was initiated by
addition of 100 mM PB and cadmium-insensitive L-lactate as an energy donor.
Effiux experiments were performed using the filtration method
It was shown that Mg 2+ protected the cells against 109Cd preloading. Mg 2+
accelerated also the initial rate of steady-state l9Cd effiux with an increase in
the resultant rate constant (k) from 0 06 to 0 092 rain-1. In contrast, Mg 2+ had
no effect on the fast initial rate of net 109Cd efflux and the resultant rate
constants (k), being 0.095 min-1 in the presence or in the absence of Mg2+
According to our earlier data [1], Mg2+ did not affect Cd2+ entry via the
Mn2+ transport system. It is suggested that Mg 2+, having a high affinity to the
negatively charged groups on the surface of biological membranes (surface
potentials) may protect the cadmium efflux system against binding of external
Cd2+, thereby preventing Cd2+ preloading of the cells and accelerating the
steady-state Cd 2+ effiux but not the net Cd2+ effiux
This work was supported by grant 6 P04C 020 13 from the State Committee
for Scientific Research
[1] Tynecka Z, Gos Z, Zaja~c J, J Bacteriol. 147 (1981), 305
[2] Tynecka Z, Gog Z, Zaja~e J, J Bacteriol. 147 (1981), 313

Multidrug resistance (MDR) is the major cause of failure of cancer

chemotherapy. Several mechanisms are involved, but the typical phenotype it
mainly due to the overexpression of membranes proteins, the most important
of which are the P-Glycoprotein (Pgp) and the MRP that are encoded by the
MDR1 and the M R P genes, respectively. We attempt to modulate this
phenotype by two means:
1- the use of tiamulin, a semisynthetic antibiotic discovered in the laboratory,
revealed a better in vitro and in vivo reversion activity than most of other
modulators, without showing their toxicity [1].
2- by a novative approach we developed transcriptional decoys to decrease
the activity of the human MDR1 promoter. An in vitro model (that consisted
of NIH 3T3 fibroblasts lipofected with a plasmid containing the EGFP gene
(Enhanced Green Fluorescent Protein) under the control of the human
MDRI promoter) was built to optimize the strategies and study the effects of
the decoys. This model proved to be fimctional. We have optimized the
nuclear vectorization of the decoys and we have shown that a decoy aimed at
the MED-1 region of the MDR1 promoter decreased the EGFP transcription.
This region is known to direct the transcription start codon in the multiple
initiation window of the MDRI promoter. Conditions have been optimized
and are being tested on MDR tumor cells. Moreover, in order to better
understand the transcriptional regulation of the MDRI gene, which is TATAless, we try to identify and to isolate the protein(s) that could interact with
MED-1. We are now developing ribozymes to destabilize the Y-terminal
region of the mRNA of the human MDR1 gene.
[1] L.G. BaggettO, et al., Biochem. Pharmaco156 (1998) 1219.

Abstracts FEBS'99

(Mo/14.2/243)

Investigation o f A T P . s e n s i t i v e K - c h a n n e l s
u s i n g a baeulovirus e x p r e s s i o n s y s t e m
M.V. Mikhailov, S.J.H. Asheroft

s229

(Mo/14.2/244)

Nuffield Dept. of Clinical Biochemistry, University of Oxford. John

Radcliffe Hospital, Headington, Oxford, OX3 9DU, UK

ATP-sensitive K-channels (K^rpchannels) play a key role in regulation of

insulin secretion from pancreatic I~-cells. We have expressed active I~-cell
K,a~ channels in Spodoptera frugiperda (Sf9) cells using a baculovirus
vector. A high yield of active channels was obtained on co-infection with
SURI and Kir6.2 engineered to contain N- and/or C-terminal tags to permit
detection by Western blotting. Channel activity was sensitive to ATP,
glibenclamide and diazoxide. Channel activity was also obtained on
expression of a C-terminally truncated Kir6.2 (Kir6.2zxC26): these channels
were blecked by ATP but were insensitive to sulphonylureas. In contrast
to X e n o p u s oocytes and mammalian cells the full length Kir6.2 also gave
rise to active channels in St9 cells when expressed alone. The highest yield
of active K^av channels was obtained on infection with a fusion protein
containing SUR1 linked to Kir6.2AC26 via a 6-amino acid linker.
SUR1 was divided at position Pro1042 into two halves - these N- and Cterminal parts were expressed separately. The constructs had C-terminal
tags and the C-terminal construct had an artificial leader sequence for
facilitation of membrane insertion. Analysis by fluorescent microscopy of
expression of variants of these recombinant proteins containing GFP as tag
showed membrane insertion of both proteins. Cells expressing either
protein separately showed low gh~cenclamide binding activity. Binding of
glibenclamide was markedly increased after co-expression of both proteins
and even more after co-expression of both proteins with Kir6.2. These data
demonstrate that: 1) the glibenclamide binding site includes ammoacid
residues from different parts of SUR1, 2) there is strong interaction
between different regions of SURI that makes possible self-association, 3)
Kir6.2 participates in K^~ channel assembly.
These studies demonstrate that expression of KArP channels and their
individual protein components using the baculovirus-insect cell system is a
powerful tool for investigation of K^~rchannel function.

Analysis of TAP genes involvement in IDDM :

a new TAP2 allele in Reunion's island population
LJ. Hoaraa, M. Cesari, F. Cadet, M. Pabion
Laboratoire de Biochimie et G~n~tique Moldculaire, Universit~ de la
R~union, 97715 Saint-Dems, France (DOM)

In this study, we have investigated the frequency of TAP1 and TAP2 alleles
in a group of 236 persons, previously HLA DQAI, DQB1 and DRBI
genotyped by us [1], constituted by 70 insulin-dependent diabete mellitus
(IDDM) patients and most of their first degree relatives (i. e., 156 parents and
full sibling subjects) living in Reunion island.
Population of this island is constituted by a particular structure of highly
crossbreeding individuals of African ancestry (35%), Indian origin (20-30%),
French origin (30%) and Chinese background (5%).
TAP typing was performed by polymerase chain reaction (PCR) amplification
refractory mutation system (ARMS). Seven among the eight TAP2 alleles
previously described in human patrimony were observed in control and
IDDM populations. Interestingly, the new TAP2 G allele, previously
discovered by us in Reunion island [21, was found to be increased in IDDM
population and the calculated HRR was shown to be relatively high (3.3).
This susceptibility to IDDM seems to be due to a positive linkage
disequilibrium between TAP2G allele and the highly diabetogenous DQA1
0501/DQB1 0201/DRB1 0301 haplotype (HRR=9). These data suggest that
TAP2G is not an additional predisposition factor of IDDM but its relativeIy
high frequency in 1DDM population allows to consider this allele as a genetic
marker of IDDM.
In addition, we show that minority alleles (TAP2D, E, F. G) are associated
with a restricted number of HLA class I1 haplotypes and each of them exhibit
a preferential linkage with one particular haplotype.
Finally, in contrast to other alleles and despite a HRR value close to 1, we
show that TAP2E allele contributes significantly to a drastic reduction of the
diabetogenic effect of DQA1 0301.2/DQBI 201/DRB7 haplotype. Indeed,
this haplotype which is usually preferentially transmitted to affected children.
is dominantly transmitted to healthy children when it is associated to TAP2E.
Therefore this allele seems to contribute to genetic protection to IDDM.
[11 Feugeaset al. Ettr J lmmunogen.. 23, (1996),459.
[2] CesariM., et al.. hnmunogenetlc,. 45. (I 997), 280

(Mo/14.2/245)

ALD and ALDR genes: of functional redundancy and

mouse model of X-Adrenoleukodystrophy
A Pujol, E Metzger, N Troffer-Chadier, C Kretz
and JL Mandel.

(Mo/14.2/246)

Glucoeortieoid regulation of P-glycoprotein in a brain

capiilay endothelial cell line
A. R~gina, I Romero, P.-O. Couraud, and F.Roux
L\rSERM L~26, 200 rue du Fbg St Denis 75475 Paris cedexlO

]nstitut de G~ndtique et de Biologic Molbculalre et Cellulaire (1GB3/[C),

CNRS/INSERM/ULP, 67404 IIlkirch Strasbour~, France.

X-linked adrenoleukodystrophy (X-ALD), is a severe demyelinating disease

associated with impaired beta-oxidation of very-long-chain fatty acids. The
gene responsible for the disorder encodes a peroxisomal half-ABC
transporter, the ALD protein. ALD is characterized by a very high
phenotypic variability within the same kindred, a fact that suggests the
existence of modifier genes. An animal model of the disease is not available
yet, since ALD knock-out (KO) mice do indeed show a biochemical
phenotype (accumulation in organs of VLCFAs) but no demyelination. We
have characterised the human ALDR gene structure, the closest homolog to
the ALD gene (66% identity at the aa level). The ALDR gene has been
shown to complement the biochemical phenotype of X-ALD patients
fibroblasts [1]. We aim at demonstrating the in vivo capacity of the ALDR
gene for functional rescue of the ALD KO mouse's biochemical phenotype.
Therefore we have constructed transgenic mice expressing the mALDR
eDNA under the control of an ubiquitous promoter (chicken beta actin); a
line showing high expression of the transgene in several tissues has been
obtained and crossed with ALD KO mice. VLCFA's levels and beta-oxidation
capacity of these mice will be determined. Furthermore we believe on the
hypothesis of some functional redundancy between the ALD and ALDR
genes, and intend the creation of a mouse model of the disease by generating a
double ALD/ALDR KO. For the ALDR KO, we have disrupted the ALDR
gene in the mouse, and F1 heterozygous mice (ALDR+/-) are already
available. Analysis of ALDR KO and double KO ALD/ALDR will help to
gain insight into the function of the ALDR gene, and possibly into the issue
of functional redundancy at the peroxisomal membrane.
[1] Kemp et at, Nature Med 4, (1998) 1261.

The blood-brain barrier (BBB) has an important role in controlling the

passage of molecules from the blood to the extracellular fluid environment
of the brain. The MDR1 encoded muhidrug efflux pump P-glycoprotein (Pgp) is highly expressed in the luminal membrane of brain capillary
endothelial cells thus forming a functional barrier to a wide range of lipid
soluble drugs notably antitumor agents. It is of interest to develop an m vitro
BBB model that stably expresses P-gp to investigate its mechanisms of
regulation in expression and activity.
The immortalized GPNT cell line was derived from a previously
characterized rat brain endothelial cell line, GP8 [l] We found by western
blot a strong expression of P-gp in GPNT monoeultures, whereas the
multidrug resistance-associated pump Mrpl was not expressed. The
transendothelial permeability coefficient (Pc) of P-gp substrat vincristine
across GPNT monolayers was close to the permeability coefficient of
bovine brain endothelial cells co-cultured with astrocytes [2] (Pe~2.6710 "3
cm/min and Pe=2.4110 "3 cm/min respectively). Furthermore, the P-gp
blocker cyclosporin A induced a large increase in apical to basal
permeability ofvincristine Thus, P-gp is highly functional in GPNT cells.
One hour treament of GPNT cells with dexamethasone (1 laM) resulted in
decreased uptake of vincristine (50% of control), without any increase in Pgp expression. TPA-mediated PKC activation induced the same effect on Pgp activity as dexamethasone, and PKC inhibition impeded dexamethasone
effect on P-gp activity Thus, dexamethasone effect on P-gp function in the
GPNT cell line may involve a PKC-dependent pathway
Taken together, th.ese results show the value of this novel GPNT cell line as
an in vitro model to study drug transport and P-gp function at the BBB
1
2.

Greenwood et at., J. Neuroimmunol, 71, (1996) : 51

Fenart et at, Pharmaceutical. Res, 15, (1998) : 993

s230

(Mo/14.2/247)

Abstracts FEBS'99

Multidrug resistance overexpression influences

the differentiation of a human colon carcinoma cell line
O. Rimet, A. Mirrione, Y. Barra

(Mo/14.2/248)

~Un~versity of Burgundy, Dijon France bLudwtg-Maximilian-Universlty INRA

Toulouse France, ,t Umversity of Vtenna, Austria

UPRESA-CNRS 6032, UFR Pharmacie, 27 bd J Moulin, F-13005 Marseille

Expression of P-glycoprotein (gp-170), the MDRI gene product, confers

multidrug resistance (MDR), i.e., resistance to several classes of
chemotherapeutic drugs. In order to study the influence of MDR on cellular
differentiation, we stably transfected a human colon carcinoma cell line
(HT29-D4) with the pHaMDR1/A eDNA construction. Indeed, HT29-D4
constitutively expresses only a very low level of MDRI/gp-170.
Resistant clones were selected by colchicine and were cross-resistant to
doxorubiein. MDR1 gene expression was associated with the expression of
functional gp-170 ; the function was reversed by verapamil and cyclosporin A.
When glucose is replaced by galactose in the cuhure medium, HT29-D4
parental cells are able to differentiate in vitro. They are organized in polarized
cell monolayers, they express at the cellular apical domain the carcinoembryonic
antigen (CEA) and release high level of this antigen in the culture medium.
Upon galactose treatment, expression of MDRI mRNA and functional gp-170
were always detectable. In MDR resistant clones, the monolayer organization,
as well as the staining of cell-surface CEA, were very heterogeneous and CEA
release was dramatically decreased (60-90%). Experiences with verapamil as
gp-170 inhibitor showed partial restoration of CEA release. It appeared
therefore that MDR1 overexpression allowed HT29-D4 to differentiate only
partially.

Is the ALDR (Adrenoleukodystrophy-related) gene regulated

by PPAR ~ ? S. Savary=,S. Albeta, M. Bentejaca, J. Perrot~, C. Mariona, D.
Ndlaye~,A. Holzingerb,A. Roscherb,T Pmeauc, J. Berger~,M Bugaut~

X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disease

characterized by progressive demyelination in the central nervous system
and biochemically associated with impaired 13-oxidation of very-long-chain
fatty acids. It results from a deficiency in the ALD protein, a peroxisomal
member of the ABC transporter family. A new gene (ALDR) which encodes
a peroxisomal membrane protein showing high homology with the ALD
gene and shares the same half transporter structure has been cloned [1]. It is
unlikely that the two proteins may form heterodimers as obligatory partners
since they display distinct tissue expression patterns [2]. Nevertheless,
ALDR is a functionally redundant gene as its overexpression in X-ALD
fibroblasts is able to normalize very-long-chain fatty acid levels.
Pharmacologic induction of the ALDR gene may thus become an effective
form of gene therapy. As the ALDR gene is inducible by the peroxisome
proliferator fenofibrate in the rat liver [2], we studied the possible
involvement of PPARo~ (peroxisome proliferator-activated receptor). It was
first observed that the induction of the ALDR gene was abolished in the liver
of fenofibrate-treated PPARc~ -/- mice. The promoter (2kb) of the rat gene
was cloned and sequenced and displayed two putative PPRE (peroxisome
proliferator response element). These results suggest that fibrate induction of
the ALDR gene depends on PPARc~. Functional analysis of the putative
PPRE sequences by mobility shift assay and cell transfection is in progress.
[1] G. Lombard-Platet et al., Proc. Natl. Acad. Sci. USA, 93, (1996) 1265.
121 S. Albet et al., FEBS Lett., 405, (1997) 394.
This study was supported by funds from ELA (Association Europrenne
contre les Leucodystrophies).
Laboratotre de Biologie Molbculaire et Cellulaire, Facult~ des Setences Gabrzel. 6 Bd
Gabrwl, 21000 Dijon, France,
bDr. Von Hauner Chddren's Hospital, Ludwig-Maximdian-Universtty, Department oJ
Chnical Chemist~ and Metabolism, Lindwurmstrasse 4, 80337 Mumch, German)',
~Laboratolre de Pharmacologic-Toxicologic, INRA, 180 ch. de Tournefeuille. BP 3. 31931
Toulouse Cdx 9. France.
dlnstttute of Neurology, Universt(v of Vtenna, Schwarzspamerstrasse 17, 1090 Vtenna,
Austrta

(M0/14.2/249)

STARIK, an Arabidopsis mutant in ABC transporter

S. Storozhenko ', E. Babiychuld, M. Davey~, G. Kispalb, R.
Lill b, M. Van Montagu ~, and S. Kushnir ~

(Mo/14.2/250)

Mtiifffunctional OCTNs Mediate Organic Cation and

~ a -Depgnaent Carnitine lransport
I.Tamm"~'..R. OhashP, K. SakamQto', J. Nezu3: ,A. Oku3 H.
Yabuuchi'", Y. Sal:'~, M. Shimane', and A. Tsuii ~'~,

aDepartement Genetica, VIB, Universiteit Gent, B 9000 Gent, Belgium

blnst Zytobiologie. Philipps-Umversit~t Marburg, D-35033 Marburg, Germany

IKanazawa Univ., Kanazawa, ~CREST. JST, Kawaguchi. and JChugai

Research lnst. Molecular Medicine, lbaragi, JAPAN.

The iron homeostasis is an important part of the normal metabolic activities

of living cells, because iron is an essential cofactor for numerous enzymes.
Fe 2+3+ that is not bound to proteins or low molecular-weight chelators is
toxic, mostly because of its ability to initiate free-radical reactions (Fenton
chemistry). Recent findings have shown that the mitochondrial inner
membrane ABC transporter in yeast, ATM1 [1] and its homologue in human
cells [2] are essential for the iron trafficking in mitochondria. A defciency in
ATM 1 causes severe oxidative damage to yeast cells, resulting in an inability
to grow on nonfermentable carbon sources [1]. We have identified an
insertion T-DNA mutant, S T A R I K , in an Arabidopsis gene (STA) encoding a
half-ABC transporter that shows significant homology to the yeast ATMI.
The STA protein is a functional homologue of ATM1 as well, because an
Arabidopsis cDNA complements the a t m l yeast mutation. The N-terminal
amino acid sequence of STA conforms to the functional requirements for the
mitochondrial transit peptide. The STA ~~Z-GFP fusion localizes to
mitochondria and probably plastids, when expressed in tobacco cells. The
intercostal chlorosis and severe growth retardation are the macroscopic
symptoms of STAR1K loss-of-function. The light microscopic examination of
leaf sections shows that cells around vascular bundles have a normal
morphology with well-developed lens-shaped chloroplasts. The distal cells,
however, possess round swollen chloroplasts distributed through the cell. T h e
degeneration and damage of membranous structures was further confirmed by
ultrastructural analysis. Our progress on biochemical and molecular analysis
of the S T A R I K mutant will be presented in more details.

Camitine plays essential roles for fatty acid metabolism and its
deficiency leads to the critical symptom, systemic camitine deficiency
(SCD). Large part of camitine in urine is reabsorbed by the active
transporter(s) to maintain physiological carnitine concentration and its
distribution into various tissues is also mediated by specific transporters.
In the present study, we identified novel Na*-dependent camitine
transporter family, OCTN and examined its relevance to SCD [1-3].
Furthermore, we studied multifunctionality of OCTNs by examining
substrate specificity of them.
Human OCTN1 and 2 were cloned from fetal liver and adult kidney
eDNA libraries, respectively. Counterparts of OCTNs in mouse were
isolated by similarity to human OCTNs. These cDNAs were constructed in
expression vector, pcDNA3 and transport function was examined by
transfecting them in HEK293 cells.
When hOCTN1 or 2 from human was transfected in HEK293 cells, Na*dependent carnitine uptake was induced. The obtained Km values were 4.6
and 4.3 ~M for hOCTN1 and 2, respectively, suggested that both of them
are classified as high affinity carnitine transporters. Especially, high
camitine uptake activity was observed by hOCTN2. In mice, three
members that show functional similarities with hOCTNs were identified.
mOCTN2 had Na-dependent and high carnitine transport activity. The
j v s mouse, isolated from the C3H.OH strain, genetically exhibits a
phenotype of SCD and low serum concentration of carnitine. In mOCTN2
gene o f j v s mouse, single nucleotide transition was found, which leads to
substitution of Leu at codon 352 by Arg. When functionally analyzed, the
mutant mOCTN2 did not show any carnitine transport activity, while
expression of the protein was confirmed by Western blot analysis.
Accordingly, OCTN2 was clarified as a physiologically important eamitine
transporter.
Interestingly, some members of OCTNs showed Na+independent transport of organic cations such as tetraethylammohium,
suggesting that they facilitate renal excretion of cationic compounds,
presumably at the renal apical membrane. Accordingly, OCTNs may have
multiple functions by contributing to active secretion of cationic
xenobiotics into urine as well as to reabsorption of physiologically essential
nutrient carnitine with distinct mechanisms.
[1] Tamal et al. F E B S Lett. 419 (1997) 107. [2] Tamal et al. J. BioL Chem.
273 (1998) 20378. [3] Nezu et al. Nature Gen. 21 (1999) 91.

[1] Kispal, G. et al., FEBS Lett., 418, (1997), 346.

[2] Csere, P. et al., FEBS Lett., 441, (1998), 266.

Abstracts FEBS'99

(M0114.2/231)

s231

is Mn 2+ a substrate for the P type Cd2+-ATPase in

Staphylococcus

aureus

Z Tynecka, A Maim, Z. Szczegniak

Department of Pharmaceutical Ahcroblolo~L .~ledm'al Academy
Lubartowska 85, 20-123 Lubhn. Poland

The P type Cd2+-ATPase of the Cd2+-resistant Staphylococcus attreus

1781 OR, belonging to the superfamily of ABC transporters, is coded by a plasmid codA genes. This effiux system extrudes Cd 2+ from the cytoplasm immediately after its entry via the Mn 2+ porter [1-2]. Due to this, the resistant cells
are protected against Cd 2+ accumulation and its toxicity. Up to now, no
physiological substrate for the Cd2+-ATPase was found. Our preliminary data
suggested that Mn 2+ might be a substrate for the Cd 2+ effiux system, as it is a
substrate for the Mn 2+ porter which transports both metals into cytoplasm.
First, 10 ~tM 54Mn transport was studied by filtration in resting cells of
plasmid-carrying, Cd2+-resistant S. attreus 17810R and in the plasmidless, derivative strain S. attreus 17810S, sensitive to Cd 2+. Cells &both strains suspended in 100 mM phosphate buffer, pH 7 (PB) oxidized endogenous amino acids,
generated A~tH+ (electrochemical proton gradient) via the respiratory chain and
maintained a high ATP pool. The Cd2+-resistant cells accumulated about tenfold less 54Mn down membrane potential (A~) than the Cd2+-sensitive one.
This suggested that only the Cd2+-resistant organism could regulate the cellular
level of Mn 2+, probably via the Cd2+-ATPase To verify this, cells of both
strains were preloaded with 54Mn under special experimental conditions
(1 mM PB + nigericin) used previously to preload cells with l9cd This
procedure resulted in inhibition of cell respiration and in rapid ATP hydrolysis
Under these conditions both strains accumulated similar amounts of 54Mn.
Restoration of the primary conditions (100 mM PB) at steady-state and
addition of Cd2+-insensitive L-lactate as an energy source allowed the cells of
both strains to respire and to generate ATP However, a rapid, energy-dependent 54Mn effiux was noted only in the Cd2+-resistant strain Since Cd 2+ inhibited 54Mn extrusion with no effect on energy generation, it is probable that
Mn 2+ efflux could proceed via the Cd2+-ATPase Mn 2 did not inhibit l9Cd
effiux, probably due to a lower affinity of Mn 2+ to the effiux system
This work was supported by grant 6 P04C 020 13 from the State Committee
for Scientific Research
[I] Tynecka Z, Go~ Z , Zaj~c J, J Bacteriol 147 (1981). 305
[2] Tynecka Z , Gog Z , Zaja,c J., J BacterioL 147 (1981), 313

(Mo/14.2/233)

Involvement of multidrug resistance proteins in

resistance to glueocorticoids.
J. I. Webster and J. Carlstedt-Duke. KarolinskaInstitute,
Stockholm, Sweden.

Acquired resistance to glucocorticoid therapy is a major problem in

the treatment of various anti-inflammatory diseases such as asthma. The
mechanisms of this resistance is as yet not well understood. It has been
known for a long time that there is an ATP-dependent export of
glucocorticoids out of cells. The multidrug resistance (MDR) proteins are a
group of ABC transporter proteins, which have been implicated in the
resistance of cells to anti-cancer drugs. A relatively large family of these
proteins is now emerging. Some work has been published indicating a
connection between glucocorticoid resistance and these proteins. The
majority of this work has been done using cell lines which have been
transiently transfected with these MDR proteins or cell lines which have
been selected because of their resistance to a particular drug. Recently, two
cell lines, L929 and a derivative of L929 cells (LMCAT) have been used
and shown to be resistant to glucocorticoids [I, 2, 3]. A detailed analysis
of the resistance of these cells to glucocorticoids is presented. A variety of
drugs to the MDR proteins, which have different mechanisms of actions,
have been used to overcome the resistance. Also various other techniques
have been implicated to draw a firm parallel between the presence of MDR
proteins and glucocorticoid resistance in these ceils.
References
1. Kraili and Yamamoto, J. Biol. Chem., 271 (1996) 17152.
2. Medh et al., Mol. Cell Endocrinol. 138 (t998) 11.
3. Marsaud et al., J. Steroid Biochem. Mol. Biol., 66 (1998) 11.

(Mo/14.2/232)

Renal organic anion transport mediated by human

inorganic nhosphate trjanspor~er NPTI
H. LVchinol~, I. Tamai ~', K. Yamashita~, H. Yabuuchi b, K.
Miyamoto ~, E. Takeda ~, A. TsujP '"

JKanazawa Umversity, Kanazm~'a, ~CREST, JST,

' Tokushima Umversity. Tokushima. JAPA N

Kawaguchl,

and

Type I Na/Pi transporter NPT1 mediates the reabsorption of inorganic

phosphate on luminal membrane of renal proximal tubules. Busch et al. and
we reported that rabbit and mouse NPT1, respectively, also transport organic
anions such as benzylpenicillin and probenecid[ 1,2]. In the present study, we
further examined substrate selectivity of NPT1 in human.
When human NPT1 was transfected in HEK293 cells, the transport of
PAH (p-aminohippuric acid), a model substrate of organic anions, was
stimulated with a Michaelis-Menten constant, 2.30 0.77 raM. Uptake of
PAH was sodium-independent, but was inhibited in the presence of chloride
ions. When the concentration of chloride ions was increased, the transport
activity of PAH by NPTI was decreased. These results indicate that NPT1
possesses the anion transport activities which are similar to those reported as
a classical organic anion transport system at the luminal membrane of renal
proximal tubules. Interestingly, PAH transport was inhibited by various
anionic compounds, indicating that NPT1 has a broad substrate selectivity.
B-Lactam antibiotics, probenecid, salicylate, indomethacin and anion
transport inhibitor DIDS reduced PAH uptake strongly and we confirmed the
transport of some of the inhibitors via NPT1. Since physiological
concentration of chloride ion is significantly higher in tubular lumen than in
the cells, PAH is presumed to be secreted via NPT1 from renal proximal
tubular cells to the lumen.
In conclusion, we demonstrated that NPT1 facilitates the transport of
various organic anions and this transporter may have a crucial role in renal
secretion of organic anions. This is the first molecular demonstration of PAH
transporter at the renal apical membrane.
[I]Busch et al., Proc Natl Acad Sci USA. 93, (1996) 5347.
[2]Yabuuchi et aI., ,Z Pharmacol Exp. Ther., 286, (1998) 1391.

s232

Abstracts FEBS'99

1.3 Gene targeting, gene therapy

(Mo/1.3/254)

Triplex-directed DNA cleavage by DNA Topoisomerase I

(Mo11.3/255)

P.B. Arimondoa; C. Bailly~; J.S. Sun~; M. PrudhommeC;

T. Garestiera; C. H616ne '~

G. Bermano ~, K. Wellsb, K. Fosterb~ D. Wells~ & J. Hesketh ~

aRowett Research Institute. Aberdeen AB21 9SB, UK,
blmperial College School of Medicine, London. W6 8RP. UK

aLaboratotre de BtoplTvsique. MNHN Parts France. bcentre Oscar Lambret.

59045 Lille, France ~CNRS URA 485 63177 Aubibre, France

Sequence-specific modulation of gene expression can be achieved by

oligonucleotides which bind to double helical DNA via triplex formation
in the "anti-gene" strategy. Cleaving or cross-linking agents have been
tethered to oligonucleotides in order to target sequence-specifically DNA.
An attractive option for biological applications would be the recruitnaent of
a cellular enzyme which would be directed by triplex recognition to
perform a sequence-specific cleavage. Topoisomerase I is a nuclear
enzyme, which alters DNA topology by reversible cleavage of DNA and
by formation of a transient phosphodiester-tyrosine linkage. Several
antineoplastic drugs have been shown to stabilize this transient reaction
intermediate. We investigated whether drugs as three rebeccamycine
analogues (R-6, R-0 and R-95), inhibitors of Topo 1, tethered to a TFO
were able to induce in a highly sequence-specific manner DNA cleavage.
We have lbund that TUC-R-6, TUC-R-0 and "fUC-R-95 conjugates arc
able in varo to direct the topoisomerase I cleavage at a drug-specific site
based upon sequence recognition by triple helix formation. The TUC-R-6
conjugate showed a higher inhibitory potency than the TUC-R-95 and
TUC-R-0 conjugates in agreement with the higher topoisomerase 1
inhibitory potent 3 of the fi'ee R.-6 derivative compared to tlic li'ce R-95
and R-0 derivatives.

Dystrophin gene therapy: is the 3'untranslated region

needed for mRNA localisation?

Most of the research on Duchenne muscular dystrophy involves the

development of gene therapy vectors which should contain not only the gene
sequence necessary to produce the protein but also regulatory sequences so
that the expression of the protein is correct in terms of amount and
distribution within the muscle. At present, it is not known how newly
synthesised large muscle proteins (e.g. myosin, titin, dystrophin) are targeted
to their sites of function but there is increasing evidence for mRNA
localisation in muscle and for the 3'untranslated region (3'UTR) of mRNAs
being involved [1].
Recently, it has been shown that dystrophin mR-NA is
localised close to the sarcolemma [2], suggesting that sarcolemmal
localisation of the mRNA allows synthesis of the protein close to where it
will function. It is, therefore, very important that any gene therapy vector
provides correct targeting of dystrophin transcripts.
The aim of this work is to investigate the role of the 3'UTR in localising
dystrophin mRNA. As dystrophin 3'UTR is very long (2.7 kb), we have
decided to focus on the 3'UTR sequence normally used in gene therapy
vectors (first 246 bp: mini-3'UTR). Chimaeric gene constructs were made in
which dystrophin mini-3'UTR was linked to a reporter gene: green
fluorescent protein (GFP). In parallel myosin heavy chain 3'UTR was used as
a positive control for localisation [3]. The chimaeric constructs were
transiently transfected into CHO cells and transcript expression analysed by
Northem hybridisation whereas mRNA loealisation by in situ hybridisation.
Our results showed that whereas myosin 3'UTR localises GFP transcripts, the
dystrophin mini-3'UTR does not, suggesting that the first 246 bp do not
contain a localisation signal. On the basis of these preliminary results, we
would predict that the mini dystrophin vector will produce dystrophin
expression with the rnRNA not localised leading to inefficient protein
localisation to the functional site inside the cell membrane. Therefore it is
very important to define the part of the remaining 3'UTR required for localisation so that it can be incorporated into gene therapy vectors in the future.
l l esketh, Expt. Cell Res. 225, (1996), 219.
2] Mitsui et al., J.Neuropath. Exp. Neurol. 56, (1997), 94.
3] Wiseman et al., Cell Biol. Int. 21, (1997), 243.

(Moll .3/256)

Integrin-mediated gene transfer.

M. Colina, M. Mauriceb, G. Trugnanb, M. Kornprobst~, R. P.
Harbottle~, A. Knight~, R.G. Cooperd, A.D. Millerd, C. Coutelle~
and M. C. Brahimi-Horn~-INSERM U402~ and lNSERM CJF 96.07b,
Paris, France; lmperlal College ~~ d London SW7 2A Y, UK.

We demonstrated previously that an integrin-binding oligolysine peptide

([K]~6RGD) condenses plasmid DNA and delivers it in a specific manner to
airway and intestinal cells with an efficiency which is 10-fold that of naked
DNA [l,2]. This efficiency could be further improved 30-fold by the addition
of a cationic lipid [2]. Here we demonstrate that the major mechanism of
entry of this lipo-oligoplex is endocytic. The vector complex rapidly (5 rain)
internalizes into early endosomes, then into late endosomes and lysosomes.
Entry involves, at least in part, clathrin-coated pit-mediated endocytosis since
different conditions or drugs known to influence this pathway modify both
uptake of DNA and its expression. These results were confirmed by confocal
microscopy, which showed colocalization of the complex with a marker of
receptor-mediated endocytosis, and by electron microscopy. The increase in
expression with addition of a Iiposome correlated with an increase in the rate
of passage of the DNA to lysosomes, a decrease in recycling and exocytosis
of the DNA and an increase in the amount of DNA in the nucleus. Trafficking
within the cell involved endosome fusion and the acid environment of the
endosomes-lysosomes was beneficial for expression. After 30 min both the
peptide and DNA localized to the nucleus and the amount of intact plasmid
DNA in the nucleus was highest with the complete vector. In conclusion we
show that: I. cell entry of this vector; composed of an integrin-binding
oligolysine conjugate, plasmid DNA and a cationic lipid, occurs by
endocytosis involving clathrin-coated pits, 2. that the plasmid DNA and
cationic lipid components localize to endosomes and lysosomes, and 3. that
the plasmid DNA and the integrin ligand passage to the nucleus. Studies into
the mechanisms of cell entry, intracellular trafficking and fate of such gene
transfer vectors should help improve further the efficiency of vectors for
human gene therapy by identifying the major barriers involved.
[1] Harbottle et al., Hum. Gene Ther. 9, (1998) 1037
[2] Colin et al., Gene Therapy 5, (1998) 1488

(Mo/1.3/257)

Modulation of p94 gene expression by RNA targeting

E. Dargelos, S. Poussard, P. Cottin
Laboratoire de Biochimie et Technologie des Aliments,
Universit~ Bordeaux I, ISTAB, UA-1NRA 429
Avenue des Faeult~s, 33405 Talence Cedex, France

P94, a Ca2+-dependent skeletal muscle specific protease has been

shown as being involved in a myopathy called Limb Girdle Muscle Dystrophy
2A (LGMD 2A). In 1995, the human p94 gene was cloned and mutations in
patients were identified. It was the first time that a lack of enzyme was
correlated with a muscular dystrophy. Moreover, previous results obtained in
our laboratory showed that a lack of p94 in primary muscle cell culture
causes a disassembly of the Z-line. In order to better understand the
biological role of this skeletal muscle specific protease, it would be of great
interest to mimic in vitro the phenotype of LGMD 2A.
Consequently, mouse myoblasts (C2C12) have been transfected with a
system of two vectors (LacSwitch II, STRATAGENE)which is able to express
(by an inducible way) a mRNA antisense complementary to the endogenous
p94 one. To be ascertain that the targeted mRNA was the p94 one, we
decided to clone into the multiple cloning site of the expression vector
(pOPRSVI/MCS), a very specific sequence (IS2) of 300 bp. The double
strand constructed DNA was generated by RT-PCR, Either the antisense or
the sense construct or the pOPRSVI/MCS alone was co-transfected with the
repressor plasmid (pCMVLacl) in the C2C12 ceils, and the different clones
were tested for differentiation. Only the transfectants wlaich were able to
differentiate fully were used for further experiments.
In our experimental model (C2Cn), we have shown that p94 mRNAs
were present since the earlier stages of differentiation and that their amounts
increase dramatically all along myogenesis. On the other hand, our results
have revealed that the rate of the protease was constant. These results agree
with the fact that the expression of p94 was regulated at a post
transcriptional level. According to all these facts and in order to prove that
p94 was implicated in the sarcomerization, we have blocked the expression
of its synthesis. Our first results revealed that, after induction of the
LacSwitch II system in the early stages of differentiation, the amount of
mRNAs was decreased about 70% after 8 hours. Studying the main
consequences of such a treatment at molecular and structural levels will lead
us to a better understanding of the implication of p94 in the LGMD 2A and
consequently of its biological role during myofibrillogenesis.

Abstracts FEBS'99

(Mo/1.3/258)

Polyethylenimine but not cationic lipid improves antisensc

activity of T-capped phosphodiester oligonuclentides
S. Dheur and T. Saison-Behrnoaras

s233

(Mo/1.3/259)

Laboratowe de BIophysique. Musdum National d'Histowe Akmlrelle,

43,Rue Cuvier, 75231 Paris Cedex 05, France

Promoter sequences which regulate ~hFR transcription

as a new tool for ovarian cancer gene therapy.
E. Galmozzi, F. Mangiarotti, M. Mazzi, S Sforzini,
S. Canevari, A. Tomassetti. Dep. of Experimental Oncologv
lstttuto Nazionale Tumori, Mdan, Italy

Lipofectin, which is the mixture of a neutral lipid with a cationic lipid has
been widely used to enhance cellular delivery of phosphorothioate. 2'sugar modified and chimeric antisense oligonueleotides. Phosphodiester
oligonucleotides delivered with Lipofectin usually do not elicit antisense
activity probably because cationic lipid formulations do not sufficiently
protect unmodified oligonucleotides from nuclease degradation. We show
that a cationic polymer, polyethylenimine (PE[), improves the uptake and
antisense activity, of T-capped 20-mer and 12-mer antisense
phosphodiester oligonucleotides (PO-ODN) targeted to different regions
of Ha-ras mRNA and to 3"-untranslated region of C-raf kinase, in
contrast, PEI which forms very stable complex with the 20-mer
phosphorothioate oligonucleotide (PS-ODN) do not enhance its antisense
activity. Using fluorescently labeled carriers and oligonucleotides, we
show that PS-ODN-PE1 particles are very efficiently taken up by cells but
PS-ODN is not dissociated from the carrier. Our results indicate that
carrier/ODN particle size, stability, ODN release kinetics varies with the
chemical nature of the oligonucleotide and the carrier being transfected
into the cells. The very, low cost of the PE1 compared to cytofectins, the
increased affinity for targeted mRNA and decreased affinity for proteins
of PO-ODNs compared to PS-ODNs make the use of PEI/PO-ODNs at
least in adhcrant cells very attractive.

(Mo/1.3/260)

Adenoviral and lipid-mediated gene transfer to human

monocyte-derived macrophages and COS-7 cells
H. Heider. E. S. Wintergerst. and R. Asmis
Untvelstt~ o/Ba,~el, Biochemtstl~, Vesalgasse 1, CH-4051 Basel

Human monocyte-derived macrophages IHMDM) efficiently express

foreign DNA when offered in an appropriate adenoviral vector. Wnh
enhanced green fluorescent protein (EGFP) reporter constructs we were
able to show that adenoviral-mediated gene transfer leads to the
expression of EGFP in more than 90% of the cells. Classical trans[ection
methods, like liposome or DEAE-mediated gene transfer, usually induce
ouly weak gene expression in H M D M Transfection of HMDM by a
lipid-mediated technique, which was recently reported to be very
effective in primary cell transfection [1], resulted in our hands in
detectable EGFP expression in less than 0.03% of HMDM. The same
technique was highly efficient in DNA-delivery and expression in COS-7
cells. More than 30% of the cells were fluorescent 24h post-transfection.
Quantitation of plasmid DNA in lipid-transfected macrophages by
quantitative PCR showed that each macrophage contained on average 2fg
of plasmid DNA 24h post-transfection, and 48h alter transfection even
higher amounts of DNA were observed. This means that more than 400
ruolecules of plasmld DNA entered each ceil compared to less than 100
viral vectors that could maximally infect one single cell. The lipidmediated transfectlon technique applied to COS-7 cells yielded around 10
fold h~gher DNA contents 24h post-transfectkm. The amount of
endogenous DNA in COS-7 cells increased lurther within the next 24h.
We conclude that HMDM take up relatively large amonnts of plasmid
DNA (more than 400 molecules per cell) when offered to them in a lipidassociated manner, but they are able to strongly express foreign DNA
only when it entered via an adenoviral vehicle.
[1] Uyttersprot et al., Mol. Cell Endocrln. 142. (1998) 35

The human cz folate receptor (<zhFR) is a membrane glycoprotein of 38

Kd that participates in the internalization of folates and antifolates and
that is over-expressed on human ovarian carcinoma cells The gene,
composed of 7 exons (ex) that span ~ 6.7 Kb, presents at least 2 distinct
promoter regions, PI and P4, located within exl and upstream ex4
respectively The open reading frame is encoded by ex4 through ex7
while the 5' untraslated region is encoded by exl through ex4 Six
o~hFR eDNA isoforms have been isolated from normal and malignant
tissues Although these cDNAs share a common 3' terminus, the 5'
terminus of each cDNA is different in length and sequence. The
molecular basis of this heterogeneity involves, in addition to the use of
the two promoters P1 and P4, an alternative splicing event between exl
and ex3. With the aim to identify an ovary carcinoma-specific promoter
with potential usefulness in therapeutic approaches, we have compared,
by RT-PCR and RNase protection assay, RNAs extracted from a panel
of ovarian carcinoma cells with that from the non-ovarian carcinoma
KB cells, that express comparable amount of czhFR protein. In ovarian
carcinoma cells we found a significantly higher expression of the
transcript which also includes ex3. Based on these data, our strategy
was to utilize the ex3 alternative splicing to enhance the tissue-specific
promotion in ovarian carcinoma cells. Therefore, the activity of various
promoter-chloramphenicol acetyltransferase (CAT) chimeric constructs
were tested by transient transfections. The results indeed show a
relevant CAT activity of the promoter constructs containing ex3 only in
ovarian carcinoma IGROV 1 but not in KB cell lines. These data raise
the possibility to develop a gene therapy for ovarian carcinoma using
the othFR promoters that include ex3 alternative splicing control
element linked to a suicide gene such as Thymidine Kinase or Cytosine
Deaminase.
Partially supported by AIRC/FIRC and Heah Ministry Special project.

(Mo/1.3/261)

Kinetics of opening of the m i n i - h a i r p i n d(GCGAAGC)

B.Joll~s, Z. Chraibi*, M. R~fr~giers, A.Laigle
LPBC, CNRS URA 7033, UPMC, 75005 Paris, France
* University Cadi Ayyad, Marrakech, Maroc

Some DNA and RNA sequences form unusually stable hairpin structures.
The DNA fragment d(GCGAAGC) forms an extraordinary stable DNA
mini-hairpin with regard to thermal denaturation, nuclease degradation and
denaturing electrophomsis conditions [I ]. It is stabilized through only two
G-C base pairs and a non Watson-Crick G-A pair. Its high resistance
against nucleases makes it useful in antisense strategy because unmodified
oligodeoxynucleotides (ODN) are rapidly degraded both in serum and cells,
primarily by 3'-exonucleases. We have already shown that attachment of
the d(GCGAAGC) hairpin structure on the T-end of a 12mer does not
hinder hybridization to a complementary DNA strand [21. Moreover, the
hairpin is by now in equilibrium between a folded and an owned structure,
with an energy minimum in favor of pairing if it is possible, even with
mismatches.
The opposition between the stability of the hairpin alone and its fast
hybridization when possible is emphasized in the present work. (i) The
kinetics of exchange of the amino groups protons of the hairpin has been
studied by resonance Raman spectroscopy by passing from a H 2 0 to a
D 2 0 medium. A tl/2 of the order of 10 days has been found. (ii) FRET
(fluorescence resonance energy transfer) allowed the mechanism of ODN
hybridization/opening of the hairpin to be studied. In that purpose, (1) an
ODN protected by the hairpin on its T-side has been labeled on the same
side with fluomscein whereas its complementary ODN has been labeled on
its 5'-side with rhodamine, (2) a single ODN protected by the hairpin Js
labeled with rhodamlne on its 5'-side and with fluorescein on its 3'-end.
Since the fluorescence spectrum of fluorescein overlaps with the
absorbance spectrum of rhodamine, the excitation of fluorescein induces
fluorescence of rhodamine while its own fluorescence decreases : this
process is called FRET. The enhancement of FRET (casel) or its decrease
(case 2) allowed times of the order of seconds or minutes to be determined.
[11 Yoshizawa et al., Nucleic Acids Res., 22, (1994) 2217.
[2] Jolles et al., Nucleic Acids Res., 25. (1997) 4608.

s234

Abstracts FEB S' 99

(Mo/1.3/262)

Interaction of Hypericin and Hypocrellin A with Biolomolecules

E. Kocisnva ~b, p. Miskovsky~ L. Chinsky b

(Mo/1.3/263)

~Depart. of htophysws P J Safarik Umver , Je~enna 5, 04154 Koslce, Slovakm

~LPBC, Uttiver~tte P, et M Cl~rte, 4 place Jtt~atett, 7~252 PartsCede.~05. F~ance

In recent years great interest has been focused on aromatic polycyclic

compounds - hypericin and hypocrellin A occuring m plants of genus
Hypericum and the parasitic fungus Hypocrella bambuase, respectively. These
photoactive substances have been long time used in folk herbal medicine but
recently many scientists have concentrated attention to them because of their
ability to inhibit the replication of several types of viruses including HIV-1 and
to display an antlcancer potency [1].
In order to understand the basic mechanism of their interactions with
possible cellular targets we have concentrated our work to study their mode of
action on biological molecules (complex of oligonucleotides with hypericin
and the complexes of human serum albumine with hypericin and hypocrellin).
For these studies different experimental technics have been used. Resonance
Raman spectroscopy enhances vibrational lines corresponding to nucleic acid
base modes (257 nm) and vibrations of the aromatic amino acides of protein,
particularly the tryptophan one (222 nm). The surface enhanced Raman
scattering techmque allowed to see the drugs in the complex structures via
strong enhancement of their vibrations and quenching of fluorescence.
Information about stability of the protein structure in presence of drugs was
obtained from the differentml scanning microcalorimetry.
According to the model for albumin/hypericin complex hypericin
interacts with tryptophan pIaced in IIA subdomain of the protein through
hydrogen bonding between its carbonyl group and N 1-H side of tryptophan [2].
In the similar model for hypocrellin A more than one binding places on the
protein surface are proposed. Hypocrellin A does not anteract with interior
binding site, its interaction is nonspecafic but it affects the general albumin
structure and induces rearrangement in the protein molecule. The AG and GA
nucleotide doublets as a specific targets for hypericin have been identified.
Moreover, the interaction with 5'AG3' target is stronger than that with 5'GA3'
one [3]. Gradually shortening oligonucleotides gwe another selectivity
information.

Modulation of m d r l gene expression by antisense

oligon ucleotides
E.Kostenko M.Dobrikov, l.Titov, M.Zenkova, V Vlassov
lnsntute o f Btoorgamc ChemlstO" 630090, Novostblrsk, Russia

Multidrug resistance ts one of the major obstacles, interfering with

successful anticancer chemotherapy. Antisense oligonucleotides offer a
molecular targeting tool for selecting arrest of biosynthesis of the pglycoprotein and overcoming cellular multidrug resistance. Use of antisense
oligonucleotides for inhibition of protein synthesis is well established,
however, factors affecting potency of mdividual antisense sequences are
complex and poorly understood. To identify human mdrl (multi drug
resistance) mRNA region suitable for targeting with antisense
oligonucleotides, secondary structure of the 5'-part of the mRNA was
probed with structure specific nucleases. 8 antisense oligonucleotides (ONs)
(16 mers) were chosen so as to target hairpms present m the RNA structure.
ON binding to mdrl mRNA was investigated using fluorometric assay.
DNA probes were 5' end labeled with fluorescent dyes. Upon the
conjugates binding with the RNA changes in fluorescence spectra occurred.
Concentration and time-dependencies of these changes allowed to study the
kinetics of the process and to determine RNAoON binding constants. The
most accessible sites for antisense ON were found within RNA regions
including the initiating AUG codon (region 398-448) and region 298-329
(the numbering system is that of the HMDR1. GB_PR gene sequence N
M14758). The obtained binding constants are in agreement with mdrl RNA
secondary structure built using computer modeling. Efficiently hybridizing
oligonucleotides were shown to cause a decrease of the mdrl mRNA level
in cultured KB (mdrl +) cells.

[1] Z. Diwu, Photochem. Photobiol., 61, (1995) 529.

[2] P. Miskovsky et ah, J. Am. Chem. Soc., 120, (1998) 6374.
[3] E. Kocisova et ah, J. Biomol. Str. Dynamics 15.(1998) 1147.
(Mo/1.3/264)

Is apoptosis involved in the regression of rat liver tumors

expressing antisense c-DNA of the insulin-like growth factor ?
C. Lafarge-Frayssinet ~, S. Enouk-Achard ~, C. Frayssinet ~,
G. Desauty ~, H.T. Duc-~and A, Sarasin ~
I. Lab. de Ggngtique Moldculaire, UPR42 CNRS BP8 94801ViHejuif'
2. Centre Hdpatobiliaire, HOpital Paul-Brou~se, 94800 Vtllejuij;
3. Lab. de Toxicologic, Fac. de Pharmacie Reng Descartes, 7_5005Paris.

The neoplastic transformation of hepatocytes is accompanied by the

reappearance of insulin-like growth factor I (IGF I) which is essential to
the establisment and maintenance of transformed phenotype.
In our laboratory we have established an hepatocarcinoma cell line
(LFCI2A) which produces voluminous tumors when injected to
syngeneic Commentry rats ; these cells synthetized high level of IGF L
When transfected with an episomal vector expressing the antisense IGF I
c-DNA under the control of metallothionein I promoter inducible by zinc,
these cells partly lose their tumorigenic properties and become able to
induce the regression of established hepatocarcinoma in syngeneic rats.
These transduced hepatocarcinoma cells which do not produce IGF I
anymore induce in vivo a tumor immunity mediated by CD8+ T ceils
and the expression of MHC class I protein was greatly increased as
compared to parental cells. Furthermore we have shown that apoptosis
appears in the transduced cells as demonstrated by the presence of
apoptotic bodies and the formation of inter nucleosomic fragments.
The purpose of the present study is to evaluate (using other apoptotie
agents as camptothecin) the relative impoJ~ance of apoptosis versus
immunogenic mechanism in this antisense strategy of gene therapy.

(Mo/1.3/265)

Reconstitution of BH4 Biosynthesis with

Triple-cistronic IRES-Containg Retroviral Vectors
S. Laufs a, S. H. Kim b, S. Kim b, N. Blau ~, B. Th~ny ~
Dtv. of Chn. (?hem. at~,,Blachem., Department of Pedlatrtcs, Untverslty
of Zurich, Swttzerlcmd: [MB(; Nanonal l lmverstty, Seoul, Korea

Tetrahydrobiopterin (BH4) is an essential cofactor for catecholamme and

serotonin neurotransmitter biosynthesis. BH4 biosynthesis is carried out
in a three-enzyme pathway involving GTP cyclohydrolase I (GTPCH), 6pyruvoyl-tetrahydropterin synthase (PTPS) and sepiapterin reductase
(SR). Treatment of genetic defects leading to BH4 deficiency requires
neurotransmitter replacement since synthetic cofactor does not efficiently
penetrate the blood-brain barrier. Autologous fibroblasts transplanted
into the brain as depository cells for drug delivery might offer an
alternative. However, normal fibroblasts do not express GTPCH, and
fibroblasts from PTPS patients lack two biosynthetic enzymes for BH4
production. Here, we engineered primary fibroblasts by the use of triplecistronic, retroviral vectors for cofactor production. Constitutive SR
activity in these cells enabled BH4 biosynthesis by transducing GTPCH
and PTPS cDNAs together with a selective marker coupled in a single
transcript with two IRES-elements in tandem. Upon reaching a critical
threshold concentration (> 400 pmol/mg protein) of intracellular BH4,
the fibroblasts effioently released cofactor even under non-dividing
conditions. The use of triple-cistronic vectors for single transduction to
reconstitute metabolic pathways or to treat multi-genetic diseases may be
useful for engineering, for instance, depository cells for various organs,
including the nervous system.

Abstracts FEBS'99

(Mo/1.3/266)

s235

Cardiac function in transgenic mice overexpressing

different types of adenylyl cyelase in heart.
L.Lipskam, N.Defer, I.Hajar, C.Cho, M.Garrel,
H.Rockman, J.Hanoune.

(Mo/1.3/267)

Antisense oligonucleo-peptide conjugate containing a

Nuclear Export Signal localizes in the cytosol.
L. Meunier, R. Mayer, M. Monsigny and A.C. Roche
Glvcobiologte, CBAI/CNRX Rue Charles Sadron, 45071 Orleans cedex 02.

INSERM U99, 94010 Crdted. France; Chapel Hill, NC 27599. USA

In cardiomyocytes, the B-adrenergic signal transduction is

dynamically controlled by the two second messengers calcium and
cAMP. Calcium and cAMP concentrations may oscillate providing both
temporal and spatial informations. Adenylyl cyclase (AC) is a membrane
bound enzyme that converts ATP to cAMP. Nine isoforms have been
identified which are expressed by genes located on different
chromosomes. They are expressed in different tissues and submitted to
different types of regulations. Among them, the cardiac specific isoform
AC5 is inhibited by low calcium concentrations, and the brain specific
AC8 is activated by calcium and calmodulin. To address the role of
adenylyl cyclase and cAMP in the regulation of 13-AR signalling and
cardiac contractility, we have generated two lines of transgenic mice
overexpressing either the canine AC5 or the human AC8 specifically in
the cardiomyocytes, using the a-Myosin Heavy Chain promoter.
Characterization of heterozygous transgenic line (AC5TM)
showed that the amount of AC5 mRNA and activity increased 10 fold and
1.7 fold respectively relative to their control littermate. Characterization of
heterozygous transgenic line (AC8TM) showed a high level of AC8
mRNA in cardiomyocytes and appearance of calciurn/calmodulin
stimulated AC activity in cardiac membranes. The contractile parameters
were measured by echocardiography and invasive cardiac catheterization.
Strong modifications in the cardiac contractility were observed in
AC8TM, whereas cardiac function of AC5TM was essentially the same
as that of control mice.
Taken together these results suggest that in vivo i) the level and
function of the g-adrenergic receptor might be the primary determinant of
the contractility ; ii) the different components of the g-adrenergic
signaling pathway need to be selectively compartimentalized ; iii) the
adenylyl cyclase activity in cardiac myocyte is strictly controled.

(Mo/1.3/268)

Defective thymocytes maturation

Kinase (erk 1) knock out mice

in p44

MAP

Pag/~s,Ga., Gurrin, Sb., GralI,Da., Bonino, F~., Smith,

A~., Auberger,P b. and Pouyssegur,J".

(a)Cemre de Btochunie. UMR CNRS 6543, ,arc Vah'ose. 06094 Nice France.(b)Faculd' ~
Medecme, CJF INSERM 96 05. Avenue de Vallombtvse, 06107 Nice France. (c)Center [or
genome resealvh, King '~ Building. West Mares Road. Edimburgh . Scotland

The p42 and p44 MAP Kinases (erk2 /erkl) signaling pathway has been
implicated in proliferation as well as in differentiation programs. To
understand the specific role of the commonly expressed and activated
isoforms p42 and p44 MAP Kinase (erk2 and erkl) in the whole animal, we
generated erkl deficient mice through homologous recombination in
embryonic stem cells. Erkl KO mice are viable, fertile and apparently
indistinguishable from wild type mice. this first result indicates that erkl is
dispensible and that the second isoform erk2 can compensate the loss of erkl.
However a closer analysis oferkl -/- mice shows that thymocytes maturation
beyond the CD4 +/CD8 + stage is reduced by half with a similar diminution
in the TcR high thymocytes subpopulation. We also observed a severe
reduction in thymocytes proliferation following activation with an anti-TcR
monoclonal antibody in the presence ofphorbol myristate acetate even if erk2
activity is not affected in these cells. Our result demonstrate that apparently
redundant kinases could play specific role in highly differentiated tissues and
also point out the specific role of erkl in thymocyte maturation and
proliferation.

Antisense oligonucleotides (odn), are potential therapeutic agents ; they are

supposed to interact selectively and in a predictable manner with a target
RNA and are routinely used to selectively prevent the expression of a given
gene. Upon endocytosis, most oligodeoxynucleotides accumulate in vesicular
compartments , a tiny amount of them cross the vesieule membrane, reach
the eytosol and, by passive diffusion, enter the nucleus where they are
entrapped. So far, the compartment in which an antisense odn interacts with
its mRNA target has not been precisely characterized. As an attempt to
answer this question, odn-peptides were designed with the aim of
maintaining them in the cytosol.
The nuclear export of many proteins depends on a short peptide sequence
called the nuclear export signal (NES). Fluorescent odn-peptide containing
either an active NES or an inactive NES analogue were synthesized and their
intracellular distribution, upon microinjection, was studied by confocal
mieroscopy. Odn-peptides containing an active NES sequence were
efficiently and rapidly exported from the nucleus to the cytosol whereas odnpeptides containing the inactive NES analogue were found to be located in
the nucleus. The nuclear export of odn-peptide bearing an NES was
temperature-dependent and was inhibited by leptomycin B, a known inhibitor
of NES-mediated export of proteins from the nucleus to the cytosol.
On these bases, the inhibitory activity of antisense odn was tested in a model
system allowing the specific transcription of a luciferase reporter gene in the
cytosol. Antisense propynylated odn-peptide-NES conjugates, directed
against the luciferase gene, efficiently inhibited (75%) the cytosolic
expression of luciferase whereas at the same concentration the peptide-free
propynylated odn or the propynylated odn-peptides containing an inactive
NES were nearly inactive. Based on the ability of an active NES peptide of
maintaining an odn in the cytosol, it will be now possible to determine
whether a given odn elicits its activity on a cellular gene by encountering its
target in the cytosol or in the nucleus. Moreover, such conjugates could be
used to efficiently inhibit viruses which have their replicative cycle in cytosol
or those which have to be retrotranscribed in the eytosol immediatly after
their penetration in cells, as in the case of retroviruses and especially AIDS
viruses
(Mo/1.3/269)

Unfolding of Yeast tRNA r ~ by Antisense

Oligonueleotides
V. Petyuk, M. Zenkova, R Giege#, V. Vlassov
Institute of BioorganicChemistry,Novosibirsk,Russia.~Institutde
Biologic Moleculaireet Cellulaire du CNRS, StrasbourgFrance

Interaction of tRNA phe with ohgodeoxyribonucleotides (ONs)

complementary to the 3' half of this RNA under physiological conditions
was investigated [1-3]. The first ON serie were 9 0 N s (12-16 mers)
complementary to the acceptor and TwC stems of the tRNA. For effective
hybridization with the tRNA, these ONs were found to have to be
complementary to the ACCA or at least to ACC sequence at the 3' tRNA
end. Probing of the tRNA Ph structure in the course of the hybridization
using RNase T1, RNase A, DMS and RNase V1 has revealed two-step
binding mechanism. At the first step, ON binding rapidly occurs with open
ACCA sequence yielding an intermediate complex. The second step is a
slow unfolding of tRNA structure and extended duplex formation. The
obtained results demonstrate that effective nucleation is a crucml event m
the process allowing the oligonucleotides to nest and invade into the tRNA
structure by displacement of the 5' strand of the acceptor stem. Second
ohgonucleotide serie were 8 0 N s complementary to the TwC arm and extra
loop of the tRNA, another area of effective oligonucleotide binding.
Complementarity to the variable loop was essential for the binding of these
ONs, The obtained results demonstrate a possibility of effective targeting of
folded RNA structures with antisense ONs complementary to the regions
with short single-stranded sequences suitable for nucleation
1 M Zenkova et al DokI.AkadNauk, 361, (1998), 260-263
2 V Petuyket al NueMozides& Nucleotides, 1999, in press
3 V Petuyk et al, FEBS Letters, 1999, in press

s236

(Mo/1.3/270)

Abstracts FEBS'99

Antitumoral vaccination by suicide gene therapy. V. PierrefiteCarleI, P. Baqu62,A. Gavelli3, M. Mala2, M. Chazal2, A Bourgeon2,

(Mo/1.3/271)

G Mdano4 and B. Rossil 1Unit~INSERM364, Nice.2Hrpita] FArchetII,

Nice. 3HdpltalPrincesseGrace, Monaco.4CentreAntoineLacassagne.Nice

Liver metastasis constitute one of the major complications of colon carcinoma

for which surgery and chemotherapy give deceiving results. Suicide gene
therapy, which consists in the transfer of a "killer" gene into tumor cells,
seems to be a promising alternative for the treatment of this kind of cancer.
Among these suicide genes, the cytosine deaminase (CD) gene of E. coli
converts the non toxic compound 5-fluorocytosine (5-FC) into the lethal drug
5-fluorouracil (5-FU). We chose to analyze the efficacy of suicide-gene
modified cancer cells as autologous tumor vaccines in a rat model mimicking
a liver metastasis situation. We have introduced a vector expressing the CD
gene in a BDIX rat colon carcinoma cell line which, after intra-hepatic
injection in syngenic animals, generates experimental single liver "suicide
tumor". 5-FC treatment induces regression of CD-expressing (CD+) tumors
and rendered treated animals resistant to challenge with wild-type tumor cells.
In a bifocal model consisting of a CD+ tumor on one lobe of the liver and a
wild type parental tumor on the opposite lobe, treatment with 5-FC results in
regression of both types of tumor. This effect, also termed distant bystander
effect, is likely due to a stimulation of the immune system by 5-FC-induced
regression of the CD+ tumor. Importantly, such bystander effect can also
induce partial or total regression of a wild type preestablished tumor after
intra-hepatic challenge with CD+ tumor cells and 5-FC treatment.
Immunodepletion of rats and immunohistological analysis of tumors indicate
that NK cells are likely to be involved in this antitumoral effect. Taken
together, these results suggest the potential use of suicide-gene modified
tumor cells as therapeutic vaccines against liver metastasis from colon
carcinoma.

Construction and functional expression of an epitopetagged human choline acetyltransferase

I. Robert, C. Quirin-Stricker
Laboratoire de G~n~tique Mol~culatre des Eucaryotes, Facuh~ de
Medecine, 11 rue Huraatm, 67085 STRASBOURG, France

The enzyme choline acetyltransferase (CHAT), unique enzyme responsible

for the synthesis of acetylcholine (ACh), is the most specific cholinergic
marker. ACh is a main neurotransmitter in the central and peripheral
nervous system and plays a crucial role in cognition. The deficit of cognitive
functions is correlated with a decrease in ChAT and ACh. These changes,
which give rise to an altered cholinergic transmission, are one of the
principal biochemical abnormalities, characteristic of Alzheimer's disease.
The cDNA containing the entire coding sequence for human choline
acetyltransferase gene (hCHAT) [1] was fused to the influenza virus
hemagglutinin (HA) epitope preceded by a Kozak sequence [2]. The
recombinant HA-hChAT was inserted into an expression vector under the
transcriptional control of the cytomegalovirus (CMV) promoter.
After transient transfection into COS-1 cells, the construction was tested for
its ability to express the recombinant mRNA by Northern blot analysis, and
the protein, by Western blot analysis and immunocytochemistry. The cells
transfected wtth pCMV HA-hChAT showed the presence of mRNA and
protein of the predicted length and molecular weight, respectively. By
immunofluorescence, using polyclonal anti- CHAT and monoclonal antiHA antibodies, we revealed a strong fluorescent signal in the transfected
cells. The enzymatic activity of the chimeric HA-hCHAT protein was
assayed and compared to the native, it behaves identically to unmodified
hChAT showing that the HA epitope does not affect ChAT activity.
This approach enables to distinguish the expression of the H A - h C h A T from
endogenous CHAT, and allows an easy detection of the recombmant CHAT
by the epitope.
In the future, engineered xenogenic and allogenic cells will allow efficient
m s a u expression of the ChAT gene and the production of ACh after their
administration in the CNS.
[1] Y. Oda and al., Mol. Brain Res.,16, (1992) 287.
[2] P.A. Kolodziej and al., Methods Enzymol, 194, (1991) 508.

(Mo/1.3/272)

A padlock oligonucleotide for labeling plasmids.

T. Roulon, C. Escud& J.S. Sun, T. Garestier and C. Hdl~ne.
Laboratoire de Btopkvs~que , Musdum Nanonal d'Ht.stoire 'Vature/le,
43 rue ('re'let 75231 PA RI8 ('EDEX 05 France,

There is no way today for labeling a double-stranded DNA

without any chemical or enzymatic modification of its sequence. A
method allowing a non-covalent Irreversible labeling of a plasmid has
been recently developed m our laboratory [11. A triple-helix forming
oligonucleotide (TFO/ binds specifically to an oligopurine sequence
carried by a plasmid. Then the ends of this TFO hybridize to an
oligonucleot~de template and arc joined through the action of T4 DNA
ligase, thus forming a padlock oligonucleotide.
The triple-helix used in this study was designed to be very stable.
Some G T ohgonucleotides are known to form triple-helix only in the
presence of specific ligands [2]. Such an oligonucleotide was succesfully
used to form a padlock in the presence of benzo[e]indoloquinolines(BelQ) ligands. The padlock formation was shown to be
sequence-specific and only appeared when ligand was prescnt. The
topologzcal state of the plasmid was not altered by padlock labeling. This
could lead to a padlock that should be free to move around the plasmid
when no more [igand is present.
This new labeling system would enable labeling with various
markers, fluorophores for instance. Moreover, attaching a chemlcal
moiety to the padlock could improve purification protocols or targeted
delivery of plasmids for applications in gene therapy.
l I] Escud6 C. & at.. submitted for publication.
[2] Escud6 C. & al.. Biochemistry. 35(18), (1996) 5735.

(Mo/1.3/273)

A yeast-based cloning system to generate large

plasmids: Application to high capacity
adenoviruses.
G. Vassaux and N.R. Lemoine
ICRF Molecular Ontology Unn, Hammersmuh Hospital, London, UK

Adenoviruses have been shown to be very efficient vectors for gene

delivery both in vitro and in vivo. In addition, large titres of recombinant
adenoviruses can be obtained. However, a "'leaky" expression of viral
proteins, as well as a low capacity of foreign D N A , limits the clinical
applicanons of the first and second generation of adenoviral vectors.
Recently, a third generation of adenoviral vectors has been described,
in which, all viral coding sequences have been deleted. This technical
development has been shown to decrease the aspecific cellular toxicity of the
previous adenoviral vectors as well as to increase the transgene capacity to 36
kb. A sustained level of expression of the transgene was also observed.
We are planning to use the advantages of this new vector for cancer
gene therapy. The large transgene capacity would allow the expression of a
battery of suicide genes driven by tumour selective promoters in combinanon
with cytokines and molecules able to selectively activate the ~mmnne system
against the tumour. To generate these "high capacity" adenovlruses, the
construction of large plasmids (between 28 and 37.8 kb) is necessary.
However, the multiple cloning steps involved make this project technically
difficult in clasmcal bacterial cloning systems.
As an alternative, we propose to use homologous recombination in
yeast to generate these large plasmids. More precisely, we have engineered
two plasmids, one carrying the genes of interest and the other some neutral.
"stuffer" sequences. After introduction of the first one into S. Cervesiae, the
directed homologous recombination in yeast with the second plasmid has
already allowed us to generate a packageable plasmid above the critical size of
28 kb, containing the Lac Z gene as the unique expressed sequence in
mammalian cells. In a current pilot study, we are amplifying the adenovirus to
obtain the high titres expected with this sort of vector.
A battery of neutral "stuffer cassette" plasmids of different sizes has
also been developed and different yeast clones stably carrying each of these
plasmids have already been obtained. This will simplify and speed up the
process as, in the future, each transgene to be packaged in a high capacity
adenovirus will simply have to be cloned into a classical Bluescript-based
vector and transformed into the yeast strain carrying the complementary
stuffer to 28 kb.
We are planning to generate versions of these plasmids that will
contain "smcide cassettes" or "immuno-st~mulatory cassettes" and we will
exploit the flexibility and versatility offered by this yeast-based system to
optimise the combination of the different agents for cancer gene therapy.

Abstracts FEBS'99

s237

3.1 Cell cycle

(Mo/3.1/274)

Study ofgec3, a gene involved in the quiescence state.

P. Adami, J.F. Musard, M. Sallot, M. Jouvenot

(Mo/3.1/275)

Laboratoire de Biochimie, UFR Sciences et Techniques Universtt~ de

Franche-Comt~, 16 route de Gray 25030 Besanon cedex France

Estrogens control cell proliferation by regulating gene expression in

target tissues. The identification of genes estrogens regulated during
stimulation of cell proliferation is an essential step for the understanding of
the mitogenic activity of these homones. Using differential hybridization, a
potentially estrogen-down regulated gene, gee3, was first isolated as a 2300
bp 3' eDNA from a L-gtl0 library prepared from guinea-pig endometrial
cells. Using a 5'-RACE method, a 2491 nucleotide gee3 sequence (U82982)
was determined. This sequence shared 83% identity with a human
quiescence-inducible Q6 eDNA [I]. Using guinea-pig northern blots, the 2.7
kb gee3 mRNA was detected in lung, ovary and endometrium but not in
brain, liver and kidney. In some human tissues (heart, placenta, pancreas
and testis), two transcripts (3.4 kb et 3.7 kb) were also detected.
An extensive study of gee3 expression is now in progress, using
normal glandular epithelial cells in secondary culture [2] or SV40 Large T
antigen immortalized glandular epithelial cells submitted to various culture
conditions. It was shown that gee3 transcript accumulated when normal
cells were forced to enter reversible quiescence by serum starvation (10
times induction after 40 hours). In immortalized epithelial cells, gee3
mRNA level did not vary during serum depletion.
A recombinant GEC3 protein was expressed in E. coli, purified and
used to produce polyclonal antibodies (PAb). In an immunofluorescence
assay on endometrium, PAb labelled strongly and specifically the luminal
and glandular epithelial cells but not the stromal cells.
It can be thought that gec3 could be involved in the negative circuit
that governs growth suppression in glandular epithelial cells. Immortalized
or cancer cells can he considered as cells that cannot enter reversible
quiescence anymore. Regulation of gee3 expression could be impaired in
immortalized or cancerous cells.

Analysis of human Polo-like kinase 1 (Plkl)

at the kinetochore during mitosis
L. Amaud, E.A. Nigg
Dpt of MolecularBiology. Universityof Geneva. Switzerland

The kinetochore is an essential mitotic structure required for the

accurate segregation of chromosomes during cell division. Interest in
the kinetochore has recently been revived due to its described role in
the mitotic checkpoint. We have previously shown that human Plkl, a
protein kinase necessary for centrosome maturation and bipolar spindle
formation, localises to the kinetochores from prometaphase to late
telophase. To determine the domain(s) of Plkl responsible for its
kinetochore targetting, we have tagged different deletion mutants with
Green Fluorescent Protein (GFP) and expressed them in human cells.
This has enabled us to localise the kinetoehore targetting domain in the
C-terminal non-catalytic domain ofPlkl. To further analyse Plkl at the
kinetochore, we performed an interaction screening of an human eDNA
expression library with its C-terminal non-catalytic domain. One of the
interactors isolated encodes the dual specificity protein kinase TTK, a
putative human homologue of the budding yeast MPSI which is
implicated in Spindle Pole Body (SPB) duplication and the mitotic
checkpoint. TTK is able to phosphorylate P ~ I in vitro and by GFP
tagging, we have shown that, similar to Plkl, TTK localises to the
kinetochores in mitosis. We are pursuing the study of the Plkl/TTK
interaction but our preliminary data suggest that Plkl is not only
required for proper bipolar spindle formation but is also involved in the
mitotic checkpoint pathway.

[1 ] Coppock et al., Cell Growth and Differentiation, 4, (1993) 483.

[2] Jouvenot et aL, Cell Mol. Biol., 36 (1990) 421

(Mo13.1/276) Cell cycle regulated expression of ribonucleotide reduetase

(RNR) genes in synchronized BY2 tobacco cell suspension.
M.E. Chabout~, B. Clement, N. Chaubet, G. Philipps.
IBMP/CNRS, Universit~ Louis Pasteur, 12 rue du G~n&al Zimmer ,
67084 Strasbourg Cedex, France

Eukaryotic ribonucleotide reductase (RNR), the enzyme involved in the

synthesis of the dNTPs, consists of two subunits R1 and R2, whose
activities and gene regulation are differentially regulated during the cell
cycle [1]. We have isolated and characterized three eDNA clones from a
tobacco S-phase library, two encoding the large R1 subunit, the first RNRI
genes cloned in plants, and one encoding the small R2 subunit [2]. RNR2
is encoded by a single-copy gene whereas RNRI genes belong to a small
multigene family. Complete gene sequences were obtained by genomic
walking PCR and IPCR. RNR2 gene contains no intron compared to the
two RNR1 genes containing several introns. The level of RNR transcripts
is cell-cycle regulated in synchronized BY2 tobacco cell suspension,
showing a maximum in S phase. In mid-S phase, RNR2 mRNA level is
higher than RNR1 mRNA level. In order to study more accurately the cell
cycle regulation of RNR gene expression, RNR promoters were studied.
Data base search analysis of RNR promoter sequences revealed the
existence of E2F-like motifs [3]. Specific interactions of nuclear proteins
with these motifs were observed in bandshift as well as in vivo
footprinting experiments. To check the functionality of these motifs, and
their putative role in the cell cycle regulation of RNR genes, constructs
were established by fusing the different promoters, with wild type or
mutated E2F motifs, to the luciferase reporter gene and transgenic BY2
cell lines were generated. The expression patterns of these lines were
investigated in exponential-growing cells, stationary ceils and in different
phases of the cell cycle. The obtained results will be discussed.
[1] Elledge S.J. et al., Bioessays, 15, (1993) 333.
[2] Chabout~ et al., Plant Mol Biol, 38, (1998) 797.
[3] K. Helin, Curr Opin Cell Biol, 8, (1998) 28

(Mo13.1/277)

Gene induction at the G1/S transition of the plant cell cycle

N. Chaubet-Gigot, B. Combettes, F. Schmitt, M. Fl~net,
W.H. Shen, M.E. Chabout6, G. Philipps
lnstitut de Biologie Mol~culaire des Plantes/CNRS-Universit~ Louis Pasteur,
12, rue du G~n~ral Zimmer, 67084 Strasbourg Cedex, France

The major interest of our group is to elucidate the molecular mechanisms

involved in the induction of gene expression at the G1/S transition of the plant
cell cycle. We are working on two alternative model-genes, the genes
encoding the histones necessary for packaging the S phase-replicated DNA
into chromatin, and those encoding the two subunits of ribonucleotide
reductase (RNR), the enzyme involved in the synthesis of the dNTPs
necessary for DNA replication. Both genes are induced at the G1/S transition
in synchronised tobacco cells but RNR genes are induced slightly before
histone genes, which suggests that the two genes are induced by different
regulatory mechanisms. Indeed, cis-clements identified by structural and
functional approaches are not the same in these promoters. The persistence of
protein footprints on the historic promoter cis-elements throughout the cell
cycle indicated that transcriptional activation is mediated by modification of
prebound proteins rather than by the direct binding of GI/S transition-specific
proteins. Band-shift experiments suggested that this modification may result
from a phosphorylation event.
Therefore, we investigated the cyclins capable of activating cyclin-dependent
kinases (cdk) at the G1/S transition. Three distinct subtypes of cyclins A were
identified whose genes are induced sequentially from late G1 to mid S phase.
Based on these results, a model is proposed for the sequenUai induction of
genes during the G I/S transition : whereas the earliest set of genes might be
induced by a mechanism involving an E2F-like factor, the second set might be
activated by phosphorylation of transcription factors by a kinase complex
involving the earliest-expressed cyclin. Antibodies were raised against the
cyclins A and used to analyse cyclin expression at the protein level and to
check for the presence of cyclins in transcriptional complexes. Surprisingly,
the amount of the three cyclins remained at a high level throughout the cell
cycle. The massive degradation of cyclin A which occurs in animal cells at
early mitosis due to the ubiquitin/proteasome destruction pathway was not
observed. This result was confirmed by immunolocalisation experiments. The
three cyclins were found in the mitotic spindle zone and in amyloplasts. In
addition, the earliest-transcribed cyclin was predominant in the nucleus, in
keeping with our hypothesis that it might be involved in transcription
activation. In order to investigate the potential participation of this cyclin to
transcriptional complexes, a method was developped to characterize large
nuclear protein complexes on native gels. Complexes of similar molecular
weights were revealed with antibodies against this cyclin or with a histone
promoter fragment. To further characterize these complexes, a method is
being developed based on immuno- or DNA-affinity columns.

s238

Abstracts FEBS'99

(M0/3.1/278)

Identification of a putative cyclin-selective ubiquitin

conjugating enzyme (E2-C) from Arabidopsis thaliana
M-C. Criqui, Y. Parmentier, A. Derevier, A. Duff, J. Fleck,
P. Genschik

(Mo/3.1/279)

IBMP/CNRS, 12, rue du Gdndral Zimmer, 67084 Strasbourg, France

Destruction of mitotic cyclins by ubiquitin/26S-dependent proteolysis is

essential for exit from mitosis and entry into G1 of the next cell cycle.
Recently, genetical and biochemical studies led to the identification of a
novel cyclin-selective ubiquitin conjugating enzyme (E2-C) and a 20S
particle termed anaphase-promoting complex (APC), both necessary for the
ubiquitination of mitotic cyclins and other mitotic proteins and their
subsequent degradation by the 26S proteasome complex [1]. These
substrates are targeted to this machinery by a nine-amino-acid motif known
as the destruction box (D-box) [2].
Here we report the identification in the EST sequence database of a putative
plant E2-C homolog from Arabidopsis thaliana, E2-C_At. It shares 69%
amino acid similarity with clam E2-C and it contains the N-terminal
extension that distinguishes this class of ubiquitin-conjugating enzymes.
Interestingly, the expression pattern in A r a b i d o p s i s is correlated to tissues
with high mitotic activity. Moreover, we show in aphidicolin synchronized
TBY-2 cell suspension cultures that the expression of E2-C_At is cell cycledependent. The accumulation pattern of E2-C At mRNAs is similar to that
of B-type cyclin (Nicta;CycB 1;1). This indicates that E2-C itself might be
subjected to cell cycle-dependent regulation. We are currently investigating
if E2-C_At is the functional plant homolog of this class of ubiquitinconjugating enzymes.
[1] F.M. Townsley and J.V. Ruderman (1998) Trends Cell Biol. 8, 238-244
[2] P. Genschik et aL, (1998) Plant Cell 10, 2063-2076

(Mo/3.1/280)

Rottlerin, a Specific Inhibitor of Protein Kinase C& Blocks

Contact-Dependent Inhibition of Growth
C. Dietrich, I. Heit, F. Oesch, R. Wieser
lnstttut fur Toxikologie, Universitat Mamz, 55131 Mainz, Germany

Growth of non-transformed ceils is regulated in vitro and in vivo by cellcell interactions. In vitro, cells are arrested in Gl-phase when they have
reached confluency (contact-inhibition of growth) which is reflected by
decreased phosphorylation of the retinoblastoma gene product (pRB), the
gate keeper of G1/S transition. We have previously shown that in human
diploid fibroblasts (FHI09 cells) the tumorsuppressor p16 and the cdkinhibitory protein p27 are involved in contact-dependent inhibition of
growth resulting in reduced kinase activities of cdk4 and cdk2,
respectively, and thus decreased pRB phosphorylation [1,2]. In order to
elucidate further the signal transduction pathway we addressed the
question if protein kinase C is involved in contact-dependent inhibition
of growth. There is evidence that PKC is not only involved in mitogenic
processes but also in negative growth control and differentiation. Cellcell contacts were imitated by the addition of glutaraldehyde-fixed cells
[3]. Down-regulation of protein kinase C by preincubation with TPA
(100 nM) for 48 h leads to decreased contact-inhibition by 37 % whereas
the inactive isomer 4~x-phorbol-didecanoate does not reduce contactinhibition significantly. Western blot analysis revealed that only the two
isoforrns ~t and 6 are down-regulated in response to TPA-pretreatment.
In the presence of Rottlerin (I p,M), a specific inhibitor of PKC &
contact-inhibition is totally abolished. In contrast, the a-specific inhibitor
G66976 (100nM) does not affect contact-inhibition. The results indicate
that PKC 6 which has recently been considered to be a tumor suppressor
might be involved in contact-dependent inhibition of growth in FH109
cells.
1. Dietrich et al., Oncogene, 15 (1997), 2743
2. Wieser et al., Oncogene, 18 (1999), 277
3. Wieser et al., J Cell Biol, 103 (1986), 361

Cell cycle progression in nematode-induced feeding sites

J. de Almeida Engler ~, S. Burssens~, M. Van Montagu~, G.
Engler b, D. Inz6b, and G. Gheysen~
" Departement Plantengenetica. and ~ Laboraroire Assocle de I'INRA. VIB.
Untversitett Gent. K L Ledegand~straat 35. B 9000 Gent, Belgtum

We have used Arabidopsis thaliana as a model host to study cell cycle

progression in nematode-induced feeding sites. The simplicity of its anatomy
combined with the translucent character of the roots allowed us to perform
observations in whole mounts to study the ontogeny of the feeding cells
induced by two sedentary parasitic nematodes. Cell cycle markers were used
to monitor how nematode-infected sites develop and what the initial signals are
that trigger certain root cells to evolve into giant cells embedded in a gall, or
into a syncytium. Tritiated thymidine incorporation experiments were applied
to monitor DNA synthesis, which was used as a marker for the S phase of the
cell cycle. Extending our analysis, the expression pattern of two
cyclin-dependent kinases (CDKs) and two mitotic cyclins were examined. The
CvcB1;1 gene was used as a marker tor the G2 phase, the Cdc2bAt and
CycA2;1 for the S and G2 phases, whereas the Cdc2aAt gene was used as a
marker for all cell cycle phases. Nuclei stained with 4',6-diamidino-2-phenylindole were used to look to chromocenters (during interphase) and for mitotic
figures, Nematode-infected seedlings were also treated with two cell cycle
inhibitors hydroxyurea and oryzalin to investigate the relevance of DNA
synthesis and mitosis on the development of these feeding sites. All
i experiments were carried out on Arabidopsis roots infected with the root-knot
nematode Meloidogyne incognita and the cyst nematode Heterodera schachtii
i at different time points after inoculation. A strong correlation was observed
i between initiation of feeding cells by both root-knot and cyst nematodes and
the induced DNA synthes~s, and expression of the cell cycle genes analyzed.
Our results show that endoreduplication and mitotic cycles play a role in this
root cell redifferentiation process caused by nematode infection.

(Mo/3.1/281)

Poly (ADP-ribosylation) in regulation of cell proliferation

M. Ebner, H. Berghammer, R. Marksteiner, B. Auer
Institute of Biochemistry, University oflnnsbruck, Peter Mayrstr la,
A-6020 lnnsbruek, Austria

Polymerizing NAD+:protein(ADP-ribosyl)-transferase (ADPRT) is found in

the nuclei of almost all eukaryotic cells. Several lines of investigation point
to an important biological role of poly(ADP-ribosyl)ation. To elucidate the
function of this enzyme in vivo, the gene was inactivated in mice via
homologous recombination. Surprisingly, ADPRT null mutants were viable
and developed normally [I]. Cell lines from ADPRT deficient mice were
established by a 3T3 protocol and characterized. ADPRT negative fibroblasts
grow significantly slower than their positive counterparts and show a 3 -4
fold higher frequency of sister chromatide exchange. Constitutive expression
of human ADPRT can rescue these phenotypes of the ADPRT knock out
ceils.
The mild phenotype observed in ADPRT(-/-) mice could be due to
genes which are not silenced or to normally repressed genes which are
induced during development to compensate for the ADPRT deficiency.
Therefore differential cDNA libraries were constructed by PCR mediated
subtractive hybridisation of cDNAs from positve and negative 3T3 cells. We
could identify two genes, which are overexpressed in ADPRT(-/-) cells as
compared to wt fibroblasts and rescued cell lines with ectopic ADPRT
expression. These genes code for the Cellular Retinoic Acid Binding Protein
1 (CRABP I) and for the Interleukin-I Receptor homologue TI (ST2). Both
genes are normally expressed during the embryonic development and
involved in the regulation of proliferation and differentiation. This results
indicate that the missing influence of ADPRT in regulation of growth and
differentiation could be replaced by other proteins such CRABP or T1.
Because of the existence of such compensatory genes the lack of ADPRT
presumably causes only mild impairment in cellular metabolism.
[ 1] Wang et al., Genes Dev., 9 (1995) 509

Abstracts FEBS'99

(Mo/3.1/282)

Cloning and Characterization of the mouse p130 promoter.

Regulation of its expression during differentiation.
L, Fajas, N Servant, J Polanowska,L. Le Cam, E Fabbrizio,C Sardet

s239

(Mo/3.1/283)

lnstitut de (;~n~tique Mol~culaire, UA/IR5535/CNRS. Montpellier, l;)~ance

. . . . Retinoblastoma family prolem,~re .major rr~l~tors of cell cycle

sharing the ability to negatively regulate the E2F mediated transcription.
Whereas pRb and p107 exert its function in the G1/S transition,
pRb2/p130 is mainly involved in mantaining and may be responsible for
the quiescent state of the cells. Exit of the cell cycle is one of the required
events in most of differentiation processes. C2C12 myoblast cell line
represents a good model for muscle cell differentiation. It is characterized
by a coordinate cell cycle withdrawal followed by the expression of
dift~rent~ation-specific genes. Consistent with the withdrawal of cell cycle
in the early stages of differentiation, an important increase of the p130
protein has been observed. In order to study whether this increase was the
consequence of a transcriptional up-regulation of the p130 gene we have
analysed by RNase protection assay the mRNA expression of p130 during
the C2C12 differentiation process. Levels of p130 mRNA were
significantly elevated after 48 hours of thfferentiation. To better analyse
the transcriptional regulation of p130 we have cloned and characterized the
murine p130 promoter. Stable cell-lines expressing the lueiferase reporter
gene under the control of the p130 promoter were incubated in
dift~rentlating medium and luciferase activity was evaluated observing an
increase of the promoter activity upon differentiation of the cells. In w v o
footprint analysis of the p130 promoter showed a diferentially ocupated
sequence when the cells were proliferating relative to cells in quiescence
suggesting the presence of a repressor complex in the p130 promoter in
proliferating cells Further analysis of the sequence revealed that the
protected sequence corresponded to a putative CCRE/CHR element which
has been demonstrated to negatively regulate the expression of several
genes implicated in cell cycle such :as the cyclin A, cdc25C, b-myb, or cdc2.
The differential ocupation of the site was further demonstrated by EMSA
assays using nuclear extracts of' C2C12 cells at different stages of
differentiation and the CCRE/CHR element as a probe. In summary, we
show that the transcriptional actwity of the mouse p130 promoter ~s
sul~lected to a strong repre.ss~on m proliferating cells and that lhe
repression is released upon induction of differentiation of C2C 12 cells.

(Mo/3.1/284)

Effects of C-terminal of N H E - I overexpression on

cell proliferation, and Na/H exchanger activity.
H. Godan; N. Touret; B. Culler; I Rubera; P. Poujeol, and L. Counillon
UMR 6548 CNRS, UNSA, Pare Valrose, 06108 Nice Cedex 2, Fratu'e

The Na/H+ exchanger NHE-I isoform is structurally divided into two

distinct domains : an N-terminal domain responsible for the ionic
transport, and a C-terminal domain which contains binding sites for
regulatory proteins involved in the signalling cascades activated by
growth factors. Therefore, overexpressing C-terminal of NHE-I may
titrate these proteins, and inhibit cell proliferation as well as NHE-1
activation by growth factors.
To test this hypothesis,we first used a fusion protein GFP-C-terminal
of NHE- 1, which allows the detection of cells expressing the protein of
interest by the fluorescence of GFP after excitation at 490 nm. Our
results show that the transient overexpression of this protein into CCL39
cell line has a high cytotoxic effect, and inhibits NHE-1 activation.
Moreover, additionnal experiments performed on DC1 cell line
transfected with the pECE-GFP-NHE-1 construction did not develop a
swelling-activated conductance controlled by growth factor.
Due to the high cytotoxicity of the C-terminal of NHE-1, and to get
more quantitative results, we used a Tet off inducible gene expression
system. We stably transfected CCL39 cell line expressing a tTa with a
bicistronic vector containing the IRES-SEAP sequence and encoding the
gene of interest under the control of a tet responsive element. The
expression of C-terminal of NHE-1 was quantified by SEAP activity
measurement, and fluorescence measurement. Our results indicate that in
these conditions, overexpressing the C-terminal of NHE-1 inhibits
significantly cell proliferation. Under the same conditions, the inhibitory
effects can not be observed when GFP alone is overexpressed as a
control.
The use of a FACS analysis will soon sort out which part of the cell
cycle is affected by the C-terminal of NHE-1 overexpression.
In conclusion, these data suggest, that overexpressing the C-terminal
of NHE-I titrates the regulatory proteins involved in the growth factors
signalling cascades.

Cell-cycle regulation in embryonic stem cell derived

cardiomyoeytes
A.C. Fijnvandraat, K.H. Lekanne Deprez, A.F.M. Moorman
Department of Anatomy and Embryology, University of Amsterdam,
Meibergdreef15, 1105,4ZAmsterdam, The Netherlands.

The inability of adult cardiomyocytes to proliferate prevents

proper regeneration of the myocardium after severe injury,
irreversibly leading to heart failure or cardiac death. Despite
research about cardiomyocyte cell-cycle regulation, not much is
known about the mechanism by which cardiomyocytes
withdraw from the cell-cycle. This is mainly due to 1) lack of
cell lines that faithfully recapitulate cell proliferation and cellcycle withdrawal, 2) no or limited proliferative potential of
primary cardiomyocyte cultures, 3) the inefficient transfection of
primary cardiomyocyte cultures and 4) difficulty to measure
cardiomyocyte proliferation in the animal. As a tool to
investigate cell-cycle withdrawal during cardiogenesis, we
investigated the applicability of a test-system based on
embryonic stem-cell derived cardiomyocytes, displaying great in
w v o resemblance. One potential pitfall of this procedure is the
cellular complexity upon ES cell differentiation. Therefore, we
are currently investigating if we can increase the cardiogenic
potential of ES cells by testing different ES cell lines and growth
conditions. To scale up the resulting cell population towards a
100% cardiomyocyte content, we want to make use of a genetic
selection procedure. To this end we want to make genetically
modified ES cells by placing the puromycin resistance gene (pac
gene) under direction of the myosin light chain 2V ( M L C 2 V )
promoter. This cell line will be used to study cardiomyocyte
cell-cycle regulation with or without the addition of candidate
factors by investigating the expression and activity of cell-cycle
regulators, FACS analysis and apoptosis. This might indicate
factors involved in cardiac cell-cycle withdrawal.

(Mo/3.1/285)

A unique pool of cdc2 phosphorylated human

nucleolin is associated with mitotic chromosomes.
D. Goldgabcr a, A. Dranovsky ~, Y. Takahashi ~, , P. Davies ~
aState Universtry of New York, Stony Brook, New York, 11794
hAlbert Einstein Medical Sch, 1300 Morris Park Ave, Bronx, NY 10461

Nucleolin is a major multifunctional phosphoprotein that participates in a

number of cellular processes and is phosphorylated by cell cycle cdc2/cyclin
B kinase in mitosis. Using monoclonal antibody TG3, we identified a
unique pool of nucleolin in human mitotic cells. TG3 immunoreactivity was
dependent on posphorylation of nucleolin by cdc2 kinase in vitro and in
vivo. In vitro phosphorylation by cdc2/cyclin of nucleolin, isolated from
cells in interphase, resulted in the generation of TG3 immunoreactive
epitope. Using laser confocal microscopy, we followed TG3
immunoreactive nucleolin in mitosis. We observed temporal and spatial
dynamic interaction of TG3 immunoreactive nucleolin with mitotic
chromosomes was observed in mitotic cells. The unique pool of TG3
immunoreactivity appeared abruptly in prophase, dissipated throughout the
mitotic cytosol after disintegration of the nuclear membrane, was associated
with chromosomes in prometaphase and metaphase, and gradually
disappeared during separation of chromosomes in later stages of mitosis.
A recombinant fusion protein, GFP-nucleolin, was created and transiently
expressed in COS-7 cells. The unique TG3 reactive phosphoepitope that
characterized this pool of nucleolin was identified using site-directed
mutagenesis. Serine or threonine residues in cdc2 consensus sequences
were substituted to alanine and mutated recombinant GFP-nucleolin was
transiently expressed in COS-7 cells. After nocodazole treatment, extracts
of transfected cells were analyzed by Western blotting using TG3 antibody.
Only one mutant failed to react with TG3. It contained alanine instead of
serine at position 67 suggesting that phosphorylated Ser67 is the key amino
acid forming TG3 epitope. Interestingly, there is no cdc2 consensus
sequence at this s~te in rat, mouse, and hamster nucleolin. Indeed, Western
blot analysis of mitotic nucleolin from these species showed no
immunoreactivity with TG3. Laser confocal microscopy of live transfected
COS-7 cells showed that GFP-nucleolin behaved like endogenous
nucleolin. The GFP-nucleolin chimera was detected in nuclei of transfected
cells and was concentrated in nucleoli. In mitosis GFP-nucleolin also
behaved like endogenous nucleolin and was found both in nucleoplasm and
in association with chromosomes.

s240

(Mo/3.1/286)

Abstracts FEBS'99

The mammalian protein NUCKS is phosphorylated on

at least one serine in the C-terminal end by CK-2 and
on Ser 182 by CDKI/Cyelin B in vitro.
K.Grundt,B.Wanvik.J H.No~m.A.C.Ostvold
NeurochemicalLaboratory,UniversityofOslo,Nor,ray

The nuclear phosphoprotein NUCKS (previously designated P1), was

recently cloned from human and rat libraries. The primary structure
revealed seven "minimum requiremenf' phosphorylation sites for cyclin
dependent kinases S/TP and nine consensus phosphorylationsites for
CK-2 (S/l" XXED).
NUCKS is indeed a highly phosphorylated protein in vivo, and an
excellent substrate for CK-2, CDK-1, CDK-2, CDK-4 and CDK-6 in
vitro. Furthermore, the in vivo phosphorylation pattern of NUCKS is
cell cycle dependent.
In order to identify the phosphorylation sites for CDK-1 and CK-2,
three peptides corresponding to some of the putative phosphorylation
sites where synthesized. They where utilized in kinetic experiments with
purified CDK1/Cyclin B and CK-2. One of the peptides corresponding
to a region close to a GRP (DNA binding) motif, is phosphorylated by
CDK1/Cyclin B with a Km of 25.6 gM, while a peptide corresponding
to 18 amino acids in the C-terminal end is phosphorylated by CK-2 with
a Km of 8.5 laM.
These results indicate that Ser-182 is phosphorylated by CDK-1 in the
G2/M boundary of the cell cycle. Immunofluorescence experiments
with anti P1 antibodies show that P1 is located in the nuclei in the
interphase of the cell cycle but distributed in the cytoplasm in mitotic
cells. This might be due to phosphorylation of Ser-182 by CDKl/cyclin
B. Previous results indicate that one or more serines are constitutively
phosphorylated by CK-2. The implication of the phosphorylation by
CK-2 is not known.

(Mo/3.1/288)

Cloning and characterization of a sea urchin CHED

Kinase homolog.. F Marqu/~s,J.L. Moreau, G. Peaucellier,
J.C. Lozano, P. Schatt, M.N. Simon, A. Picard and
A.M. Genevi/~reLaboratoireArago, Banyuls-sur-mer, France

The CHED (cholinesterase-related cell division controller) protein is

one of the less well known member of the cyclin dependent family of
kinases (CDKs). The human CHED eDNA was cloned by serendipity while
screening for cholinesterase-related proteins involved in hematopoiesis [1].
Although antisense oligonucleotide inhibition experiments have implicated
the CHED protein in the development of specific blood cell lineages, the
underlying mechanism remains elusive and the CHED gene product was not
characterized in this work.
We have unexpectedly cloned a CHED homolog from sea urchin
embryos in a PCR-based screen for novel CDKs involved in cell cycle
control during early embryogenesis. Both human and sea urchin CHED
proteins display an unusual sequence, being longer than the other known
CDKs at their N- and C-termini.
We have shown that CHED mRNA is present in unfertilized sea
urchin eggs as well as in two-cell stage embryos and larvae, strongly
suggesting that CHED is not only involved in late differenciation but plays a
more general role in cell cycle control and/or development as well.
Furthermore, immunofluorescence experiments using an antibody raised
against a characteristic motif inside the kinase domain and a second one
against the entire recombinant kinase domain have allowed us to visualize
the sea urchin CHED protein. The study of the regulation of CHED
expression and kinase activity during the cell cycle and development is in
progress and will provide crucial insights in the function of this original
molecule.
[1] Lapidot-Lifson Y. et al., PNAS USA, 89, (1992) 579

(Mo/3.1/287) Serial analysis of enriched-expressed sequence tags to identify

differentially expressed cDNAs in fetal and adult heart
R Lekanne Deprez, M van den HolT, M Lombardi, M Luijermk, M
Markman, A Buffing; W Lamers, A Moorman.Departmentof Anatomy
& Embryology, Universityof Amsterdam, Meibergdreef15, The Netherland

In contrast to fetal cardiomyocytes, adult cardiomyocytes are not

able to divide anymore. Therefore, heart diseases (e.g. myocard
infarction) that result in cardiomyocyte death will lead eventually
to heart failure. The controlled induction of cell division in adult
cardiomyocytes could be beneficial for cardiac performance.
However, this requires knowledge about cell-cycle regulation in
these cells, which is at present limited. To be able to identify gene
products involved in cell-cycle regulation we want to compare
gene expression between actively dividing cardiomyocytes (fetal)
and non-dividing cardiomyocytes (adult) using a differential
expression screen. For this purpose we are currently developing
the Serial Analysis of Enriched-Expressed Sequence Tags (SAEEST) method. SAE-EST uses a PCR based subtraction followed
by a fast identification of enriched cDNAs by including a serial
analysis of 16 bp expressed sequence tags. Each tag will be
derived from the 3'end of an enriched cDNA and contains
sufficient information for unique identification.

(Mo/3.1/289)

REQUIREMEIqT OF THE BUDDING YEAST MPSI/RPKI

PROTEIN KINASE FOR SPORE MORPHOGENESIS
A. Camasses, F. De Fraipont & RP. Martin
CNRS-UPR 9005, IBMC, 15 me R. Descartes, 67084 StrasbottrgFrance

In the budding yeast Saccharomyces cerevisiae, centrosomal function is

provided by the spindle pole body (SPB), a trilaminar structure embedded in
the nuclear envelope which nucleates microtubule arrays in the nucleus and
cytoplasm. The MPSI/RPK1 gene encodes an essential dual-specificity
protein kinase [1,2] which is required for SPB duplication during the G1
phase of the cell cycle [3] and for a checkpoint which is activated to arrest
progression through mitosis when the integrity of the mitotic spindle has
been compromised [4].
We show here that this kinase has also a developmental role during
meiosis-sporulation. MPS1/RPK1 mRNA levels are sharply increased in the
middle period of the sporulation program, with maximal accumulation when
cells are completing meiosis II. A homozygous rpkl temperature-sensitive
diploid, although defective in SPB duplication during vegetative growth, can
progress through both meiotic divisions at the restrictive temperature.
However, the mutant is unable to form mature spores. Electron microscopy
revealed that the mutant asei are defective in organizing proper spore wall
assembly. MPS1/RPK1 function is required for normal levels and timely
expression of developmentaUy-regulated genes of the sporulation program.
In fact, sporulation-specific genes are both retarded and expressed at lower
levels in rpkl cells and this effect is the most severe for middle-late and late
genes involved in spore wall formation and ascospore maturation.
t. Pooh et al. (1994) Mol. Gen. Genet. 243 : 641
2. Lauz6 et al. (1995) EMBO J. 14 : 1655
3. Winey et al. (1991) J. Cell. Biol. 114 : 745
4. Weiss & Wlney (1996) J. Cell. Biol. 132 : 11 t

Abstracts FEBS'99

(Mo/3.1/290)

s241

Analysis of Rad3 and Chkl checkpoint protein kinases.

Isolation of suppressors of rad3.ts HU sensitivity.
R.G. Martinho, N.J. Bentley, H.D. Lindsay and A.M. Carr
MRC-Cell MutationUnit, Universityof Sussex,Brighton,BNI 9RR, UK

Most eukaryotic ceils respond to both DNA damage and S-phase replication
blocks by arresting cell cycle progression. We are using S pombe as a
model system to identify and order the various components of the DNA
structure-dependent checkpoint pathways. The Chkl protein kinase is
phosphorylated "after DNA damage and is essential for the mitotic arrest
checkpoint. During S-phase, a second protein kinase Cdsl is activated in
response to DNA damage and DNA replication blocks. The response of both
Chkl and Cdsl requires the six "checkpoint Rad" proteins. Cdsl is not
activated in response to DNA damage in non-S-phase cells. We demonstrate
that the phosphorylation of the Chkl protein kinase in response to DNA
damage is cell cycle specific, occurring primarily in late S-phase and G2,
but not during Gl/early S-phase or mitosis. To understand the significance
of the cell cycle specificity of checkpoint responses, we isolated an allele of
tad3 that is temperature-sensitive for function. The Rad3 kinase is essential
for all DNA structure-dependent checkpoints. Characterisation of the rad3ts
allele showed that in response to DNA damage, rad3 is primarily required to
initiate the Chkl dependent mitotic arrest checkpoint, but is not required to
maintain the arrest. Conversely, rad3 is required to both initiate and
maintain mitotic arrest when DNA replication is inhibited. We see a strong
genetic interaction between rad3 and cdsl and biochemical evidence
suggests a physical interaction between Rad3 and Chkl and between Rad3
and Cdsl protein kinases. In order to isolate new genes involved in the DNA
replication checkpoint response a multi-copy suppressor screen of HU
sensitivity of the rad3ts at the semi-permissive temperature was performed,
the results of which wilt be presented.

(Mo/3.1/292)

Evidence for a caspase-cleaved form of p27 ~K',

involved in GI growth arrest.
N. Rocheta, A. Loubat~, L Turchi~, R. Rezzonico~, D. FarahiFar~,
P Aubergerh, B. Rossl~and G. Ponzio'~
,t I N S E R M U364, I~CJF INSERM 96 0 5 Nice, Fraltce

p27[K1PII (p27) is a cyclin dependent kinase inhibitor involved in the

negative regulation of G1 progression in response to a number of
antiproliferative signals. In this study we show that growing mouse
hybridoma (7TD1) and human myeloma (U266) cell lines highly
express p27 but this protein is only slightly upregulated when cells are
arrested, regardless to the phases of the cell cycle. In contrast, the
specific blockade of these cells in early G1 phase reveals the induction
of a protein of 23 kDa (p23) specifically recognized by polyclonal antip27 antibodies raised against the NH2 terminal part of p27 but not by
anti-p21[ CIP1] antibodies. Experiments using caspase inhibitors
strongly suggest that p23 results from the proteolysis of p27 by a
"caspase-3-1ike" protease. This cleavage leads to the cytosolic
sequestration of p23 but does not alter its binding properties to CDK2
and CDK4 kinases. Indeed, p23 associated in vivo with high molecular
weight complexes and coprecipitated with CDK2 and CDK4. We
demonstrate by transfection experiments in SaOS-2 cells that p23
induces a G1 phase growth arrest by inhibition of cyclin/CDK2 activity.
In summary we describe here a caspase-cleaved form of p27, induced
in absence of detectable apoptosis and likely involved in cell cycle
regulation.

(Mo/3.1/291)

Cyelin E expression is regulated by a bipartite repressor

element bound to a novel E2F complex.
J. Polanowska, L. Le Cam, E. Fabbrizio, A. Philips, C. Sardet
lnstBut de GdndtiqueMoldculaire. UMR5535/CNRS,Mon~ellier. France

The transient expression of cyclin E gene in late GI is one of the

critical events that gate the progression of mammalian cells into S phase.
Based on detailed in vivo and in vitro analyses of the cyclin E promoter in
mouse fibroblasts (Swiss 3T3) we present novel evidence that the timing of
its activation is controlled via Cyclin E Repressor Module (CERM), a
regulatory element located immediately downstream the transcription start
site. CERM is a bipartite element composed of a variant E2F binding site
and the upstream AT-rich sequence. Their coorporation during G0/GI
causes the delay of cyclin E transcription until late G1 CERM binds a
cellular complex, termed CERC, which gradually disappears upon
progresssion through G0/G 1. This event correlates kinetically with the loss
of occupation of the CERM module in vivo and with activation of cyclin E
transcription. CERC is a novel high molecular complex containing E2F4/
DPI, a pocket protein and at least one additional unidentified protein.
Analyses performed in nullyzygous for members ofpRB family cells show
that pRB, p107 and p130 are interchangable in CERC complex and that
CERM-dependent repression is functional Also, previously described
deregulated expression ofcyclin E in pRb-deficient cells corresponds to the
premature dissociation of CERC from CERM in mid G1. Moreover, in
contrast to the classical E2F complexes which do not vary in exponentially
growing cells, the analysis of the promoter activity in homogenous
elutriated populations of human hematopoetic cells (K562) reveals the
implication of CERM/CERC in active repression of the cyclin E promoter
during each G1 phase.
Altogether, we identify a new regulatory element that controls
repression of Gl-specific genes and we unambiguously demonstrate that
the mentioned repressor complex represents a novel E2F complex that
differs in size, binding behavior and role from previously described E2F
complexes.

(Mo/3.1/293)

Effect of MAPK pathway inhibitors on the progression of

mitotic cell cycle in Xenopus
A. A. Tokmakov, K-I. Sato, Y. Fukami
Laboratory of Molecular Biology, BiosignalResearch Center, Kobe
University, Nada, Kobe657-8501, Japan

Mitogen-activated protein kinase (MAPK) is known to be activated

during mitosis in several mammalian cell lines and cycling Xenopus egg
extracts. It is involved in the control of mitotic spindle formation in both
Xenopus cell extracts and a Xenopus tadpole cell line. Recently, it has been
demonstrated that MAPK is activated downstream of Cdc2 kinase during
mitosis in cycling Xenopus egg extracts and that its activity is essential for
maintaining the mitotic state after Cdc2 activation [1]. However, there is
little or conflicting evidence whether MAPK is activated in the mitotic M
phase of cleaving Xenopus embryos and whether it is indispensable for the
normal cleavage of the embryos.
In the present study, we investigated the effect of two recently
described inhibitors of MAPK pathway, PD098059 [2] and a synthetic
peptide corresponding to the activation segment of Xenopus MAPK, termed
IDA MAPK peptide [3], on the progression of mitotic cycle in cycling
Xenopus egg extracts and in Xenopus eggs after fertilization. Both
PD098059 and IDA MAPK peptide at 200 pM were found to effectively
inhibit MAPK pathway in cytostatic factor-arrested and cycling Xenopus egg
extracts with little effect on the activity of Cdc2 kinase. The duration of
mitosis in both PD098059 and IDA MAPK peptide-inhibited extracts was
noticeably shorter than in non-treated control extracts, as judged by nuclear
morphology of added demembranated sperm, indicating that the spindle
assembly checkpoint is abolished in the presence of the agents.
Microinjection of the inhibitor peptide into Xenopus eggs at 30-70 rain after
insemination inhibited the f'trst cell division and blocked the following early
development of embryos. Biochemical analysis showed that the peptide
prevented transient MAPK reactivation on the onset of the first division. On
the other hand, normal cleavage was observed when eggs were treated with
PD098059 after insemination. The inhibitory effect of PD098059 on the
activity of MAPK pathway in the unfertilized eggs could be registered only
after several hours long incubation with the drug. Therefore, poor
permeability of PD098059 can account for the lack of its effect on the
cleavage of Xenopus embryos.
References:
[1] T. M. Guadagno and J. E. Ferrell Jr., Science 282, (1998) 1312.
[2] D. R. Alessi et al., J. Biol. Chem. 270, (1995) 27489.
[3] A. A. Tokmakov et al., Biochem. Biophys, Res. Commun. 252, (1998)
214.

s242

(Mo/3.1/294)

Abstracts FEBS'99

Identification and characterization ofgecl :

a novel early estrogen-regulated gene
S. Vernier, A Fraichard, M. Sallot, J Radom, M Jouvenot
Lahora~cnre de 13tochlmte, Btologte31oldculazre, 25030Be~an~on, ~'anc'e

Estrogens control cell proliferation by regulating gene expression in target

tissues Estrogen-regulated genes have been classified into three groups
primary response genes, secondary response genes and delayed primary
response genes [1]. In an effort to identify the genes that may play a role in
the early action of estrogen, a cDNA library has been constructed from
guinea-pig endometrial glandular epithelial cells (GEC) stimulated for 2 h
w~th estra&ol-17[3 (Ez) plus cycloheximide (Chx) By differential
hybridization, an early estrogen-induced gene, calIed gecl, has been isolated
as a 833 pb 3' cDNA A two fold increase in the 1 8 kb gecl mRNA level
has been observed at 120 rain after E2 treatement [2]
A gecl-specific probe was used to investigate not only expression of
guinea-pig gecl but also expression ofgecl human counterpart. The I 8 kb
gecl mRNA was detected preferentially in endometrium, lung, liver and
weakly m the other guinea-pig tissues examined In most of the human
tissues, lung excepted, the gee1 probe revealed one transcript of I 9 kb,
The screening of a ), FIX guinea-pig genornic DNA library was performed
using a digoxlgenin-labeled gecl probe It allowed isolation of a 20 kb
clone A 3190 pb fragment, hybridizing with the cDNA probe, was
subcloned, entirely sequenced and analysed by "All in One seq Analyser"
software This gene did not show similarity with well characterized genes
The sequence analysis suggested that the gecl gene could be organized m 2
exons and I intron Gecl would encode a 16 5 kDa protein with a leucine
zipper domain suggesting DNA/protem interact)on Studies are now in
progress to characterize the GECI protein and to identify the human gecl
counterpart
[I] Dean D M and Sanders M M , Mol Endocrinol 10 (1996) 1489
[2] Pellerin 1 e t a [ , MoI Cell Endocrinol 90 (1993) 401

Abstracts

FEBS'99

s243

5.3 Architecture, design and evolution of RNA

(Mo/5.3/295)

Synthetic ribonucleases as probes for investigations of

RNA structure

(Mo/5.3/296)

N.Beloglazova, E.Kostenko, V.Sil'nikov, M.Zenkova,

V.Vlassov

UPR 9002 - CNRS IBM(', 67084 St~a~hol.;~,. [-'t(dll(c

Institute o f Bioorgamc Chemistry, 630090, Novostblrsk, Russta

Mimicks of the ribonuclease A active center have been designed.

Bicationic diazabicyclo[2.2.2.]octan was conjugated to constructs bearing
imidazole residues and carboxylic groups. RNAse mimicks (compounds
DI, D2 and D3) have in their catalytic domain histamine, methyl ester of
h~stidine and histidine, respectively. Hydrolytic activity of the conjugates
was investigated in experiments with yeast tRNA ph~ and in vitro transcript
of influenza virus M2 RNA in physiological conditions (50 mM imidazole
buffer, pH 7,0; 0,2 M KCI, 0,1 mM EDTA, 100 gg/ml RNA-carrier) at
37C. It was found that the compounds display high structure- and
sequence specificity. They cleave RNA primarily at Py-Pu sequences
within single-stranded regions of RNA structure and regaons with internal
loops and mismatches. The rates of phosphodiester bond hydrolysis by DI
- D3 in different sequences decrease in the order CA>UA>>CG. Results of
probing of the structure of different RNAs with chemical nucleases D I D3 are in accordance with the data of the probing with established
structure probes. Phosphodiester bonds m all CA and UA sequences were
cleaved in tRNA Ph~ incubated with constructs D1 - D3 for 18 h at 3T'C in
semi-denaturing conditions (0,5 p_M RNA, 0,1 mM D1 - D3, no
magnesium). In the case of M2 RNA, 2 h incubation with the conjugates
resulted in 80% cleavage of CA motifs (incubation conditions: 0,01 mM
D3, standard imidazole buffer with 5 mM MgCI2, 37C). No cleavage at
UA sites was observed m these conditions.. The designed RNA cleaving
compounds represent a new faintly of tools for investigation of RNA
structure in solution

(Mo/5.31297)

Catalysis of Carbon-Carbon Bond Formation by a Small

Ribozyme
A. J~.schke & B. Seelig
lnstitut fiir Biochemte der Frelen Umversttat Berhn
Thtelallee 63, D-14195 Berhn

The most successful strategy for the selection of new RNA catalysts is direct
selection, which was, however, limited to self-modifying reactions of RNA
until recently [1 ]. To expand this strategy to bimolecular reactions of small
reactants, we have developed direct selection with linkcr-coupled reactants.
We have applied this technology to the catalysis of the Diels-Aldcr reaction
of anthracene and maleimide. Our strategy involved generating a
randomized pool of RNA conjugates, in which anthracene is attached to the
individual pool molecules via a poly(ethylene glycol) (PEG) tether [2]. This
pool was then incubated with biotinylated maleimide to select RNA
molecules that catalyze C-C bond formation between the two reactants. The
newly formed biotinylated Diels-Alder product would then be covalently
attached to the RNA via the linker. After removal of all non-biotinylated
members of the pool by affinity chromatography, active RNA molecules
were amplified and used in iterative selection cycles.
After 10 rounds of selection, the activity of the enriched pool was increased
by four orders of magnitude. After cloning and sequencing. 35 different
RNA species were found that catalyzed the Diels-Alder reaction. Secondary
structure analysis identified a common 38 nucleotide long RNA motif with
only 11 conserved nucleotides. This motif accelerates as a true catalyst the
reaction of a small anthracene-oligonucleotide substrate with biotin
maleimidc about 20.000-fold [3].
We have identified a small RNA motif that can solve the complex task of
tbrming two C-C bonds between substrates that are not templated by basepairing. This is the smallest and fastest RNA-catalyst for carbon-carbon
bond formation reported to date. The catalytic activity described here
reveals the potential of ribozymes for performing chemical transformations,
which is not only essential for recreating prebiotic reaction pathways but
also for the application of RNA catalysts in bioorganic synthesis.
I.
2.
3

Tarasow, T.M el al Nature 1997. 389, 54-57.

Seehg, B. & J~ischke, A. Tetrahedron Left 1997, 38. 7729-7732.
Seelig, B, & Jfischke, A, Chem. BioL 1999, 6, 167-176

Crystal structure of the first m a m m a l i a n tRNA :

tRNA(3Lys)
P. B6nas. G. Bec, G. Keith. B. Ehresmann and P. Dtnnas

We have solved the X-ray crystal structure of natural tRNA(3Lys) at 3.3 ~,

resolution. This is the fir,,t structure determination of a mannnahan tRNA.
tRNA(3Lysl is the natural prnner tor reverse transcription in hmnan
Immunodeficlency virus type I (H1V-I). and ,ts anticodon loop has been
implicated in playing a role in both its placement onto the HIV-I genome and
its interactions with HIV- I reverse transcnptase (RT) [ 1].
In ad&tion, it has been shown by ~,everal groups that the modified bases in the
anticodon loop play an important role in fi'amc~,hiftmg to," correct translation
and in premature termination of tran,dation.
Our structure reveals that the overall lblding of the rihose-phosphate
backbone is similar to that ftmnd for other tRNAs. The cry,,tal packing
com, isb, of a right handed superhelix which involve,, two anticodon loop,, and
two 3' tel'mlnal CCAto t i t a n a new motif of interaction
Ahhough our structure is in the carly stcp, of refinement, it does not confn'm
previous structural work,, [2.31. a,, the anticodon loop i,, apparently lest
dtstorted than suggested with all three anticodon bases a;.ailable for codon
pairing.
I.
2.

3.

(1993).

lsel et al., J. Biol. Chem,, 2 6 8 . 25269

Agriseta/...RNA, 3.420(1997).
Durant et at.. J. Mo/. Biol., 285, I 15 (1999).

(Mo/5.3/298)

[Rp] methanephosphonamidate DNA backbone

promotes triplex formation
B. Nawrot, M. Boczkowska and W.J. Stec
Polish Acad. o f Sci., SienMewicza 112, 90-363 Lddk, Poland

Novel P-chiral internucleotide linkage has been constructed [1]. Diastereomeric

dithymidine methanephosphonamidates (X) were used as building blocks to
prepare chimeric oligomers by means of phosphoramidite automated solid phase
synthesis Here we present hybridization properties of four stereodefined 20mers
of two sequences T6X4T6 and TX9T, of either [R~] or [So] absolute
configuration, with complementary DNA, liaNA or hairpin dsDNA
o
crl,

NH

I--o :~ o

[Rp]

-J-

Methanephosphonamidate linkage
Thermal stability of all duplexes is lower than that of the umodified ones, and
strongly depends on the type and number of introduced intemucleotide bond
modifications However, oligo(deoxyribonucleoside methanephosphonamidate)s
with incorporated [Rv] dinucleoside unit exhibit higher ability for triplex
formation with d(A21C4T20 hairpin oligomer than ones originated from [Sv]
counterpart as well as the parent T20 oligonucleotide. Triplex ~ hairpin
transition is a salt dependent process. We are able to demonstrate higher triplex
stability if ammonium chloride is used instead of sodium chloride. Triplex
formation was confirmed independently by lowering of 220 nm CD absorption
and the presence of slowly migrating bands at native PAGE.
[1] Nawrot B. et al N u c l e i c A c M s R e s 1998, 26, 2650.

s244

Abstracts FEBS'99

(Mo/5,3/299) Positive control of ribozyme activities by the

addition of anti-loop oligonucleotides
Yasuo Komatsu, Shigeko Yamashita and Eiko Ohtsuka
Faculty of Pharmaceutical Sciences. Hokkaido
University, 060-0812, Japan

(Mo/5.3/300)

SEOUENCI~SrECIFIC Rgase Tg4

PrzbwtpgJ, J Lipedm~, A. Edelmsnb, A. Ptzykocska*
Ncnckl Insn'$te of F.Jrt~,rtmentolBiolo~,O2-OgJ Wor~w, Poland,
"INSEP,A Unite 467, C.H.U. Necker, 7J730Parts Cea~x IJ. Fr~Ae
lnslltute of Bloeheraistr~ and Biophysics, 02-106 IVarsw~ Poland

Inactive hammerhead ribozymes containing a loop at the

stem II region have been constructed and activated by the addition of
oligonucleotides complementary to the single stranded region of the
loop. The antidoop oligonucleotide increased the rate of cleavage of a
target RNA by 30-fold. A T-O-methyl anti-loop oligonucleotide
induced higher activity. A hammerhead ribozyme containing
G 10.1 G 11.1 and lacking an active G 10.1 C 11.1 was also activated
by the addition of the complementary strand with a terminal cytidiue.
Thus these anti-loop oligonucleotides are assumed to act as a coenzyme of ribozymes and seem to induce a pseudo-half-knot
structure [ 1 ] within the active ribozyme. This technique has been
applied to control two ribozymes targeting two different RNA
sequences which were described previously using modified hairpin
ribozymes [2, 3], and selective activation was obtained by the
addition of different anti-loop oligonucleotides. This regulation
method should be applicable to controlling the achwt]es of RNA
molecules with a loop structure.
References
[1] Ecker, D. J. e t d . (1992) Science 257,958-961.

The enzymatic p~otein was purified from human colon c~rcinomz T84
cells and was free of other R.Nase activities as was shown by
SDSlpc~yacn/lamide-gel electrophoresis, activity staining, and mapping of
RNase Tg4 cleavage positions |I] This endon~lcleasehas a molecular
weight of 19 kDz and catalyses the cleavage of tKNAs in a dinucl~tidespecificmanner, KNas "I"84 reeo~zes 5'PupU3' sups and atalys~s thehydrolysis of the phosphodiester bonds at the Y side of A or G, and the $'
side of U yielding YA(OH) or G(O[-D and 5' pU terminated products. The
yeast tKNA'~' and yeast tRNAv'* substrales used in our experiments to
establish the sequence speczficity of KNase T84 have clearly established
secondary and terlia~ structure, which therefore ecabled us to study the
structural preference of RNase T84 The Rl~ase T84 bydrolyzed both
single and double-stranded regions of tRNA, hut its activity was facilitated

by denaturation of tKNA Using two naturally occurring yeast tlINATM

species differing by tingle base-pair substitution allowed us to show that
ApU or GpU dyad sequences are absolutely required for the enzymatic
activity of R.Nase T84 and that the ApU dinocIeotide is much better
reeo nized by the en~me than GpU at the sarae longer sequenceconte~_.
Thc~hange of ApU present in tR/qA~n"Minto ApC present in tlLNATM[2] resulted in complete inaccessibility ef this position to the RaNase, while
the change of ApU to GpU resulted in over 10 times lower cleavage rate.

[2] Komatsu, Y. etal. (1997) Biochemistry 36, 9935-9940.

[3] Komatsu, Y. etal. (1997) Bioorg. &Med. Chem. 5, 1063-1069.

(Mo/5.3/301)

Cis-acting elements in mengovirus

RNA replication / G. Shatskaya, T. Dmitrieva, E.
Tolskaya/A. N Belozersky Institute of Physical and Chemical
Biology, Moscow State University, 119899, Moscow, Russia

The 3' non-translated region (3' NTR) of picomaviral genomic RNA is

essential for replication by RNA-dependent RNA polymerase.
Functional and structural dements of mengovirus (representative
ofpicomaviridae family) YNTR were studied. According to RNA
structure probing data the most part of 3'NTR of mengovirus genome
RNA along with the terminal region of coding sequence represents a
large stable hairpin structure with two main loops. To explore the
functional role of such structural organization several clones
containing mutations within YNTR had been obtained by sitedirected mutagenesis, and the infectivity of mutant RNA in COS
cells was examined. The mutations in hairpin loops were obtained to
verify the assumption whether these regions play role in maintaining
the three-dimensional conformation of 3'NTR and binding to
proteins of replication initiation complex. The replacement of
nucleotides in hairpin apex loop did not cause the decrease in mutants
infectivity. Thus the nucleotide sequence of this loop does not play so
crucial role as in an analogous structure in 3' NTR of some other
picornaviruses. The mutation causing hairpin destruction results in
complete loss of RNA infectivity, suggesting that this structure is
necessary for replication and probably contains cis-acting signals for ()RNA replication initiation. Mutants with enlarged hairpin loop or
reduced double-stranded region were of low infectivity and presented
the plaque phenotypes differed from that of wild type viruses.
However the efficiency of viral reproduction in last cases was
recovered after several passages while the mutations in genomic RNA
were retained. This reestablishment may be achieved by generation of
compensatory mutations in coding sequence. Taking together, these
results suggest that the structural organization of mengovirus 3'NTR
provides its functional properties.

II] przewlocki, G c t al, Nuel Acids gas., 26, 0998)4047.

[2] Keith, G et at, Biochem Biophys Res. Cornmun., 142, (1957), 183.

(Mo/5.3/302)

Cellular factors interacting with well defined structural

regions in Potato Spindle Tuber Viroid RNA
V. Thiel, A. Schmitz, D. Riesner
lnstitut fiir Physikalische Biologie, Heinrich-Heine-Universit~t Diisseldorf,
Universitiitsstrafle 1, 40225 Diisseldo~ Germany

Viroids are infectious RNA molecules causing serious diseases in higher

plants. Viroid RNAs are small (246-399 nt), single stranded and circularly
closed. They do not encode any protein but display characteristic structural
features that enable them to use host systems for their replication and systemic
distribution. The molecular mechanisms underlying viroid pathogenicity are
still unknown. We investigated the structural motifs that determine pathogenicity, and characterized cellular factors interacting with viroid RNA.
Sequence variants of Potato Spindle Tuber Viroid (PSTVd) that drastically differ in their ability to induce symptom expression show corresponding
differences in their threedimensional structure: Employing native PAGE we
found that the so-called Virulence Modulating (VM) region is bent and that the
degree of its bending increases with the pathogenicity of the sequence variant
[1]. Bending of the VM region might modulate the affinity of the viroid
molecule to the dsRNA-activated protein kinase (PKR), an enzyme involved in
the control of translation initiation. We found that the degree of phosphorylation and hence activity of PKR does indeed correlate with the severity of
symptoms in the infected plant.
Viroids replicate and accumulate in the host cell nucleus and have
repeatedly been suggested to interact with cellular RNA at some stage of their
replication cycle. We performed psoralen crosslink-experiments which revealed that PSTVd does interact with host cell RNA in vivo. The position of
the RNA-binding region within the viroid molecule suggests that this
interaction might be of functional importance in viroid processing. Further
results indicate that the cellular RNA involved might be a small nuclear or
nucleolar RNA (snRNA/snoRNA).

[1] Schmitz and Riesner, RNA 4 (1998) 1295.

Abstracts FEBS'99

(Mo/$.3/303)

s245

Structure and folding of the hairpin ribozyme:

An ion switch
F. Walterab, A.I.H. Murchicac, D.M.J. Lilleya
aDept of Biochemistry, DDI 4HN Dundee, UK, blBMC/CNRS, 15 rue R
Descartes, 67084 Strasbourg, F. CRiboTargets, CBI2JX Cambridge, UK

The hairpin ribozyme is a self-cleaving motif found in the negatives strand of

the satellite RNA of some plant viruses. In its natural form it consists of a
four-way RNA junction of which the single-stranded loop-carrying helices are
adjacent arms.
Fluorescence resonance energy transfer (FRET) was used to analyze the
conformation and folding of the hairpin ribozyme [1 ] and compared to the
four- way junction in isolation of the interacting loop A and B [2]. In the
absence of added metal ions both the hairpin ribozyme and the isolated 4-way
jtmction adopt a square configuration of coaxia|ly stacked arms, based on A on
D and B on C stacking. In the presence of magnesium, calcium, or strontium
ions, the junction of the ribozyme undergoes a ion-induced transition into a
distorted antiparallel geometry, creating close physical contact between the
two loops [3]. Manganese ions induce the same global folding, but no catalytic
activity; this change in global conformation is therefore necessary but not
sufficient for catalytic activity. Fitting the dependence of the conformation on
ionic concentration to a two-state model suggests that cooperative binding of
two ions is required to bring about the folding, with an apparent association
constant in the range of 2* 109 M-I. However, further ion binding is required
for cleavage activity. Only one divalent metal ion is observed in the case of the
junction alone, with an apparent association constant in the range of 500 M-I.
Cobalt hexammine ions also bring about global folding, while spermidine
generates a more symmetrical form of the antiparallel structure. Cadmium ions
generate a different folded form, interpreted in terms of close loop-loop
association while the junction arms are unfolded. Sodium ions were unable to
induce any folding of the ribozyme or the junction, which remained slightly
parallel. These results are consistent with a folding process induced by the
binding of two group IIA metal ions, distributed between the junction and the
loop interface.
[1] Murchie, A.I.H., Mol.Cen, 6, (1998), 873pp.
[2] Walter, F. et al., Biochemistry, 37, (1998), 14195pp.
[3] Walter, F. et al., Biochemistry, 37, (1998), 17629pp.

s246

Abstracts FEBS'99

8.2 Structure and function of chaperones

(Mo/8.2/304)

Involvement of oxidative reactions and protein chaperones

the rescue of misassembled in the follicular lumen
F.~,lu, P-J.I.q~, L.Vi~t,P,fml,n, B.Ilall~t

(Mo/8.2/305)

AIIosteric regulation of an archaeal chaperonin

by adenine nucleotides
I. Gutsche, O. Mihalache, W. Baumeister
Max-Planck-lnstitut f~r Biochemie. 82152 Martinsrted, Germany

Reactive oxygen species (ROS) are involved in many pathological

processes through modifications of structure-activity of proteins.
Generally ROS are generated by various systems producing H202 in
presence or not of peroxidases. But, ROS also participate in
physiological pathways such as thyroid hormone biosynthesis, which
proceeds through oxidation ( H202 generating system + thyroperoxidase)
of the prothyroid hormone (thyroglobulin; Tg 19S) and iodide.
Regarding to the colloidal insoluble multimerized Tg (m-Tg),
which bears dityrosine and disulfide bridges, and represents #30% of the
total colloidal Tg. We noted that a mild oxidative system generated two
different soluble forms ofm-Tg, more or less compacted by hydrophobic
associations and linked with protein chaperones (Grp78 and Grp94). The
first soluble form was represented by several anits of Tg (Tgl2S) linked
to Grp78 (ratio Grp78/Tg12S = 0.6). The second type of soluble form
was represented by the association of a limited number of Tgl2S
essentially linked with Grp94 (ratio Grp94/Tg 12S = 2.6).
Our results show that protein chaperones are able to escape from
the endoplasmic reticulum (ER) and are involved with ROS, in the
follicular lumen, for thyroid hormone synthesis. Accordingly, two
hypothesis can be considered: i/misassembled Tg associated with protein
chaperones escape to the retention signal from the ER and are secreted
altogether in the follicular lumen or ii/ Tg molecules secreted in the
follicular lumen are aggregated by successive reactions of oxidation,
according to the physiopathological statut of the gland, then secondary
the thyrocytes secrete different protein chaperones to rescue this m-Tg.
Therefore, we propose a model of roles of m-Tg in the follicular lumen.

Chaperonins are ubiquitous toroidal protein assemblies which bind

nonnative proteins within their central cavity and promote their ATPdependent folding to the native state. The archaeal chaperonin, commonly
referred to as the "thermosome" is an attractive working model in attempts to
elucidate the mechanism of action of the so-called group II chaperonins
comprising in particular the eukaryotic cytosolic chaperonin TRiC. They are
distantly related to the group I chaperonins typified by GroEL from E. coli
which hitherto provided the paradigm for chaperonin action. Our current
understanding of the group II chaperonins is hampered by the absence of
information about their allosteric regulation by adenine nucleotides, which has
only been established for the group I chaperonins. In the case of GroEL, its
partner GroES binds to the ATP-charged chaperonin to seal off the folding
compartment. As for the thermosome, cryo-electron microscopy reveals an
open "donut"-like substrate-acceptor state [11, whereas its crystal structure
shows a closed "ball" conformation, in which protrusions from the apical
domains occlude the central cavity of the protein [2]. Comparing these data
with those obtained for the GroE system, it is tempting to suggest that the
crystallisation conditions have captured the protein in a folding-active state,
even though ATP was not present I3]. The question arises whether the
observed open and closed states are relevant and if the interconversion could
indeed be initiated by ATP binding. Therefore, we started a detailed
investigation of the allosteric consequences of nucleotide binding and
hydrolysis by homo- and heterooligomeric thermosomes from Thermoplasma
acidophilum. The changes in intrinsic protein fluorescence axe used to
monitor the nucleotide binding to the thermosome and the infuence of
different parameters such as temperature, pH and ionic composition of the
solution. Enzymological analysis of the ATP hydrolysis provides further
insights into the functional cycle of the chaperonin. Finally, small angle
neutron scattering is used to probe the structural organisation of the protein in
the functional states defined by the biochemical studies. The results will be
discussed in the light of the current models of action of the group II
chaperonins.
1. Nitsch, M. et al., J. Mol. Biol., 267, 1997, 142
2. Ditzel, L. et al., Cell, 93, 1998, 125
3. Horwich, A. L. and Saibil, H. R., Nature Struct. Biol., 5, 1998, 333

(Mo/8.2/306)

Expression of the ibpAibpB operon

in the rpoH15, rpoH165 and ArpoH mutants of E.eoli.
D.Kuczyflska-Wi~nik, E. Laskowska, A. Taylor
Dep. of Biochemistry, Univ. of Gdahsk. Kladki 24, GdaSsk, Poland

The ibpA and ibpB genes code for the two small heat shock proteins,
IbpA and IbpB. These proteins were described as capable of recognising
endogenous proteins aggregated intracellularly by heat shock [1] and
inclusion bodies of heterologous proteins [2]. The ibpA and ibpB genes form
the operon regulated by the rpoH gene product, o 32 protein. The presence
of the active, o 32 dependent promoter upstream of the first operon gene ibpA, was documented [2,3]. However, our results indicated that some
unidentified factors also participate in the ibpAibpB operon regulation.
Surprisingly, we have found that the IbpA and IbpB protein level was higher
in the rpoHl5 and rpoH165 strains (which are unable to induce the heatshock response) than in wild type and rose after heat shock [ 1]. Induction of
the other heat shock genes, dnaK and dnaJwas blocked as expected, in these
conditions. No IbpA/B protein production was observed in the ArpoH strain.
We examined transciption products ofibpAibpB operon by Northern
blotting using specific ibpA probe and by primer extension. In both, wild type
and rpoH15 strains subjected to heat shock, we found two transcripts
identified as ibpAibpB mRNA and ibpA mRNA which begin at that same
transcription start point. In ArpoH strain no transcripts was observed. These
results suggest different interactions of the o32-RNA-polymerase
holoenzyme with the cr32-dependent promoter upstream of the ibpAibpB
~l~eron than those with the known heat shock promoters recognisable by o
z. Supposedly, unknown factor was needed in the case of interaction with
this promoter. If so, binding o f o 32 changed by mutation, together with this
factor might be sufficient for the transcription initiation. However, there was
no transcription in the total absence o f o 32 in the ArpoH mutant.
1. Laskowska E., Wawrzyntw A., Taylor A., Biochimie 78 (1996): 117
2. Allen SP., Polazzi JO., Gierse JK, Easton AM., JBacteriol. 174
(1992):6938
3. Chuang SE., Burland V., Plunkett GIII. Daniels D.L., Blattner F.R, Gene,
134(1993):1

(M0/8.2/307)

Identification of in vivo substrates of the

DnaK chaperone system
A. Mogk,P. Goloubinoff,T.Tomoyasu,H.Langen*,D.Roder*,B. Bukau
Institute of Biochemistry and Molecular Biology, UniverstO"of Freiburg,
D-79104 Freiburg; *Hoffmann-LaRocheAG, Ch-4002Basel

The DnaK (Hsp70) chaperone machinery constitutes a protein repair system of

E. coli that prevents aggregation and assists refolding of misfolded proteins
and that is essential for cell survival under stress conditions such as heat shock
to 4T'C [1]. In addition, this system facilitates the degradation of shortlived
and misfolded proteins by proteases. While the protein repair function of the
DnaK system is well established for model substrates, the nature of the
misfolded protein substrates which accumulate in E. coil under heat shock
conditions is largely unknown and was analyzed in this study.
When soluble total extracts of E. coil cells were subjected to severe heat shock
treatment a significant fraction of proteins aggregated. Among several
exogeneously added chaperones of the E. coli cytosol tested for their ability to
prevent protein aggregation in these extracts, only the DnaK system was
found to be highly effective. This finding correlates well with the essentiality
of this system in vivo at 42C. To identify the heat labile protein substrates of
DnaK, we analyzed the cellular proteins which aggregated in a DnaKdependent fashion in both, cell extracts and in vivo (in AdnaK52 nutants),
using 2-D gel electrophoresis and mass spectroscopy. More than 50 different
proteins aggregated in dnaK null mutants upon shift to 42C, and a similar
protein pattern was found in heat-shocked cell extracts. These protein
substrates exert a multitude of cell functions and show no specificity towards
their pI value. However, large and oligomeric proteins were more abundant
while small proteins (< 25 kD) were largely excluded, showing that
multidomain and oligomeric proteins are particularly heat labile and require the
chaperone activity of the DnaK system. For one natural substrate identified in
our analysis, the MetE protein, we verified with purified components that the
DnaK system acts directly to prevent its aggregation. Together, these results
provide for the first time information on the nature of the heat labile proteins in
E. coli that require the chaperone function of the DnaK system.
In a second approach we investigated the ability of the DnaK system to
cooperate with other chaperones of the E. coli cytosol in the disaggregation of
aggregated proteins. Data will be presented which document such activity.
[1] Hesterkamp and Bukau, EMBO J. 17, (1998) 4818

Abstracts FEBS'99

(Mo/8.2/308)

s247

Ct truncated hsp90 : in vivo distribution and origin

V Montel, M. Dols-Lafargue, J. L Azanza, J Raymond

(Morn.2/309)

ISTAB, Laboratoire de biochtmte et Technologw des Aliments

M R C Group in Membrane Biology, Departments o f Medicine and

Biochemistry, University o f Toronto, Toronto, Canada.

UR.4-1N/Z4. Ui~iversaO Bordeaux L Avenue des Facultes, 33405

Talence, Cedex France.

Analysis of crude extracts from either different rat tissues or

myoblasts cell cultures reveals the presence, in addition to full-length
hsp90, of specific products reacting with anti-hsp90 mAbs A 73 kDa
protein is predominantly found in the majority of the tissues analyzed
It corresponds to a truncated form of hsp90 lacking the C-terminal
fragment as revealed by immunodetection Ct truncated hsp90's are
found as dimers in tissues and cell cultures. Our results suggest that, in
addition to the stabilization of transcription factors and tyrosine kinases
(of dimeric fulMength hsp90s), the truncated 73 kDa dimer is a further
functional partner in cell metabolism A double pool comprising fulllength and truncated hsp90 is evidenced In addition, in primary cell
cultures the presence of ubiquitinated hsp90 is demonstrated
When tested on Suc-Leu-Leu-VaI-Tyr-MCA as substrate,
purified full-length hsp90 from either rat muscle or commercial bovine
brain display a low <<chymotrypsin-like >~ peptidase activity which is
activated by Ca ~- and M g " ions. On the other hand, using long-term m
wtro experiments, we demonstrate the ability of hspg0 to convert into
a 73 kDa truncated product This autocatalytic degradation proceeds
from the C-terminal end of the full-length hsp90 This corresponds to
an intermolecular process since addition of exogenous 73 kDa product
speeds up the maturation kinetics The peptidase activity is enhanced in
the 73 kDa product and is sensitive to peptide aldehyde inhibitors and
to lactone compounds Maturation appears to be uncoupled with the
peptidase activity because it is refractory to MG 132, PSI and clastolactacystin [3 lactone inhibitors We checked that 20S proteasome and
m-calpain were not responsible for the observed activities. Indeed, the
peptidase activity and self-processing appear to be intrinsic properties
of the chaperone. The truncated hspg0 product obtained m vttro
resembles to the 73 kDa product previously described m vtvo Taken
together, these data suggest that, in addition to the maintenance
function of the dimeric full-length hsp90s, this protein may enable
regulation which, after truncation may shift the function.

(Mo18.21310)

Evidences for a chaperon-like function of the prodomain of

cathepsin L like cysteine proteases
K. Schilling, M. Fehn, G. Maubach, S. Waldmann, S, Kreusch,
W. Rommerskirch, and B. Wiederanders
lnst f Btochemte 1, Khmkum FSU, Nonnenplan 2, D-07743 dena, Germany

One of the most striking differences between the two subfamilies ofpapain-like
cysteine proteases, cathepsin L- and B-like endopeptidases is the extended proregion of the L-like subfamily, exceeding the length of the corresponding B-like
structure by about one third. Common structural motifs in the proregions of
both subfamilies were detected by X-ray crystallography of the proenzymes.
The C-terminal part spans the active site cleft. The adjacent strand-turn-helix
hairpin anchors to the propeptide binding loop of the mature enzyme. Only in
the L-like subfamily, the N-terminus folds to a mini-domain harboring some
highly conserved amino acids [l]. To elucidate the function of this structural
specificity of the L-like subfamily we studied the consequences of Ala exchange of some of the most conserved amino acids in the prodomain of cathepsin S.
Based on i) far UV-CD measurements of recombinant propeptide mutants,
ii) Trp fluorescence studies, and iii) the loss of the mutants inhibitory capacity
to the parent enzyme we ranked the individual contribution often conserved
amino acids to the structural stability of the prodomain at pH 6.5. We concluded
from CD spectra that secondary structure is not considerably affected by all mutations, whereas Trp fluorescence shift measurements revealed that the ternary
structure depends on the conservation of three interdigitating Trp residues in the
core region of the prodomaln. Regarding the inhibition kinetics towards cathepsin S, the propeptide mutants with molten cores show i) Ki-values about two
orders of magnitude higher than the Ki's of the other mutants and ii) a two step
reaction (E + I ~ EI <--->EI*). The initial low affinity binding step is followed
by a slow one, saturable with increasing concentration of the mutant propeptide,
indicating that these mutants undergo enzyme induced conformationaI changes.
Considering i) the reciprocity of such interactions between protein domains,
ii) the cotranslational domain folding, i.e. the prodomain establishes first,
m) the failure of all folding attempts with proregion truncated L-like cathepsins
in-vivo and m-vitro, and iv) successful refolding of proregion truncated cathepsin B, these results strongly suggest that the chaperon function may be a special
one of the prodomain of the cathepsin L like proteases.
[1] Coulombe, R. et aI., EMBO J. I5 (1996) 5492
S u p p o r t e d by D F G Wi 1102/1-4

Band 3 alone, or with calnexin chaperone?

M. Popov and R.A.F. Reithmeier

The interaction of the endoplasmic reticulum chaperone, calnexin, with Nglycosylation mutants of a polytopic membrane glycoprotein, the human
erythrocyte anion exchanger (AEI, Band 3), was characterized by cell-free
translation followed by immunoprecipitation using anti-calnexin antibodies. AE1
contains 12-14 transmembrane segments and has a single site of N-glycosylation
at Asn642 in the 4th extracytosolic loop (EC4). This site was mutated (N642D)
to create a non-glycosylated protein. Calnexin showed a preferential interaction
with N-glycosylated AEI relative to non-glycosylated AE1, both in cell-free
system and in transiently transfected HEK293 cells. This interaction was blocked
by inhibition of glucosidases I and II with castanospermine. Caknexin had access
to novel N-glycosylated sites created in other EC loops in AEI by site-directed or
insertional mutagenesis and the interaction with AEI was enhanced when multiple
sites were introduced into the same loop or into two different loops. An
association of calnexin with truncated versions of N-glycosylated AEI was
detected after release ofthe nascent chains from ribosomes with puromycin. The
results show that the interaction of calnexin with the polytopic membrane
glycoprotein AEI was dependent on the presence hut not the location of the
oligosaccharide. Furthermore, calnexin was associated with AE1 after its release
from the translocation machinery. The interaction of calnexin with AE1 may
assist in the post-translational folding and oligomerization of the AEI membrane
protein. (Supported by the Medical Research Council of Canada.)

(M0/8,2/311)

The C h a p e r o n e Effect of Lens C r y s t a l l i n s

F. Skouri, F. Bonnet6, K. Prat, A. Tardleu
UMR 7590 - CNRS, Universitrs Parts 6 &7
Laboratotre de Mmdralogie - CristaUographte
Case 115, 4 Place Jussteu, F 75252 Parts Cedex 05, France

c~-Crystallins from eye lens belong to the small heat chock protein family.
They are polydisperse ohgomers of about 800 kD. We have previously
shown that in vivo, the short range repulsive cc-crystallin interactions
account for eye lens transparency [ 1].
At high temperature, the association of a-crystallins to partially denatured
proteins is known as a chaperone effect (although not followed by
renaturation of the denatured species). It was therefore hypothesized by
different groups that in eye lens, the associatmn of ct-crystalhns to
denatured 13- and ~,- crystallins, thus preventing further denaturation,
precipitation and therefore light scattering, protects against cataract. The
structure of the ct-13--crystallin assemblies is not known, although some
cx-crystalhn peptides have been reported to be specifically involved in the
interactions. We have started to design a chaperone assay in order to
classify the ability of the vartous 13- and ,/-crystallins, rather native or
recombinant, to associate to ct-crystalhns in different physico-chem~cal
conditions. The method combines size exclusion chromatography and
small angle X-ray scattering and allows us both to measure the complex
molecular weight and to quantify the changes in ct-crystalhn repulsive
interactions induced by the 13-and ~'-crystallin associatmn.
[1 ] Vrrrtout F., Delaye M. & Tardleu A. J M o l Biol 205 (1989) 713
keywords : chaperone, crystallins, interactions

s248

(Mo/8.2/312)

Abstracts FEBS'99

Heat shock in Rhodothermus marinus

E. Thorolfsdottir, V. Backman, T. Blondal, H. Hauksdottir,
S. H. Thorbjarnardottir and G. Eggertsson.
Institute of B~ology, University of Iceland, Grensasvegur 12, Reykjavik

Over 25 proteins are induced during heat shock in the thermophilic

eubacterium Rhodothermus marinus as was seen in two dimensional gel
electrophoresis analysis of proteins fi'om heat shocked cultures. Among these
are proteins which are comparable in size to the main heat shock proteins of
other eubacteria. Amino acid sequencing of proteins induced during heat
shock is underway. The genes for four of the main heat shock proteins,
GroES, GroEL, DnaK and DnaJ, have already been cloned and sequenced.
Conserved inverted repeats (CIRCE) were found upstream the
groESL operon. These repeats have been found upstream ofgroESL and
dnaK operons in many bacteria and have been shown to interact with a
repressor protein. The groES gene codes for a 100 amino acid long protein
with calculated molecular weight of 11.1 kDa. This protein showed 42%
amino acid identity with E. coli GroES. The groEL gene codes for a 540
amino acid long protein with calculated molecular weight of 57.7 kDa. This
protein showed 65% amino acid identity with E. coli GroEL.
In many bacteria the dnaK and dnaJ genes are together in an operon,
but this is not the case in R. marinus. No CIRCE sequences were found
upstream of these genes. The dnaK gene codes for a 640 amino acid long
protein, with calculated molecular weight of 70.2 kDa. This protein showed
57% amino acid identity with E. coli DnaK. By cutting the R. marinus
genome with restriction enzymes that cut upstream and downstream of the
dnaK gene and Southern blotting with a dnaK probe, we were able to
determine that there is only one dnaK gene in the R. marinus genome. The
dnaJ gene codes for a 316 amino acid long protein with calculated molecular
weight of 35.9 kDa. This protein showed 26.9% amino acid identity with E.
coli DnaJ. A 68 amino acid deletion is seen in R. marinus DnaJ when
compared to E. coil This deletion is also seen in the DnaJ proteins of
Thermus aquaticus, Deinococcus proteolyticus, Synechococcus sp and the
DnaJ like protein CbpA of E. c o i l This deletion spans the zink-f'mger region
of DnaJ, which has, along with the C-terminal domain, been proposed to take
part in substrate binding. Southern blotting of the R. marinus DNA, using the
dnad PCR product as a probe, showed that there is only one dnaJ copy in the
R. marinus genome.

(Mo/8.2/314)

Quaternary structure of the Hsc70 Interacting Protein

M. Velten and M.M. Ladjimi
UMR 7631 CNRS-UniversitO P et M Curw. Part~. France

Hsc70 Interacting Protein (Hip) is a cytoplasmic co-chaperone which

regulates the chaperone activity of Hsc70 during folding of polypeptide
chains and maturation of the progesterone receptor. Hip binds to the
ATPase domain of Hsc70 and stabilizes the ADP-bound form of Hsc70,
which has a high affinity for polypeptide substrates. Hip, hke most
chaperones, forms homo-oligomers and this oligomeric structure may be
important for its co-chaperone activity. Because the degree of association of
Hip is unclear, we have undertaken rigorous determination of the quaternary
structure of the protein by using several biochemical and biophysical
techniques.
In raze exclusion chromatography, Hip eluates as a single species having a
Stokes radius (Rs) of 56 A. Sedimentation velocity experiments by
analytical ultracentrifugation indicate the presence of a single species whose
sedimentation coeffiment (s20.,d is 4.4 S. The combination of the determined
Rs and S%.wgives a molecular mass of 105,000 Da, a value close to that of
the theoretical molecular mass of a dimer (.87,090 Da). Moreover,
sedimentation equilibrmm data fit best to a single ideal species model givmg a
molecular mass of 88,284 Da. Therefore, we clearly demonstrate that Hip
exists as a dimer in solution. In addition, the f/fo value of Hip dimer obtained
by sedimentation velocity is 1.9, indicating that this dimer does not have a
globular shape, but rather adopts a strongly elongated conformation. Circular
dichroism analysis, together with secondary structure predictions, indicate
that Hip is mostly an alpha helical protein and is able to form coiled-coil
structures. These structural features (elongated form and high percentage of
alpha hehxes) are similar to those of BAG-l, another co-chaperone that
competes with Hip for binding to the N-terminal domain of Hsc70 and
affects nucleotide exchange. The fact that HIP and BAG-1 share a similar
overall shape suggest the existence of a common mechanism of interaction
with HscT0, presmnably at the same site.

(M0/8.2/313) Regulation of IFN-'/folding in CHO cells by the ER

chaperones Grp78/Bip, Grp94 and calretieulin
K. Vandenbroeck, I. Ronsse, E. Martens and A. Billiau
Rega lnstaute jol" Medical Research. B-3000 Leuve~I. Belgmm

Refolding of acid-denatured IFN-7 under physiological conditions

(35C) is hampered by off-pathway aggregation reactions. Using an
in vitro approach we have recently demonstrated that both acid- and
heat-denatured IFN-,/ can be refolded efficiexatly by the E. coli
GroEL/ES ill and DnaFdDnaJ/GrpE [2] chaperone systems in the
presence of ATP. We have now used CHO cells expressing high
levels of IFN-7 as a tool tc~analyse the in vivo folding pathway of this
cytokine. Either ATP depletion or tunicamycin (Tn) treatment
decreased the level of IFN-y secretion, and induced massive IFN-7
aggregation in a pre-Golgi compartment. In either of both cases, the
aggregating protein species was found to be the unglycosylated 17kD foma. Treatment with castanospennine (CST) did not alter the
secretion kinetics, and neither did it induce aggregation of IFN-7.
Grp78, Grp94 and calreticulin were identified as major components
of cross-linked IFN-7 complexes. The amount of Grp78 and Grp94
co-~mmunoprecipitating with 1FN-v was markedly increased under
conditions of ATP depletion. Tn-treatment or ATP-depletion - but
not CST-treatmenl -, blocked the interaction between IFN-7 and
calrcticulin. Two other chaperones, known to interact with nascent
glycoproteins through carbohydrate side chains, Le. calnexin and
Grp58/ER-60, were not found in complexes with IFN-y. Thus, our
results point to a crucial role for calreticulin in inhibiting aggregation
during folding of IFN-7, lltrough interaction with otigosaccharide
side chains. Previous reports have demonstrated that glycosylation
can modulate the biological activity, or increase the protease
resistance of lFN-7. Our results demonstrate for the first time that
cytokine glycosylation is required for efficient folding and secretion.
[1] Vandenbroeck, K., Martens, E. and Bilhau, A. (1998) Eur. J Biochem
251,181-188.
[2] Vandenbroeck, K and Bllhau, A. (1998) Biochzmie 80. 729-737

Abstracts FEBS'99

s249

13.3 Phosphorylation/dephosphorylation in regulatory processes

(Mo/13.3/315)

Expression and localization of Raf-1 and B-raf proteins in

adult rat brain.

(Mo/13.3/316)

J. V. Barnier C. Morice, F. Nothias, S. K6nig, P. Vernier, B Guibert IAF,

UPR 2212 CNRS, 1 av. de la terrasse, F-91198 Gif sur Yvette France.

The Raf kinases play an important and specific role in the activation
of Extracellular signal-Regulated Kinases (ERK) cascade. Beside its role in
the control of proliferation and differentiation, the ERK cascade has also
been implicated in neuron-specific functions. In order to gain clues on the
function of Raf kinases in the adult central nervous system, we
performed a comparative analysis of the distribution and subcellular
localization of the different Raf kinases in rat brain with antibodies
specific for the different Raf kinases.
We show that B-Raf and Raf-I proteins are present in the brain
whereas A-Raf is not detected, lnterestingly~ the two Raf proteins are
detected in the same brain areas and have an approximately similar
pattern of distribution with a rostro-caudal decreasing gradient of
expression. These two kinases are localized mostly in neurons and likely
in the same cells but they showed differences at the subcellular level.
Raf-1 is localized mainly around the nucleus whereas B-Raf is largely
distributed in the cell bodies and in the neuritic processes. In addition, we
demonstrated that numerous B-Raf isofomas are present m the brain.
These isoforms have a differential pattern of distribution, some of which
are ubiquitously expressed whereas others are localized to specific brain
areas. These isoforms also have a clear differential subcellular locahzation.
Thus these results suggest that each Raf protein could have a distmct
function in the brain.

(Mo/13.3/317)

Evidence for a role of protein kinase CK2

in cell proliferation
L. Bianchini, C. Cochet, E. M. Chambaz, F. Lebrin
1NSERM U244,CEA,DBMS-BRCE. 17 rue des Martyrs, F-380~4 Grenoble

CK2 (formerly known as casein kinase 2) is a ubiquitous serine/threonine

protein kinase whose regulation and function still remain enigmatic. CK2
holoenzyme consists of two catalytic subunits (c~ or c~') and two
regulatory subunits (13); recent evidence, however, suggests that
independent CK2 c~or CK213 subunits might be present in cells. We have
investigated the potential involvement of CK2 in mitogenic signalling
using transient transfection experiments allowing specific overexpression
of each CK2 subunit in NIH 3T3 fibroblasts. For these experiments,
DNA replication was monitored by BrdU incorporation using GFP
(Green Fluorescent Protein) as a marker for transfected cells. We found
that overexpression of either subunit of CK2 is unable to impair cell
proliferation. We then used a kinase-inactive mutant of the CK2 catalytic
subunit in an attempt to interfere with the functions of CK2. We first
demonstrated that this kinase-inactive CK2 c~mutant is capable of
recruiting endogenous CK2B regulatory subunit to generate inactive oc2132
tetramer. Interestingly, we then found that overexpression of this
dominant-negative mutant results in an inhibition of G1/S progression in
NIH 3T3 fibroblasts demonstrating an important role for CK2 kinase
activity in cell cycle progression.

Effect of platelet-derived growth factor on

adhesion of smooth muscle cells to fibronectin.
E. Berrou and M. Bryckaert. U. 348 INSERM, IFR 6, 8, Rue
Guy Patin, 75475 Paris Ceder 10, Frame

Convergence of intracellular pathways activated by growth factors and

integrirl-mediated cell adhesion is required for normal cell growth. We
investigated the effect of platelet-derived growth factor (PDGF-BB), on
adhesion of smooth muscle ceils (SMC) to fibronectin.
Adhesion of SMC to fibronectin was inhibited by pre-treatment with PDGFBB. Maximal inhibition occurred at 2 ng/ml of PDGF-BB and required a 30minute pre-treatment. This inhibition decrease (from 80 % to 50 %) with
increasing concentrations of fibronectin (from 0.5 to 5 gg/ml). Moreover,
when activation of extracellular signal-regulated kinase (ERK) was inhibited
by PD 98059, remaining inhibition of cell adhesion ( 50 %) was still observed
and was independent of fibronectin concentration. Effect of PDGF-BB on
inhibition of cell adhesion by RGD peptides was investigated. Cells treated
with PDGF-BB was more sensitive to low doses of peptide inhibitor than
control cells: 45 % of PDGF-BB-treated cells versus 80 % of untreated cells
adhere to fibronectin in the presence of 0.2 mM RGD peptide, indicating a
decrease in integrin avidity. These results suggested that 1) PDGF-BB might
induce inhibition of SMC adhesion by two mechanisms, 2) ERK might act as
a negative regulator of cell adhesion.
Inhibition of cell spreading by PDGF-BB was also observed, suggesting
modifications of protein-protein interactions within focal adhesions. Since
paxillin, a focal adhesion protein, functions as an adaptO,.,r protein involved in
forming multiprotein complex, its tyroslne phosphorylation was assessed by
immanobloting after immunoprecipitation. Pre-treatment of cells with PDGFBB diminished the adhesion-induced tyrosine phosphorylation of paxillin.
Inhibition of tyrosine phosphorylation of paxillin might alter the structural and
functional link between integrins and the actin-cytoskeleton and thus mediate
action of PDGF-BB not only on ceil spreading but also on cell adhesion.
In conclusion, PDGF-BB might inhibit adhesion of SMC to fibronectin by
two mechanisms: by decreasing avidity of integrins, and by altering the
organisation of focal adhesion.

(Mo/13,3/318)

ANALYSIS OF TEE TIME COURSE OF ERK ACTIVATION IN THE

NEURONAL DIFFERENTIATION OF PC12 CELLS
Boulgri. C~, Szeber~nyi, J.
University Medical School of P~cs, Hungary
Department of Biology

Long-term
activation
of
ERK
(extracellular
signal
r e g u l a t e d kinase) enzymes induced by NGF (nerve growth factor)
is t h o u g h t to be a key event in neuronal differentiation of
PC12 cells occurring in response to this agent. Moreover, the
translocation of ERKs from the cytoplasm to the nucleus can be
detected exclusively upon treatments eligible causing neuronal
d i f e r e n t i a t i o n of PC12 cells. Trying to establish a firm causal
r e l a t i o n s h i p between these remarkable events as well as explain
their biological signicance we examined wild type PC12 cells in
c o m p a r i s o n with a mutant PC12 subclone expressing a dominant
negative Ha-Ras Asn-17 protein. In these cells - named Z-M17-5
cell line - MGF is not able to cause neuronal differentiation,
however, this block caused by the interfering Ras mutant can be
bypassed by combined treatment with NGF+dbcAMP or NGF+ionomycin
but not with NGF+TPA.
In our experimental system both wild type PC12 cells and
the m u t a n t Z-M17-5 subclone were treated with different growth
factors (EGF and NGFi alone or in combination with the second
m e s s e n g e r analogues mentioned above. Besides, we examined the
effect
of
phosphatase
inhibitors
(okadaic
acid
and
orthcvanadate) on ERK phcsphorylation and nuclear translocation
too. The time course of phosphorylation was detected by Western
blot analysis using phospho-specific ERK antibody; the chasing
for p r o t e i n movement was carried out performing immunoc y t o c h e m i s t r y using the streptavidin/biotin method.
Our results in detail are presented in the poster.

s250

(Mo/13.3/319)

Abstracts FEBS'99

NIPPI and MYPTI as subunits of PPI in liver cytosol

A. Boudrez, K Evens, M. Beullans, E Waelkens,
W. Stalmans and M. Bollen

(Mo/13.3/320) SAPK induced transcriptionnai regulation during

myogenesis
C.Cabane, W.Englaro, B.Cuiller, B.D6rijard
UMR CNRS 6548, UNSA, Parc Valrose, 06108, Nice, France

Afdeling Btochemie, Faculteit Geneeskunde, KbZeuven. Belgtum

Various studies have provided evidence for the existence of cytosolic

species of protein phosphatase 1 We report here the purification of these
enzymes from rat liver eytosol by successive ehromatographies on
microcystin-Sepharose and Resource Q. Two isolated enzymes were
identified by western analysis, by far-western blotting with digoxygeninlabelled catalytic subunit and by peptide sequencing as heterodimers of
the catalytic subunit and either the inhibitor NIPP1 [1] or the myosinbinding subunit MYPT1 [2]. The hepatic MYPT1 was PCR-cloned using
a rat liver cDNA library as template and primers based upon the known
sequence of MYPT1 from rat aorta These investigations revealed that rat
liver expresses two isoforms of MYPTI that only differ from each other
by the presence or absence of a 56-residue insert in the central domain.
Northern blot analysis showed that a 6.1 kb transcript of MYPT1 was
expressed in all rat tissues investigated, except pancreas and skeletal
muscle. This MYPT1 transcript was also expressed in rat hepatocytes and
FAO cells. An additional 3.8 kb transcript was expressed in testis. The
adopted purification protocol also enabled the large-scale purification of
the free A-subunit (PR65) of protein phosphatase 2A.
[1] Jagiello, I. et al. (1995), J. Biol. Chem 270, 1725
[2] Hartshorne et al. (1998), J. Muscle Res. Cell Motil. 19, 325

(Mo/13.3/321)

SAPK induced transcriptionnal regulation during

myogenesis
C.Cabane, W.Englaro, B.Cuiller, B.D~rijard
UMR CNRS 6548, UNSA, Parc Valrose, 06108, Nice, France

Muscle cell differentiation is controlled by extraceltular

growth factors whose signals are transduced into the nucleus.
Stress Activated Protein Kinase cascade are involved in this
process and members of these pathways are potently expressed
in skeletal muscle.
My research project is based on the mouse skeletal muscle
C2C12 cell line as a model system. This cell line is able to
undergo a differentiation program in vitro such that the major
steps in myotubes formation are identifiable. Several stable
clones expressing various elements of the stress-induced
MAPK cascades have been isolated: some C2C12 clone
express a constitutive inactive form of the MAPKK MKK3
(p38 pathway) or the MAPKK MKK4 (.INK pathway), others
express the wild-type p38 or JNK1.
A myoblast differentiation affecting phenotype was observed
with the MKK3 dominant negative stable clone. These cells
have been used side by side with wild-type cells in a
differential screening so as to study gene regulation involving
MAPK signalling pathways during differentiation.
Genes exhibiting differential regulation using cDNA grid array
have been confirmed by Northern Blot. The promoters of these
differentially regulated genes are currently under investigation
using reporter genes.
Other differential screenings involving MAPK pathways
inhibitors SB 203580 (p38-MAPK) and PD 098059 (MEK1) as
well as other stable clones will be performed.
Finally, we are planning to isolate and characterise muscle
specific transcription factors required for the regulation of
these genes involved in myogenesis.

Muscle cell differentiation is controlled by extracellular

growth factors whose signals are transduced into the nucleus.
Stress Activated Protein Kinase cascade are involved in this
process and members of these pathways are potently expressed
in skeletal muscle,
My research project is based on the mouse skeletal muscle
C2C12 cell line as a model system. This cell line is able to
undergo a differentiation program in vitro such that the major
steps in myotubes formation are identifiable. Several stable
clones expressing various elements of the stress-induced
MAPK cascades have been isolated: some C2C12 clone
express a constitutive inactive form of the MAPKK MKK3
(p38 pathway) or the MAPKK MKK4 (JNK pathway), others
express the wild-type p38 or JNK1.
A myoblast differentiation affecting phenotype was observed
with the MKK3 dominant negative stable clone. These cells
have been used side by side with wild-type cells in a
differential screening so as to study gene regulation involving
MAPK signalling pathways during differentiation.
Genes exhibiting differential regulation using cDNA grid array
have been confirmed by Northern Blot. The promoters of these
differentially regulated genes are currently under investigation
using reporter genes.
Other differential screenings involving MAPK pathways
inhibitors SB 203580 (p38-MAPK) and PD 098059 (MEK1) as
well as other stable clones will be performed.
Finally, we are planning to isolate and characterise muscle
specific transcription factors required for the regulation of
these genes involved in myogenesis.

(Mo/13.3/322)

Structure and splicing of the human gene encoding sds22

H. Ceulemans, A. Van Eynde, E Pdrez-Callej6n, M Beullens,
M. Bollen and W. Stalmans
AfdelingBlochemie,Faculteitder Geneeskamde,KULeuven,Belgium

Sds22 is a regulatory subunit of protein phosphatase-I that is required for the

completion of mitosis in yeast. It consists largely of 11 tandem leucine-rich
repeats of 22 residues that are expected to mediate interactions with other
polypeptides, including phosphatase-1. We determined the structure of the
human gene encoding sds22, designated PPPIR7. This gene (33 kb)
comprises 11 exons, but these do not coincide with the sequences encoding
the leucine-rich repeats. Up to 6 splice variants can be generated by exon
skipping and alternative polyadenylation, as revealed by EST data base
analysis, RT-PCR and northern blot analysis The sds22 transcripts are
expected to encode 4 different polypeptides. Sds22cq corresponds to the
variant that was previously cloned from human brain [1] Sds22131 is
truncated within the ninth repeat and has a short and different C-terminus.
Both variants also exist without the sequence corresponding to exon 2, and
these are termed sds22c2 and sds22112. The Y-flanking region of P P P I R 7
contains two NF-Y binding CCAAT-boxes near the transcription start site,
and potential binding sites for the transcription factors e-Myb, lk-2 and NF1, that are conserved in the mouse gene.
[1] Renouf et at., FEBS lea., 375, (1995) 75

Abstracts FEB S' 99

s251

(Mo/13.3/323) LMW-PTP is involved in Rho-mediated cytoskeleton

rearrangement after PDGF stimulation.
Chiarugi, P, Cirri, P., Taddei, L., Canucl, G., Raugel, G, RanapomG.

(Mo/13.3/324)

Dlparttmento di Scienze Btochimtche, Umversltgtdegh Studt dt b)renze, wale

Morgagn150, 50134 Ftrenze, Itaha.

The low molecular weight protein tyrosine phosphatase (LMW-PTP) is able

to specifically bind and dephosphorylate activated PDGF receptor thus
modulating PDGF induced mitogenesis. In particular LMW-PTP is involved
in pathways that regulate the transcription of the immediately early genes
myc eros in response to growth factors stimulation. Recently we have found
that LMW-PTP exists constitutively in cytosolic and cytoskeletonassociated localization and that, after PDGF stimulation, c-Sre is able to
bind and to phosphorylate LMW-PTP only in the cytoskeleton-associated
fraction. In this study we have investigated the function and the regulation
of the cytoskeleton associated LMW-PTP in consequence of integrin and
PDGF stimulation To address this issues we have transfected a mutant form
of LMW-PTP where the phosphorylation sites (Y131 and Y132) were
mutated and we have studied the effect of this mutant in cell adhesion and in
cell migration Firstly, we have established that both wild type and mutant
LMW-PTP are present in the cytoskeletric fraction only after integrin
stimulation. Then we have shown that LMW-PTP phosphorylation by c-Src
after PDGF treatment strongly influences both the capability of the cell to
adhere to the substrate and to migrate in response to PDGF gradient In fact,
cells overexpressing wild type LMW-PTP possess an higher adhesion and
migration capability with respect to control cells Cells expressing the
double mutant Y131A-Y132A behave almost like the non-transfected cell
with respect to both parameters. Finally we have discovered a new LMWPTP substrate localized in the cytoskeleton after PDGF treatment, p 190RhoGAP The Y131A/Y132A LMW-PTP mutant shows a lower activity on
phosphorylated pl90Rho-GAP with respect to wild type LMW-PTP
demonstrating that, also in vivo, LMW-PTP phosphorylation leads to an
increase of its phosphatase activity. The action of LMW-PTP on
phosphorylated pl90Rho-GAp could explain the above mentioned effect of
LMW-PTP on cell adhesion and migration. In conclusion, LMW-PTP plays
multiple role in PDGF-R mediated mitogenesis since it can bind and
dephosphorylate PDGF-R and, at the same time, the cytoskeleton-associated
LMW-PTP pool through the regulation of pl90Rho-GAP phosphorylation
state controls the cytoskeleton rearrangement in response to PDGF
stimulation.
(M0/13.3/325)

Molecular characterization of a reversible protein

tyrosine phosphorylating system in bacteria.
A.J. Cozzone, C. Grangeasse, C. Vincent, E. Vaganay,
M. Riberty, R. Preneta, P. Doublet, B. Duclos.
IBCP-CNRS, 7passage du Vercors, 69007 Lyon, France.

In prokaryotes, even though the occurrence of phosphotyrosine in

proteins was repotted in a series of bacterial species, there has been, so far,
neither characterization nor identification of the enzymatic activity that modifies
these proteins at tyrosine.
In this work we have shown that modification at tyrosine occurs in a
82-kDa protein of the bacterial strain Acinetobacter johnsonii. This protein,
localized in the inner-membrane fraction, has been purified to homogeneity and
shown to autophosphorylate at several tyrosine residues.
The gene encoding this protein, termed ptk for "prokaryotic proteintyrosine kinase", has been cloned and sequenced. Theoretical analysis of the
deduced amino acid sequence has revealed several conserved features of
eukaryotic protein kinases. However, structural studies of protein Ptk, using
site-directed mutagenesis experiments, have shown that the ATP binding site of
this bacterial kinase is different from that generally used by eukaryotic enzymes
and consists of two amino acid sequences that closely resemble the Walker
motifs A and B.
In addition, sequencing of the region immediately upstream of the ptk
gene has led to the characterization of a second gene, termed ptp, encoding a
protein similar to low-molecular-mass phosphotyrosine-protein phosphatases.
We have demonstrated that protein Ptp can catalyze the dephosphorylation of
phosphorylated substrates with a strict specificity for phosphotyro~ine, by using
the same catalytic mechanism as that of eukaryotic phosphatases.
The identification of these two genes, localized in the same cluster and
coding for two opposing activities has led us to study the reversible nature of this
phosphorylation reaction and the control it may play in cell physiology. For that
purpose, overproduction and purification of the two corresponding proteins have
been performed and, interestingly, Pip has been found to specifically
dephosphorylate Ptk.
We have therefore genetically and biochemically identified, for the first
time, a bacterial protein-tyrosine kinase and the cognate phosphotyrosine-protein
phosphatase. It is noteworthy that the two corresponding genes are orgamzed
like a number of clusters involved in the biosynthesis of cell surface
polysaccharides. It has been widely demonstrated that these glycoconjugates are
important virulence factors in bacteria. Consequently, our results suggest the
existence of a relationship between the pathogenicity of bacteria and the
reversible phosphorylation of proteins at tyrosine.
Ref : Grangeasse et al. (1998) J.Mol.BioL 278, 339-347
Doublet et al. (1999) FEBS Lett., in press.

The interaction between p97 repressor and AIdA-NRE

regulates transcription of aldolase A L-type promoter
P. Costanzo., A. Lupo, L. Medugno, C. Zevino and P Izzo
Dip Biochzmicae Biotec. Med, Univevsit3"FedericoI1". Napoli,ITALY

The human aldolase A gene directs transcription of three major mRNAs

species, differing in their 5'-untranslated regions, by usage of multiple
promoters (pL, pM and pF) and alternative splicing of leader exons. We
have found within the pL promoter a negative regulatory element (AIdANRE), highly homologous in human and murine genes, that is recognized
by a 97kDa nuclear protein (p97) [1]. The L-type mRNA transcription is
modulated during cell-cycle and cell differentiation by the interaction
between AIdA-NRE and p97. This interaction is weakned in serumdeprived NIH3T3 cells [2] and in differetiated CaCo-2 cells compared to
the interaction observed in proliferating NIH3T3 cells and undifferentiated
Caco-2 cells, respectively. Here, we demonstrate that the decreased
binding between p97 repressor and AIdA-NRE during the differentiation
of Caco-2 cells, as well as during alternating stages of the cell-cycle in
NIH3T3 cells, is due to a phosphorylation of the repressor. We
demonstrate that phosphorylation is PKC-dependent. Infact, stimulation of
PKC in vivo largely abrogates the binding of p97 to AIdA-NRE, while
inhibition of PKC rescues the binding. Finally, we demonstrate that PKC
downregulation in cells stably expressing an AIdA-CAT fusion construct
increases the binding of the 97kDA repressor to AIdA-NRE and drastically
reduces trascriptional L-type promoter activity. These findings suggest
that PKC is a mediator of the binding and silencing function of the
p97/AIdA-NRE repressor complex
This work was supported by grants from MURST (Cofin '97) and CNR
(target project: Biotechnology)
References
[1] Lupo, A. et al. (1995) Biochem. Biophys. Res. Commun. 216, 69-77.
[2] Lupo, A e t al (1997),/. Biol. Chem. 272, 31641-31647.

(Mo/13.3/326)

Transitory Increase of a EF-1 kinase activity during the

early development of Sea urchin Sphaerechinusgranularis
C. Delalande, A. Mormier, P. Cormier, J.Morales, O.
Mulner-Lorillon and R Bellr.Station biologique de Roscoff,
CNRS-UPR9042, BP74, 29680 Roscoff~

Fertilization triggers in the first hours of sea urchin development

an amplification in protein synthesis wich is associated with an increase
(2.5 fold) in the translation elongation rate.
The elongation phase of protein synthesis is regulated by the
elongation factor-1 (EF-I) composed of a protein complex EF-113~/5,
associated with EF-Io~. EF-1 [~7~5catalyses the exchange of GDP/GTP on
EF-lct wich transfers aminoacyl-tRNA to the ribosome.
We have isolated the cDNA of the sea urchin Sphaerechinus
granularis encoding for the EF-15 subunit of the complex EF-1 (EF-15)
and we have synthetized and purified a fusion protein (GST- EF-18) used
for the analysis of the phosphorylation of EF-15.
Several highly purified protein kinases were used to analyse the
phophorylation of EF-15 in vitro. EF-15 was a substrate for casein kinase
II (CKII) and protein kinase A (PKA). Conversely, EF-15 was not
phosphorylated by the mitotic kinase CDK1. Therefore, sea urchin EF15 behaves like higher eukaryotes counterpart regarding CKII
phosphorylation and not CDK1 phosphorylation suggesting that the
latter is a process established during evolution.
In order to study the protein kinases activities during early
development activity, we have used the fusion protein as a substrate. We
have shown that global phosphorylation activity fluctuates during
development, increasing approximately four fold between 10h and 24h
post fertilization. To determine the protein kinase(s) involved in the
phosphorylation activity, we have used different inhibitors and
stimulators of CKlI and PKA. PKI had no effect on the transitory peak.
In opposition, the transitory activity was inhibited by heparin or 1,3 diPglycerate (inhibition higher than 50%) and stimulated in presence of
spermidine (1,5 to 2 fold).
We conclude that a transient EF-15 kinase activity occurs after
fertilization and could be ascribed to CKII.

s252

Abstracts FEBS'99

(Mo/13.3/327)

SAP kinases and muscle cell differentiation

B.D6rijard, W.Englaro, C.Cabane, B.Cuiller

(Mo113.3/328) Phosphorylation of RelB during cellular activation

E. Derudder ~, D. K. Ferris ~', M. K6rner ~
~CNRS UMR 7627, CERVI, Pitid-Salp@tribre,
83, boulevard de I'hOpital, 75013 PARIS, France.
bNCI-FCRDC, Fredertck, MD 21702, USA.

UMR CNRS 6548, UNSA, Pare Valrose, 06108, Nice, France

Stress Activated Protein Kinases (.INK and p38) have so

far been involved in a lot of inflammation- and stress-induced
transcriptionnal activations. More recently, several differentiation
processes such as neuron, lymphocytes, keratinocytes and vascular
smooth muscle cell differentiation have been shown to depend on
SAPK signalling. Since most components of these cascades are
potently expressed in skeletal muscle cell, we investigated whether
they might be involved in the differentiation and/or proliferation of
this cell type.
Indeed we showed that the terminal differentiation of
C2C12 skeletal muscle cells characterised by myotubes formation
is inhibited in stable clones expressing a dominant negative form of
MKK3, a p38 activator. Other stable clones expressing wild type
JNK or p38 exhibited an abnormal differentiation as well. The
expression of skeletal muscle differentiation markers such as
myogenin, p21 and p27 was impaired in all these clones.
These observations were confirmed by transient
transfection assays using reporter gene expression under the control
of muscle specific gene promoters (myogenin, MCK and MLCK)
as well as stress cascades inhibitors.
We are currently (i) constructing inducible C2C12 clones
expressing various stress cascades mutants to further characterise
the precise role of these cascade in muscle cell differentiation, (ii)
trying to characterise genes whose transcription is dependent upon
SAPK activation during the differentiation process (iii) screemng a
skeletal muscle cell expression library using inactive JNK and p38
kinases.
These experiments should lead to a better understanding of
the precise role of SAPK and MAPK in the proliferation versus
differentiation balance in muscle cells.

(Mo/13.3/329)

Characterization of a novel Cct isoform of PKA

J.-L. Desseyn*, K. A. Burton & G. S. McKnight

Members of the NF-rd3/Rel family are transcriptional regulators of

numerous genes playing roles in the immune response and
inflammation. In resting cells, NF-rd3 is held in an inactive form in the
cytoplasm by members of the I~:B inhibitor family. Following cellular
stimulation, l~Bs are degraded, releasing NF-rd3/Rel dimers which
translocate into the nucleus where they initiate transcription of target
genes. RelB is an atypical member of the NF-rd3/Rel family: although it
is widely expressed in different cells and tissues, its activity seems to
be restricted to certain cells of the immune system. Moreover, dimers
containing RelB are not inhibited by I~:B family members under
physiologic conditions. Consequently, RelB is a predominantly nuclear
protein, even in the absence of cell stimulation. We observed that
nuclear RelB complexes have different abilities to interact with K:B
target sequences depending on which cell lines are examined. In the
human T cell line HPB-ALL, RelB is expressed and located in both the
cytosol and nucleus. Despite its nuclear localization, RelB DNAbinding activity is not constitutive but rather is induced after 6 hours of
stimulation. Currently the mechanism regulating RelB DNA binding is
unknown, but here we will present data indicating that phosphorylation
of RelB is an important step in its activation.

(Mo/13.3/330)

Carbon catabolite repression in Grampositive bacteria

V.Dossonneta, A.Galmicrb, M.Zagorccc and .1 Deutschera
,111//'L tit' ( ;i;nt'llqlle lh'~' i~[ll toor~rlni~olt '~, 1' 7+l~8fi0Thil'crval ( ;ri~,mm.
t'lll~l. (h" Ihohtgle el C'htmte de~ l'roh;itw.~. I: 6t)3t57 ! ~,t+.
, l.ab. de Rt, e herrhe ~ttr la ~ tallth', INRA. 1"-7,~32l) h~llv ell .lo~ls.

Department of Pharmacology, University of Washington+

Seattle, Washington 98195, 7750 - USA

Bacteml ha\c developed a mcchamsm which alhiws them to lake up I?om a

The cAMP-dependent protein kinase (PICA) holoenzyme is a

n l i x t u r c o f tv.,o o r m o r e

sugars only tile preferred carbon s o u r c e , I e tile s u g a r

heterodimer containing two regulatory (R) and two catalytic (C) subunits.

v, htch Js moq easiI3 and most rapidly metabohzed. Despite tile presence ot

Two mouse C subunit isoforms (Ca and CI3) and a pseudogene (Cx) have

the other sugars in the growth medium, the synthesis of the enzymes

been previously cloned. Three splice variants of the murine C!3 gene (C,31-3)

necessary lbr tile uptake and metabohsm of these sugars rcmams repressed

have been characterized and arise from the use of alternate promoters. Cc~ and

until tile preferred carbon sources exhausted This phenornenon Is called CCR.

C131 are N-myristylated and expressed widely whereas the CI32 and C~3 are

In Gram-po~.itive bacterta, a signal transduction cascade is revolved m CCR

brain specific and are not myristylated.

beginning v,'Jth a metabohte-actr,.ated protein kinase. The increase of the

Using a degenerate primer deduced from the amino-terminal sequence

concenmmoa of fructose-l.6-bisphosphate (FBP) accompanying the uptake

of a novel ovine sperm C subunit, we show that the mouse Ca gene encodes

and metal~ohsm of a rapidly nletabolisablc carbon source activates the t-IPr

a second isoform we call C~2. The complete cDNA of this new isoform has

kinase "l'hEs klnase phosphorylatcs l lPr, a phosphocarrier protein of the

been cloned and sequenced and we have mapped the transcriptional start site.

bacterial pho,;photransferasc system (PTS), at seryl resldtle 46 l lPr kinase is

Cod (formerly called Ca) and Ca2 isoforms differ only in their aminoterminal sequence. The Ccd contains a consensus sequence for Nmyristylation whereas the Ca2 does not contain this consensus sequence. The
Ccc2 is the major isoform expressed in mouse testis and using Northern blot
we do not detect any C~x2isoform in brain or heart. Based on its abundance in
adult testis, we speculate that the Cc~2 isoform is the major C subunit in
developing sperm and may play a specific role in sperm maturation or
motility.
* J.-L. D. is supported by a fellowship of the "Human Frontier Science
Program".

a I',/funcllonal enzyme which catal3ses also the dephosphorylatmn of P-SerltPr

The tx,.o opposing acttvlties are regulated by FBP and inorganic

phosphate P-Ser-llPr was Ibnnd to specifically interact w~th the catabohte

control protein A (CcpA), which belongs to the LacllGalR finmly of
trancnptmnal regnlators llnphosphorylatcd HPr or t tPr phosphorytatcd at
Ills-15 does not interact v.~th CcpA The P-Ser-HPr/CcpA complex binds to
tile catabolite responsive element (ere), a czs active operator site located m
(roar of most catabohte-represscd operons and genes in Gram-positive
bacteria 13inding of the P-Ser-HPr/CcpA complex to the ere can either prevent
or, as observed in a few cases, shmulate expression of the following operon.
Most recently, expressmn of genes encoding glycolytic enzymes has also been
lbund to bc regulated by the CCR nlcchanism
\\'C IK!\C /OCt'ill[\ clnllCl.I lhc l~l~]] :lnd /lprlx gCllCS ofitlt'lt~fitll'I]/llt I'H~'('I.

confi~t~,k,t !hill P-~cr-lllh i~ m \ o l \ c d

In

CL'R of/..

t,scl

and

Abstracts FEBS'99

(Mo/13.3/331)

Pulsatile administration of GnRH to gonadotrope cell lines

(aT3-1 and LIST2) selectively affects PKC/t
B. Junoy, H. Maccatio, B. Poulin, J. L. Mas, A. Enjalbert,
and S. V. Drouva

s253

(Mo/0.1/332)

Franz Volhard Chnic and Max-DelbrLick Center for Molecular

Medtclne. Humboldt University at Berhn, German)

I C N E , Fac. de Mddecine Nord, Bd P. Dramard, 13916 Marseille, Fram'e

Protein Kinase C (PKC) comprises a family of ser/thr kinases implicated in

signal transduction and mediates a range of intracellular functions. Previous
studies performed in either normal gonadotrope cells or cell lines have
indicated the involvement of PKC in the regulation of GnRH-induced
signalling events. We have shown that specific PKC isoforms coordinate the
sequential coupling of GnRH receptor with phospholipases C, A2 and D in
c~T3-1 gonadotrope cell line [1]. In the present study, we have examined the
effects of the GnRH on the regulation of several PKC isoenzymes (ct, [3 II, 6,
E, ~) characterized in ctT3-1 (non secretory) and LIST2 (secretory)
gonadotropes cell lines. We demonstrated by SDS PAGE Western blot and
confocal microscopy techniques that in both cell lines GnRH selectively
stimulated (5-30 min) 5, e, ~ isoforms within specific intracellular
compartments. Prolonged GnRH agonist treatment (2-24h) significantly
down regulated in a time and dose (10-7-10-1o M) dependent manner the
expression of PKC 5 and ~ in both cell types. PKC ~ isoform was not altered.
In addition persistent stimulation of these cells with TPA (10 -7 M), a direct
activator of several PKC isoforms, highly diminished at different rates and to
different extents for each isoenzyme, the PKC ct, [3 II, e and 6. Thus,
selective isoforms might be implicated in the process of GnRH-induced
homologous desensitization in these cells. Interestingly, pulsatile
administration of the neuropeptide to the cells selectively down regulated
only the PKC 6. The effect was dose dependent (10-7-10-10 M) and was
observed even after the second GnRH pulse. These results underline the
importance of the individual PKC isoenzyme involvement to different
GnRH-induced cell responses, and demonstrate that the neuropeptide,
depending on the mode of its administration to the cell, selectively regulates
the expression of specific PKC isoenzymes in aT3-1 and LI3T2 gonadotrope
cells.

Urokinase
induces
activation
and
formation
of
Stat
complexes
in human
vascular
smooth
muscle
cells
I. Dumler. A. Kopmann. K. Wagner, H. Hailer. and D.-C. Gulba

Urokinase (uPA) and its specific receptor (uPAR), act in concert to

stimulate cytoplasmic signaling machinery and transcription factors
responsible for cell migration and proliferation. Recently we demonstrated
that uPA activates the Jak/Stat signaling in human vascular smooth muscle
and endothelial cells, namely Janus kinases Jakl and Tyk2 and signal
transducer and activator of transcription Statl. However, the important
question whether other transcription factors of the Stat family, in addition to
Statl, are involved in the uPAR-related signaling has not been addressed. In
this study, we demonstrate that Star4 and Stat2, but not Stat3, Stat5 or
Stat6, are rapidly activated in response to uPA. We demonstrate further
using electrophoretic mobility shift assay and confocal microscopy study,
that Stat4 and Stat2 rapidly and transiently translocate to the cell nuclei
where they bind specifically to the regulatory DNA elements. Analysis of
Stat complexes formed in response to uPA, revealed Stat2-Statl heterodimer
which lacks p48, a DNA binding protein known to combine with StatlStat2. This new uPA-induced Stat2-Statl heterodimer binds to interferon
(IFN)-y activation site (GAS) distinct from the lFN-stimulated response
element (ISRE) to which the p48 protein containing complexes generally
bind. We conclude that uPA activates a specific and unusual subset of latent
cytoplasmic transcription factors in human vascular smooth muscle cells that
suggests a critical role ofuPA in these cells.
Our
finding
that
physiological concentration of uPA induces Star4 activation is, to our
knowledge, the first indication that there is at least one more natural ligand
for the Stat4 protein beyond IL-12.

[1] Poulin et al, Mol. Celt. Endo., 122 (1996) 33

(Mo/0.1/333)

Alterations of insulin signaling in chicken muscle as

compared to rat : evidence for chicken insulin resistance
J. Dupont, C. Dagou, M. Derouet, M. Taouis
lnstitut National de la Recherche Agronomique, 37 380 Nouztlly. France

Upon insulin binding, insulin receptor (IR) undergoes

autophosphorylation and becomes an active tyrosine kinase which
phosphorylates specific cellular substrates such as IRS-1 and Shc.
Phosphorylated substrates are docking molecules for several SH2
containing domain proteins including PI 3'-kinase. Recently, we have
cloned the coding region of IRS- 1 gene 11] and a fragment of She cDNA [2]
in chicken. Both IRS-1 and Shc protein have beet~ also characterized in
chicken liver and muscle. Our previous results also showed a stricking
muscle insulin resistance as compared to liver regardi:'~.q endogene insulin
variations resulting from changes in nutritional status [2, 3]. In order to
understand this phenomena, we have compared insulin signaling cascade in
chicken and in rat (where muscle is known to be highly insulin sensitive) in
response to insulin injection. We have examined tyrosine phosphorylation
oflIL IRS-I and Shc as well as PI 3'-kinase activity in the muscle and liver
of both species. In liver, in the basal state, IR phosphorylation and PI 3'kinase activity were almost twofold higher in rat than in chicken. IRS-1
phosphorylation was similar in both species whereas Shc phosphorylation
was increased in 152% in chicken. After insulin stimulation (10 or 1000
mU/kg), IR, IRS-I and Shc tyrosine phosphorylation were parallely
increased in both species. In muscle, in basal state, IR phosphoryiation and
PI 3'-kinase activity were respectively twofold and thirtyfold higher in
chicken than in rat. After insulin stimulation, IR and IRS-1
phosphorylation and the PI 3'-kinase activity were increased in rat whereas
Shc phosphorylation was unchanged. Interestingly, in chicken, only Shc
phosphorylation was increased by 144% after 1000 mU of insulin. In
conclusion, these results clearly show a muscle insulin resistance in chicken
as compared to rat whereas chicken liver is highly sensitive to the hormone.
[11 Taouis et al., Gene, 178, (1996), 51-55
[2] Dupont et al., Am. J. Physiol., (1998), 274, E309-E316
13l Dupont et al., Biochem J., (1998), 335,293-300

(Mo/0.1/334)

The Stress-Activated Protein Kinases (SAPKs) are

involved in myoblast differentiation
W. Englaro, C. Cabane, B. Cuiller, B. D6rijard.

A TIPE "Signalisation et Diffdrenciation Musculaire % UMR

6548, Facult~ des Sciences, Parc Valrose, 06108 Nice, France.

The stress-activated protein kinases (SAPKs) JNK and p38

belong to the mitogen-activated protein kinases (MAPKs) family.
They are strongly activated by envirormemental stresses such as
UV, septic or osmotic shocks, but also by pro-inflammatory
cytokines including TNFct and IL-1. The skeletal muscle specific
expression of these kinases and their activativators, prompted us
to investigate their role in the proliferation and differentiation of
muscle ceils. We first observed that treatment of C2C12
myoblasts with SB 203580, a specific inhibitor of p38 kinase, lead
to an inhibition of the fusion of myoblasts into multinucleated
myotubes characteristic of muscle differentiation. By gene
reporter assays, we showed that dominant negative mutants of
p38, MKK3 (MAP Kinase Kinase 3) and MEKK1 (MEK Kinase
l) down-regulate the expression of myogenin, myosin light chain
kinase (MLCK) and muscle creatine kinase (MCK) which
expression is increased during myogenesis. On the contrary,
constitutive active forms of these kinases stimulated expression of
these markers. Moreover, C2C12 cells stably expressing a
dominant negative mutant of MKK3 are blocked in their capacity
to form myotubes in differentiating conditions. Therefore, these
results clearly demonstrated that the SAPKs pathways play a
critical role in the muscle differentiation process.

s254

Abstracts FEBS'99

(Mo/13.3/335) Straetural effects of phosphorylafion at the N-terminus of

LHC II
J.Forsberg, A. Nilsson, K.Alexciev, H. L.Race and J. FAllen.
Plant Cell Biology, Lund University, LUND, Sweden

Light harvesting complex-II (LHC lI) is the major antenna complex of

plants. To date there is no detailed information on the three-dimensional
structure of the N-terminal domain which is post-translafionally modified
by a protein kinase active under reducing conditions. Spectroscopic studies
of LHC II have shown that phosphorylation initiates structural changes
within this region of the protein [1]. Such changes could provide a
mechanism for regulating interactions between LHC II and photosystem II
thereby controlling the flow of excitation energy into the photosystem.
We are currently investigating the details of the phosphorylationinduced change in structure of the N-terminal domain of LHC II. To
simplify interpretation, experiments are being carried out using a peptide
corresponding to residues 2-71 of the mature Lhcbl protein from pea,
which is over-expressed in E. coil as a GST-fusion protein. This is extracted
from the cell lysate by GST-affinity chromatography and, after cleavage of
the GST tag, the peptide is purified by size exclusion chromatography. The
N-terminal peptide is phosphorylated by isolated thylakoid membranes or
by a protein kinase-enriched preparation [2]. Spectroscopic studies of the
non-phosphorylated and phosphorylated forms of the N-terminal peptide
are in progress. The site(s) of phosphorylated amino acids will be identified
unequivocally by mass spectroscopy. We aim to crystallize and determine
the three-dimensional structure of both forms of the N-terminal peptide.
[1] A. Nilsson et at., J.B.C., 272, (1997) 18350
[2] H. L Race & G Hind, Biochem., 35, (1996) 13006

(Mo113.3/337) An Alkylated Peptide That Selectively Inhibits MAPK

Activation by MEK in vitro and in vivo
S. A. Goueli, K. Hsiao
Signal Transductton, Promega Corp . Madison, W153711. USA.
and U WJsconstn, Madtson. WI. USA

Mitogen activated protein kinases (MAPKs) are a group of enzymes

whose dual phosphorylation by upstream protein kinases (MAPKKs)
results in their activation. Each consists of a three-kinase module
which includes a MAPK that is activated by a MAPK/ERK kinase
(MEK), which in turn is activated by a MEK kinase (MEKK). Three
main modules are known in mammalian cells, these are ERKs, JNKs,
and p38 MAPK (p38) pathways. They act in concert and with other
cell signaling systems. Therefore, mechanisms must exist to organize
the correct repertoires of enzymes into individual signaling pathways
in order to coordinate cellular responses to extracellular stimuli. This
can be achieved by tethering molecules to their subcellular targets via
anchoring molecules. The phosphorylation of ERK by MEK is a
major step of the ras signaling pathway and requires the interaction of
both enzymes before phosphotransferase reactions commence. We
hypothesized that blocking this interaction will halt the activation of
ERK1, 2 by MEK1, 2. Towards this goal, we have designed a peptide
that disrupts this interaction and consequently inhibits the activation of
ERK. The peptide inhibited phosphorylation and activation of ERKs
by a constitutively active MEK mutant in a dose dependent manner
with 50% inhibition attained at 50 p.M of peptide. Similar results were
obtained with wild type MEK but higher concentration of the peptide
was required. The percent inhibition of ERKs phosphorylation
decreased with increasing ERKs concentration indicating its
competitive mode of inhibition. An alkylated form of the peptide (100
and 200 ~M) was capable of inhibiting the NGF-stimulated activation
of ERK in PC12 in 15 minutes. The nonalkylated form of the peptide
had no effect on ERK activation in vivo although it was as effective as
the alkylated peptide in inhibiting ERK activation by MEK in vitro.
Inhibition of other MAPKs such as JNK and p38 required
significantly higher concentrations. Thus, the alkylated peptide maybe
useful in investigating the role of ERK activation in vivo and serves as
a prototype molecule to design more potent inhibitors of ERKs.

(Mo/13.31336) Identification of the Sites Phosphorylated in PKB B in

Response to Insulin in Primary Rat Adipoeytes
O.Grranssona, L.Rrnnstrand b, V.Manganiello and E.Degerman a
"LU, Lund, Sweden, hCMB, Uppsala. Sweden ,'NIH, Bethesda, MD, USA

The activation of protein kinase B (PKB) in response to insulin and

growth factor stimulation is believed to occur in a two-step process
includingphosphatidylinositol-3,4,5-P3-mediated translocation of
PKB to the plasma membrane followed by phosphorylation and
thereby activation by upstream kinases. The sites reported to be
phosphorylated are Thr 308 and Ser 473 in PKBct and the
corresponding amino acids, Thr 309 and Ser 474, in PKB[L The
hypothesis regarding the mechanism of activation for PKB is based
on work performed almost exclusively using transfected cell lines,
producing a need for the study of PKB and its regulation in a
physiological target cell for insulin. We previously demonstrated an
insulin-induced translocation of PKB to the plasma membrane in
primary rat adipocytes, and we have now proceeded to also study
the phosphorylation of PKB[3 in these cells. In response to insulin
PKB~3 was phosphorylated in a wortmannin sensitive manner.
There was no phosphorylation of PKB[3 in the basal state.
Identification of the sites phosphorylated has been commenced
using two dimensional tryptic phosphopeptide mapping. Two
phosphopeptides appeared after insulin stimulation, both of which
contained mainly phosphoserine.
The identity of the
phosphopeptides
is currently under investigation using
radiosequeneing, secondary cleavagewith specific endoproteinases
and comigration with synthetic phosphopeptides. Preliminary
radiosequencing dataindicate that other sites than Thr 309 and Ser
474 are phosphorylated in PKB[~ in response to insulin stimulation
of primary adipocytes.

(Mo/13,3/338) The C-terminal part of the rbPRL-R negatively

modulates MAP-Kinase activity in transfected CHO cells.
O. Goupillea, J.V. Barnierb, B. Guibertb, J. Palya and J. Djianea
a UmtJ d'Endocrmology Moleculatre. LVRA. "8352Jouv en Josas ceder. France
b [nsttlzlt Alfred bYessard. CARS 91198Gtfsur }ette. France

Prolactin induces cell proliferation and differentiation through MAPK

and JAK/STAT pathways, depending on the cell line [1]. In recent
studies potential crosstalk between these two pathways has been
suggested which culminated in MAPK dependent serine phosphorylation
of STAT5 protein. We previously reported that, in CHO cells. PRL
signaling to milk-protein gene promoters via the JAK/STAT pathway,
could only partially occur without the C-terminal part of the rbPRL-R,
thus implicating this region of the receptor in the amplification of the
PRL induced milk-protein gene transcription [2].
We investigated here whether MAPK activation could interfere with such
amplification. Using MEK (PD 98059) and PI3 kinase (LY 294002,
Wortmannin) inhibitors in CHO cells stably expressing the WT rbPRL-R,
we found that PRL activates MAPK through MEK dependent and MEK
independent pathways. However, addition of PD 98059 did not modify
transcription of the LHRE-TK-luciferase reporter gene (LHRE represents
six copies of STAT5 binding domain from rat B-casein), making, thus,
unlikely the involvement of the MAPK pathway in STAT5 amplification
of milk-protein gene transcription.
By the use of various C-terminally truncated receptor mutants, we next
showed that the MAPK activation by PRL requires the receptor
membrane proximal cytoplasmic domain, including boxl and box2 and is
negatively regulated by the C-terminal part of the rbPRL-R. We tested
the contribution of tyrosine/serine phosphatase, PKC, transcriptional
induced protein and calcium in this negative regulation of MAPK
activity, using various inhibitors. Our results clearly demonstrate the
involvement of a tyrosine phosphatase in this negative regulation. The
effects of the other inhibitors are discussed.
[1] Carey G.B. et al., Arch. Biochem. Bioph., Vol. 316, (1995) pp. I79.
[2] Goupille O. et aI., Mol. Cell. Endocrinol. Vol. 127, (1997) pp. 155.

Abstracts FEBS'99

(Mo/13.3/339)

Phosphorylation and GLUT expression during

erythroid differentiation
M. Grdi,~aa and M. K. White b
Div. Mol Med, Ruder Bogkovt6 Institute, 10000 Zagreb, Croatia
bDept Pathology, Jefferson Medical College, Phdadelphta, PA 19107

The chicken erythroblast cell line HD3 has high glucose transport activity,
which is lost upon differentiation to the red cell phenotype. HD3 ceils,
when incubated under conditions where maturation occurs, show
substantial loss of glucose transporter (GLUT) mRNA. To assess whether
the cAMP or phosphorylation status could sustain losing of GLUT
proteins and GLUT mRNA, the HD3 cells were incubated in the presence
of different phosphatase inhibitors.
Besides decreasing of GLUT transporter activity and mRNA level, the
differentiation of HD3 cells is characterized by increasing of total amount
of pbosphorylated proteins and cAMP level. By using different
phosphatase inhibitors (okadaic acid and sodium vanadate) and a cAMPelevating agent (tBMX), remarkable changes in GLUT transporter activity
and mRNA expression were observed.
In the presence of okadaic acid, the cAMP level did not change
significantly and was maintained stable during experiment. The GLUT
transporter activity decreased and synthesis of Hb was even higher than in
the presence of heroin and butyric acid. The mRNA increased with time of
induction.
Sodium vanadate increased phosphotyrosine-containing proteins, the
cAMP level decreased and synthesis of Hb was very low. The amount of
GLUT mRNA significantly increased, as well as the activity of GLUT
transporter.
IBMX, a cAMP elevating agent, increased the GLUT mRNA level, but
not the GLUT transporter activity. The synthesis of Hb was low.
The presence of phosphatase inhibitors seems to stabilize GLUT mRNA,
but not the proteins. The change of intracellular cAMP level did not affect
the activity of GLUT transporter neither GLUT mRNA. It appears that
phosphorylation/dephosphorylation is closely associated with the
regulation of GLUT expression.

(M0/13.3/341) Effect of insulin and pcroxovanadate on 3-phosphoinositide-dependent protein kinase localization in adipocytes
S Grdlo , JF Tanti,T Grtmeaux , A Casamayor,~ D Alessi* Y
Le Marchand-Brustel " INSERM E99- I 1 and U 145 Nice France. "~
Umrersity of Dundee, Dundee, Scotlan~L U.K.

Recent data indicate that protein kinase B (PKB), a physiological target of

phosphatidylinositol 3 kinase (PI 3K) could play a role in the regulation of
the translocation of the glucose transporters Glut 4 to the plasma membrane
[1, 2]. PKB possesses a Pleckstrin homology domain (PH domain) which
binds to phosphatidylinositol 3-4-5 triphosphate (PI 3,4,5 P3) and is
activated following phosphorylation on both threonine 308 and serine 473 by
two upstream kinases. PDKI (3-phosphoinositide-dependent protein kinase
1), which phosphorylales the threonine 308 possesses a PH domain and
phosphorylates PKB only in the presence of Pl 3,4,5 P3 [3]. To better
understand the mechanism of PKB activation we studied the effect of insulin
and peroxovanadate, an insulinomimetic agent for glucose transport, on the
subcellular localization of PDK1, Freshly isolated adipocytes expressing a
myc epitope-tagged PDKI were fractionated in cytosol and total membranes
and localization of both PDKI and PKB was studied by immunoblotting. In
non stimulated adipocytes, mycPDKl and PKB were localized mostly m the
cytosol. Following pcroxovanadate stimulation, PDKI and PKB were
redistributed to the membrane fractions, while insulin had no effect on this
distribution. Inhibition of P[ 3K activity by wortmannin treatment or deletion
of the PH domain of PDKI (APH PDKI) prevented the peroxovanadateinduced translocation of PDK1 to the membrane. Thus, the PH domain of
PDKI by its binding to Pl 3,4,5 P3 seems to play a role for the localization of
this protein to the membrane. When adipocytes were cotransfected with APH
PDKI and PKB, the translocation of PKB to the membranes in response to
peroxovanadate was markedly reduced. This suggests that PDKI interacts,
with PKB in the cytosol and triggers the translocation of PKB to the
membrane. Peroxovanadate also induced the tyrosine phosphorylation of
both PDKI and 6PH-PDKI on tyrosme 299 and 373 hut the role of this
phosphorylation on PDKI localization and/or activity remains undetermined.
Further, overexpression of PDK I in adipocytes was able to stimulate Glut 4
translocation. Interestingly, the expression of APH PDK1 blocked the
induction of on Glut 4 translocation by insulin. These results strengthen the
idea that PDKI/PKB pathway plays a role in Glut 4 translocation.
I. Kohn, A. D. el al (19961 J. Bwl. Chem., 271. 313722. Tanti, J.-F et al. (1997) Emhwrmology. 138.2t)05-.
3 Aless~.D. R et al (1997) Curt. Biol.. 7. 776-789

s255

(Mo/13.3/340) qnorganic pyrophosphatase from E.coli phosphorylation by

phosphoric acid esters.
O.V. Grigorieva
"Departmenlof Chemistr),Mosco~ State Uniyersity,Moscow%Russia.
Inorganic pyrophosphatases catalyze hydrolysis of the phosphoanhydnde
bond of ihorganic pyrophosphate, which synthesized as a by-product under
the biosynthesis of important biopolymers ( for example proteins and
nucleic acids) According to X-ray analysis, 9 of 15 amino acids carting
functional groups in the active site of PPase are Asp and Glu.
Inorganic pyrophosphatase Iom E colt consists of six chemically identical
subunits and exhibits unusually high reactivity toward phosphoric acid
esters (methylphosphale, phosphoryletanolamine, phosphoglycolic acid and
others) which are affinity inhibitors It is supposed that inactivation kinetics
consists in formation of an enzyme-inhibitor complex followed by
phosphorylati on of the protein
The enzyme displays half-of-the-sites reactivity, with respect to the affinity
inhibitors and produces an inhibition maximum of 50%. In the range of
pH 6 5-9 5 the dependences of inactivation rate on the concentration of
methylphosphate don't submitted to Michaelis-Mentem kinetics and
probably they reflect the subunit interactions Phosphoric acid monoesters
interact with one of the active site of dicarbonic residue It follows from
competitive character of inhibition of methylphosphate interaction with
phosphate, pyrophosphate and sulphate The sulphate binding in the active
site of PPase was confirmed by X-ray analysis Moreover methylphosphate
binding in the active site is proved by a significant decrease in modification
rate of Arg residue in the active site
The formation of covalent bond between the inhibitor and the protein was
proved by MALDI-TOF mass spectrometry. The difference between
the mass of native and methylphosphate modified proteins coressponds to
modificator weight. We suppose that this covalent bond is acylphosphate
one Relative lability of the forming bond (due to this the enzyme is able to
reactivate in time-depended manner) and formation of stable to reactivation
derivative of modified protein alter hydroxylamine treatment favor
this assumption
To locate the modified amino acid the following mutants of active site
o f E.coh inorganic pyrophosphatase were obtained in out laboratois b220Q.
E3 IQ. D65N. D67N. D70N, D97N. DI02N It appeared that the onI?
mutant (D70N) was not macti~ ated by affinity mhibitors

(Mo/13.3/342)

Hyporesponsiveness of T cells in rheumatoid arthritis

due to redox-sensitive subcellular localization of L A T
S.L Gringhuis C.C. Reparon-Schuiit, E.A.VLPapendrecht-vander
Voort, A. Leow, E.W.N. Levarht, F.C. Breedved, C.L. Verwe~

Deportment of Rheumatology, Le~denUnJversi~,Medical Center, P.O. 8~x 9600,

2300 RC Leiden, The Netherlonds

In rheumatoid arthritis (RA), the synovial fluid (SF) T lymphocytes present

in the inflamed joints, display hyporesponsiveness upon engagement of the
TCR/CD3 complex despite phenotypic evidence of former activation.
Previously, we have provided evidence that the hyporesponsiveness of the SF
T lymphocytes is due to the membrane displacement and consequently
deficient phosphorylation of the adaptor protein I_AT (linker for activation of
T cells), which plays a central role in the T cell receptor (TCR)-mediated
signaling pathways.
In healthy T lymphoeytes, LAT is present in the membrane and becomes
heavily phosphorylated on tyrosine residues by ZAP-70 upon TCR engagement,
which is required for the activation of PLC~'I and the subsequent influx of
calcium, and also for the activation of the Ras-RafI-ERK pathway.
SF T lymphocytes exhibit several features of oxidative stress, e.g. severely
decreased intracellular levels of glutathione (GSH). We have previously shown
that the subcellular localization of I_AT is sensitive to changes in the
intracellular GSH levels.
In this study, we set out to examine how the decreased GSH levels induce
the membrane displacement of LAT. We show that the disruption of the
microtubuli and actin cytoskeleton, as has been shown to occur in an
environment of oxidative stress, is not involved in the membrane displacement
of LAT. However, using mutational analysis, we demonstrate that the cysteinto-serine substitution of Cys26/Cys29 and/or Cysll 7 creates a LAT mutant
which is resistant to reduced mtracellular GSH levels. GSH-depleted T
lymphocytes expressing these LAT mutants show membrane localization of
LAT and respond normally to T cell activation.
We conclude that in GSH-depleted T lymphocytes, due to oxidation of the
cystein residues, LAT is unable to adapt the correct conformation to allow
trans-membrane localization, which hence leads to the abrogation of TCRmediated signaling pathways and hyporesponsiveness of the cells.

s256

(Mo/13.3/343)

Abstracts FEBS'99

PKC isotype-speciflc activation of the Na+/H~- exchanger

H. Grumcke, K. Strcsc, S. Kampfer, K. Hellbert, F. [Yberall
and K. Maly

(Mo/13.3/344)

lnsutute 9f Medlcal Chemlst,~v and Btochemistry, Umvers~O, o,

lnn~b uck, Frttz-PregI-Str 3. A-6020 lnnsbruck. Austma

The ubiquitously expressed Na+/H+-exchanger (NHE1) plays an

important role in the regulation of the intracellular pH . Acidification,
hyperosmotic shock as well as growth factors, hormones and integrins
have been shown to stimulate the NHE activity. Growth factor
stimulated activation of the NHE is associated with phosphorylation of
serine residues suggesting a kinase dependent regulation. Evidence is
accumulating indicating that protein kinase C (PKC) is involved in the
activation and regulation of the NHE. It has remained unclear, however,
which PKC isotype is involved. In order to address this question,
representatives from each of the three major sub-families of PKC, i.e.
classical (cPKC), novel (n PKC) and atypical (aPKC) were investigated.
The studies revealed that expression of a kinase-deficient, dominant
negative mutant ofcPKC alpha (cPKC alpha K/R) inhibits EGF-induced
stimulation of the NHE, whereas the corresponding constitutively active
mutant (cPKC alpha A/E) enhances the activity of the NHE by an EGFindependent manner. EGF-induced activation of the NHE is also
depressed by a dominant negative nPKC epsilon mutant. Overexpression
of a constitutively active nPKC epsilon (A/E) mutant, failed to enhance
the NIlE activity. EGF-mediated activation of the NHE was not affected
by expression of a dominant negative mutant of aPKC lambda (PKC
lambda K/W) and expression of the constitutively active aPKC lambda
?dE mutant did not affect the NHE activity. The MEK inhibitor PD98059 did not interfere with EGF-induced activation of the NHE.
It is concluded that in NIH3T3 cells, EGF employs cPKC alpha for the
activation of the NHE and that the activation of cPKC alpha is sufficient
for the stimulation of the NHE. In addition to cPKC alpha, nPKC epsilon
appears also to be involved. However, in contrast to cPKC alpha,
activation of nPKC epsilon is not sufficient for the activation of NHE by
EGF. Neither atypical PKC lambda nor Erkl/2 are implicated m the
EGF-mediated activation of the NHE in this system.

(Mo/13.3/345)

IDENTIFICATION AND CHARACTERIZATION

OF PKCtt IN HUMAN PLATELETS
S. Haussermann, F. Marks, and M. Gschwendt

T lymphocyte activation can be triggered by stimulation of the CD3-TCR

complex or the CD2 molecule. Beside common biochemical events, we
have previously shown that a PLCy-I- and p21'~GAP-associated 62-kDa
protein appeared strongly and specifically tyrosine phosphorylated by
CD2 [1l. We demonstrate here that this protein corresponds to p62 ak that
was recently cloned in human chronic myelogenous leukemia cells and in
marine src-transformed fibroblasts [2,3l. Indeed, the use of specific
antibodies demonstrates that p62ak is rapidly tyrosine phosphorylated
after CD2 but not after CD3 stimulation of Jurkat T cells, p62dk
associates with several proteins after CD2 triggering, whose
characterisations are progress. Furthermore, p62 ak tyrosine
phosphorylation is specific of the CD2 pathway, appearing independently
of the CD3-TCR complex membrane expression. Indeed, CD3-TCR
intemalisation with the anti-CD3 UCHT1 mAb suppress most of the CD2induced tyrosine phosphorylation events, but interestingly, enhances
p62a~ tyrosine phosphorylation. Under these experimental conditions,
p62 ak still associates with PLCy-1 and p21r~GAP although calcium influx
and MAP kinase shift are reduced. Otherwise, kinetic experiments showed
that this enhanced p62dk tyrosine phosphorylation appears early after
CD3 down-modulation, within 5 minutes after UCHTI binding. Thus,
since CD3 stimulation is known to enhance CD2 avidity to its ligand and
to induce CD2 binding to the CD2AP protein, these results also supported
the idea that the CD3-TCR complexprimes the CD2 pathway and mainly
one of its specific components, p62at(
It] Hubert,P. et al., J Exp.Med, 178 (1993), 1587
[2] Carpino.Net al, Cell, 88 (1997), 197.
[3] Yamanashi,Yet al, Cell 88 (1997), 205

(Mo/13.3/346)

Inhibition of the protein kinases by ECAP.

A.Hobta, I.Lisovskiy, M. Soldatkina, N.Markeeva, P.Pogrebnoy
Department of Tumor Cell Btology, R.E.Kavetsky Institute of
Experimental Oncology, 45. Vasylkivska St., Kyiv 25202Z Ukraine

German Cancee Research Center, Hetdelberg, Germany

PKCp_, a novel PKC, differs from all other PKC isoenzymes in that it
contains a putative transmembrane domain and a pleckstrin homology
domain, and lacks an autoinhibitory pseudosubstrate. Moreover, no
physiological substrates have been identified and well-known PKC
substrates are just poorly phosphorylated by PKCo. in vitro. Therefore,
the role of PKC~t in signal transduction is still obscure.
We have started to establish human platelets as a model system for the
investigation of PKCp. functioning. Expression of PKCI.t in human
platelets was demonstrated by immunoblotting of platelet extracts using
specific anti-PKCp, antibodies. Unstimulated platelets contained larger
amounts of the enzyme in the cytosol than in the particulate fraction.
Stimulation by the phorbol ester TPA caused translocation, and
phosphorylation of PKCp_, as demonstrated by a characteristic mobility
shift [1]. Upon TPA-stimulated phosphorylation of platelet extracts in
the presence of G66983, which is known to inhibit all PKC isoforms
except PKCp_ [2], we were able to identify three putative substrates of
PKC/a with apparent molecular weights of 52, 62, and 97 kDa. However,
the major PKC substrate present in platelets, pleckstrin, was found to be
phosphorylated just very weakly by PKC~. Regarding a possible role of
PKC~a in apoptosis of platelets, we found that PKC~t contains a
recognition site for caspase 3 and was specifically cut at this site by the
protease. Moreover, we could demonstrate significant expression of
caspase 3 in platelets.
[1] Rermecke, J., Johannes, F.-J., Richter, K.H., Kittstein, W., Marks, F.,
and Gschwendt, M. (1996) Eur. J. Biochem. 242,428-432.
[2] Gschwendt, M., Dieterich, S., Rennecke, J., Kittstein, W., Mueller,
H.-J., and Johannes, F.-J. (1996) FEBS Lett. 392, 77-80.

p62 ~ , a spoeific intermediate of the CD2 activation

pathway in T lymphocytes
J. Harriague, G. Bismuth, and P. Hubert
Labo d'hnmunologieCellulaireCNRS UMR7627, Paris, France.

During the last decade the antimicrobial peptides were revealed

to play the key role in innate immunity. They were found both in plants
and in animals of different phylogenic groups. The production of
antimicrobial peptide ECAP was shown in A431/1522 human
epidermoid carcinoma cell subline [1]. In addition to its antimicrobial
activity ECAP demonstrated the ability to reduce the EGF-dependent
auto-phosphorylation of the epidermal growth factor receptor (EGF-R)

[2].
Here we report the inhibitory activity of ECAP against the
membrane (EGF-R) and cytoplasmic (PKC!-t, Syk, Lyn) protein kinases.
The peptide reduced the phosphorylation by all investigated kinases with
half-inhibitory concentrations in submicromolar concentrations range.
Besides that ECAP was found to cause erythrolysis and to affect the
growth of cultured eukaryotic cells. Based on the results represented
here we also suppose the ability of ECAP to interact with plasma
membranes leading to their permeabilization.

[1] EV.Pogrebnoy et al., New Microbiol, 21, (1998) 269.

[2] EV.Pogrebnoy et al., Exp Oncol., 19, (1997) 296

Abstracts FEBS'99

(Moll3.3/347)

Vanadate protects osteoblasts from growth inhibition by

cortieosteroids in vitro and in vivo.
PA Hulley, MM Conradie, CR Titus, F Gordon and FS Hough

s257

(Mo/13.3/348)

Department of Endocrinology and Mretabolism, Universctv of Stellenbosch

Medical School. Tygerberg, South Africa

Chronic glucoeortieoid therapy causes rapid bone loss and clinical

osteoporosis. We have found that dexamethasone markedly inhibits the
proliferation of a mouse immature osteoblast cell line (MBA 15.4). This
correlates with a 30-40% decrease in PKC/TPA stimulated MAPK (ERK)
activity. Steroid treatment must take place for 6-24hr for inhibition to
occur, suggesting that de novo synthesis of proteins is involved Inhibition
of tyrosine phosphatases with sodium orthovanadate, either concurrently
with the steroid or 5 min before TPA stimulation, restored a normal ERK
response. Vanadate also markedly improved cell proliferation when coadministered with dexamethasone. Sodium fluoride (inhibitor of
serine/threonine phosphatases) was ineffective.
Osteopenia was induced in 3.5 month old female Sprague-Dawley rats by
sub-cutaneous injection with 5 mg/kg/day methyl-prednisolone for 9 weeks.
Rats were treated with steroid alone or in combination with 0.5 mg/ml
sodium orthovanadate, administered a d libitum in drinking water. Both
vanadate and normal drinking solutions contained ascorbic acid and were
pH 7.0. Bones were tetracyclin double-labelled by injecting all rats with 25
mg/kg intramuscular terramycin 13 days and 3 days prior to termination.
Steroid-treated rat bones were significantly shorter (10mm, P<0.01),
thinner (0.1ram, P<0.05) and less dense as measured by DEXA (5
g/cm2x 10"3, P<0.01 ) than controls. Quantitative bone histology showed that
they had a significant decrease in osteoblast activity as seen by impaired
bone formation and osteoid content Rats treated with steroid plus vanadate
were physically, densitometrieally and histologically indistinguishable from
untreated controls. These results suggest that steroid-induced bone damage
is, at least in part, caused by upregulation of tyrosine phosphatases and that
these effects can be reversed by sodium orthovanadate.

(Mo/13.3/349)

Synthesis and phosphorylation of RNA polymerase I

transcription factor UBF in PHA stimulated lymphocytes
I. Kalousek, D. Jandovfi, P. Kfi~&ovfi
[nstttute of Hematology. U nemocnice 1, 12820 Prague 2.
Czech Republic

The nucleolar transcription of ribosomal genes requires besides RNA

polymerase I and topoisomerase I additional protein factors: the selectivity
factor SL-1, the upstream binding factor UBF, required for enhancer function,
and elongation and termination factors. Recent studies have shown that
phosphorylation reactions are involved in adapting ribosomal gene
transcription to cell proliferation and that the phosphorylation state of certain
transcription factors is important in modulating their transcriptional
properties. Lymphocyte stimulation by phytohemagglutinin (PHA), converting quiescent lymphocytes into rapidly proliferating lymphoblast-like
cells, is a convenient model system for investigating the process of cell
division. In this study we examined a correlation between expression and
phosphorylation of UBF and transcription of ribosomal genes during the
lymphocyte mitogenic stimulation. Using the nuclear Run-On transcription
assay we demonstrated, that after PHA stimulation the ribosomal gene
activity increased rapidly during the first 48 hours. Within 64 hours it reached
its maximum. In contrast the UBF intracellular level, examined in immunoblots, paralleled cell
proliferation activity monitored by BrdU cell
proliferation ELISA. During the first 48 hours the lymphocytes did not
proliferate and the UBF level corresponded to that found in quiescent cells.
However the UBF intracellular content increased by nearly 2-fold in the
interval from 48 to 64 hours following PHA addition. The metabolic radiolabeling with [32p] orthophosphate for 16 hours, followed by immunoprecipitation and autoradiography enabled us to examine UBF phosphorylation states. The results showed that cellular content of UBF in underphosphorylated state was likely rate-determining factor of UBF phosphorylation. We suggest that UBF phosphorylation observed within the first 48
hours after PHA stimulation corresponded to the transformation of
underphosphorylated intracellular UBFI and UBF2 pool and predominated
the phosphorylation of neo-synthetized UBF molecules. Subsequently, within
the time period from 48 to 64 hrs the phosphorylation of neo-synthetized
UBF predominated. We speculate that the phosphorylation ofunderphosphorylated intracellular UBF pool present in quiescent lymphocytes was
implicated in the ribosomal RNA synthesis acceleration.

Mutations in the RSK2 gene are responsible for

syndromic and non- syndromic mental retardation.
S Jacquot, K Merienne, S. Pannetier, J L. Mandel,
P. Sassone-Corsi, A. Hanauer
lnstitut de G~nettque et de Biologie Mol~culaire et Cellulmre (IGB3/[C),
CNRS/INSERA~/ULP, 67404 lllklrch Strasbourg, France.

X-linked mental retardation (XLMR) is a highly heterogeneous condition

including more than 100 distinct disorders. Two subtypes are distinguished:
non-specific forms (MRX), in which mental retardation is the only clinical
manifestation, and syndromic forms in which mental retardation is one
clinical manifestation of a complex syndrome. We have recently shown that
mutations in the gene encoding the growth-factor induced kinase RSK2 are
responsible for a syndromic mental retardation, the Coffin -Lowry
syndrome. Affected males exhibit characteristic facial and digital
dysmorphisms and progressive skeletal deformations, and mental handicap is
generally very severe. Over 45 different mutations have so far been identified
in Coffin-Lowry patients. They are distributed throughout the RSK2 gene
with no clustering and almost all are unique. A very high rate of de novo
mutations (68%) is observed.
Three large families with non-specific mental retardation, in which the
disease locus encompassed the RSK2 gene, were also analysed for the
presence of a mutation within RSK2. In one family a missense mutation was
found. Functional studies of the mutant protein demonstrated that the
mutation impaired severely the signalling efficiency of RSK2 in these
patients. The majority of CLS mutations completely inactivates RSK2 but in
this case, RSK2 has a residual activity (15-20%) which leads to a mild
phenotype. This mutation pinpoints an autopbosphorylation site necessary
for optimal kinase activity. These findings suggest that genes like RSK2
already implicated in syndromal MR)( may also be mutated in cases of nonspecific MRX indicating a more complex genetic and phenotypic
heterogeneity in MRX phenotypes.

(Mo/13.3/350)

Melatonin-indueed organelle movement in melanophores

is coupled to tyrosine phosphorylation of a HMW protein
A. Karlsson and S. Svensson
Dept. of Medicine and Care, Div. of Pharmacology, Faculty of Health
Sciences, SE-581 85 Linkd}oing, Sweden

Melanophores, brown or black pigment cells, from X e n o p u s laevis are

well suited for studies on organelle movement. These cells are
specialized for intracellular transport of melanin filled organelles,
melanosomes, along microtubules in order to change the hue of the
animal skin.
We have studied the signal transduction pathway leading to centripetal
movement of pigment granules, after melatonin stimulation.
Melanophores were preincubated with genistein, an inhibitor of
tyrosine phosphorylations, and PD-98059, an inhibitor of MEK (MAP
kinase kinase) and thereafter stimulated with melatonin. Aggregation
of pigment granules was inhibited by genistein in a dose-dependent
fashion as determined by spectrophotometry at 650 nm. Preincubation
with 100 p.M PD-98059 almost completely inhibited aggregation of
pigment granules but 50 p.M PD-98059 had no effect.
SDS-PAGE and western blots were performed on cell lysates from
melatonin stimulated and control cells. Melatonin stimulation induced
tyrosine phosphorylation of a protein with a molecular weight of more
than 250 kDa. Cells preincubated with genistein, followed by
melatonin stimulation, showed a lower degree of tyrosine
phosphorylation of the same protein. Given these results, we suggest
that melatonin stimulation of melanophores leads to tyrosine
phosphorylation of a high molecular weight protein, an event that is
essential for centripetal movement of pigment granules.

s258

(Mo/13.3/351)

Abstracts FEBS'99

Identification of phosphorylation sites of B-Raf: implication for

specific regulation of the kinase among Raf family.

(Mo/13.3/352)

Phosphorylatiun of MAG and the effect of MAG

on protein kinase autophosphorylation in vitro
Petri Kursula, Veli-Pckka Lehto, and Anthony M. Heapc

S K6nig, B. Guibert, C. Morice, P. Vernier, J.D. Vincent and J -V. Barmer

Institut A. Fessard / CNRS - Gff-sur-YvetteFrance.

The B-Raf member of the Raf family of serine/threonine protein

kinases, acting as a MAP kinase kinase kinase, is responsible for the
activation of the MAPK/ ERK pathway in PCI2 cells and the mammalian
brain, to which B-Raf expression is essentially confined. Since recent
advances have emphasized the role of phosphorylation events in the regulation
of the ubiquitously expressed R a f 1 kmase, we wondered if B-Raf could "also
be regulated by phosphorylation and if such events could prowde an
additional mechanism for differential regulation of Raf kinases.
Out results showed that the tryptic digestion of the 95-kDa B-Raf
isoform immunoprecipitated from metabolically labeled PCI2 cells, revealed
seven phosphopeptides. Five of them were observed on the two-dimensional
phosphopetide map of the kinase-defective 95-kDa B-Raf protein which was
phospborylated in vitro by PKA, thus providing an in vitro system of
phosphorylation that facilitates the identification of these sites. The in vitro
phospbol2clatton of deleted B-Raf constructs, the recognition of consensus
motifs of phosphorylation and finally the site-directed mutagenesis of the
putative phosphorylation sites allowed us to demonstrate that serines 364,
429,446, 604/606 and 728 of B-Raf were phosphorylated in vitro by PKA.
RP-HPLC analysis further confirmed the co-elution of the in vitrophosphorytated tryptic peptides with five of the tryptic peptides
phosphorylated in PC 12 cells, strongly suggesting that the serine residues we
identified in vitro will correspond to in vivo phosphorylation sites of B-Raf
protein. If four of these sites are homologous to Raf-I residues which have
been identified as regulator sites lbr Raf-I protein, one of them (serine 429)
corresponds to a B-Raf-specific phosphorylation site. We are now assessing
the consequences of the phosphorylation of this latter residue on B-Raf
activity and on the ability of the phosphorylated kinase to drive M A P K / E R K
activatton.

UmverstQ" of Oulu and Ouhl Lgliverstty tIospital, Oltlu, Finland

The myehn-asstviatcd glvcopmtem is a tmnsmembmne cell adhesion

protein belonging to the tmmunoglobulin superfamd 3. Its two Jsolbrms,
S- and L-MAG, are expressed exclusl~ ely by mychnatmg glial cells of the
ncrxous s)stem, and they dfflcr onl 3 b 3 their respccmc c3toplasmic
domains. Wc ha',e studied the phosphor3latlon of recombinant MAG
c~toplasmtc domains in vitro by protein klnases A and C, and b3 casein
klnase I1. Protein kinase C phosphorblated both MAG isoforms, ~ hile
protein kinasc A phosphorylated L-MAG much more strongly than SMAG. A major threonme phosphowlatam site for protcm kmase A, Thr(O7, uas identdied m the L-MAG-specific domam. In addition, serine
phosphor31atton of the L-MAG c3toplasmtc domain b 3 protein kmasc A
~xas s~gnificantl 3 decreased in the presence of the SI(X)['I protein. SI()t~
is an EF-hand calcmm-bmdmg protein that we have recently idcntdicd as
the first non-kmasc cytoplasmtc hgand for L-MAG. Furthermore, the
presence of the MAG c3toplasmic domains had effects on the
atttophosphorxlation of all three kmases. The presence of L-MAG
dot~ nrcgulated the autophosphorylatmn of protein kmases A and C, ~ hlle
the autophosphorylat~on of casein kinase 1I ~as increased severullbld b3
D~th MAG Jsoforms. The results suggest a role for both MAG lsoforms
m the rcgulatum of stgnalhng path~ays m mychnating ghal cells.

Our results reinlbrce the prevtous observatmns indicating that Raf

kmases would share comrnon regulatory mechanisms but also possess
specific ways to be regulated. The ability to differentially regulate these
kinases could implicate B-Raf in specific cclluIar processes governed by
M A P K / E R K activation, notably in the nervous system.

(Mo/13.3/353)

Fyn membrane localization controls the constitntive

phosphorylation of Sam68 in the nucleus of T cells.
V. Lang, M. Semiehon, F. Michel+, H. Gary and G.
Bismuth. CNRS UMR 7627, HOpital Pitid, Paris, France + lnstitut
Pasteur, Paris, France

A close relationship between Sam68, a tyrosine and proline-rich

RNA binding protein, and Src protein tyrosine kinases (PTK) has already
been established, also in T lymphocytes. A constitutive phosphorylation of
the molecule has also been documented in various transformed T cells
which probably reflects an increased expression of PTK of the Src family.
Using the hybridoma T cell line, T8.1, or Jurkat T cells, we investigated the
respective contribution of the two Src kinases Fyn and Lck, expressed in T
cells, in this phenomenon. We show that the constitutive phosphorylation
of Sam68 in vivo directly correlates with cellular Fyn levels but not with
Lck expression, despite the capacity of the PTK to strongly phosphorylate
the molecule in vitro. By analysing the distribution of phospho-Sam68
molecules in T cells overexpressing Fyn, we show that phosphorylated
Sam68 molecules are expressed in a membrane-enriched fraction but also,
predominantly, in the nucleus. Overexpressed Fyn is exclusively localized
in the cell membrane compartment but we found that Sam68
phosphorylation in the nuclear fraction is lost with a delocalized Fyn
mutant deleted of its N-terminal membrane-anchoring domain. Finally we
demonstrate, using a cons~uct encoding a Sam68 molecule without its
nuclear localization signal, that nuclear expression of Sam68 is not
required for phosphorylation, We conclude that the constitutive
phosphorylation of Sam68 in T cells is a Fyn-dependent process occuring
in a cell-membrane compartment from which phospho-Sam6g molecules
can thereafter accumulate into the nucleus.
[1] Richard, S.et al. Mol. Cell. Biol. 15, (1995) 186.
[2] Fusaki, N. et al. J. Biol. Chem. 272, (1997) 6214.
[3] Lang, V. et al.. Eur. J. Immunol. 27, (1997) 3360.

(Mo/13.3/354)

Spatio-temporal regulation of p42/p44 MAPK activation.

P. Lenormand, V. Volmat, D. Grail, and J. Pouyss6gur
Centre de BmchimleCNRS UMR 6543 Facultd"des Smcnces
Pare Valrose 06108Nzce

Growth factors activate of the p42/p44 MAPK cascade during the entire
G0/GI period of the cell cycle. We had shown that this activation is
absolutely reqtured for fibroblast cell growth. The two upstream kinases of
the module. Raf-1 and MEK are exclusively cytoplasmic, whereas p42/p44
MAPKs translocate to the nucleus during mitogenic stimulation. Recently,
we have shown that preventing p42/p44 MAPKs nuclear translocation by
sequestering them in the cytoplasm severely reduced grov, th factor induced
cell cycle progression. Therefore nuclear translocation of p42/p44 MAPKs is
a critical step in the growth signaling cascade.
The nuclear translocatlon of p42/p44 MAPKs is controlled by the strict
activation of the p42/p44 MAPK cascade suggesting that p42/p44 MAPKs are
retained in the cytoplasm of resting cells via a MAPK-sensitive 'anchor'.
During the initial steps of mitogenic signalling (less than 30 minutes),
p42/p44 MAPKs tmnslocate to the nucleus independently of protein
synthesis. However, in the middle of the GI phase of the cell cycle,
activation of p42/p44 MAPK triggers the neosynthesis of short-hved nuclear
anchoring protein(s) that retain p42/p44 MAPKs m the nucleus.
The temporal activation is another key issue for determining biological
responses In this respect, expression of MAP kinase phosphatases (MKP1,
2. 3) is also strictly controlled by the activation of file MAP kinase module,
providing an autoregulatory mechanism. Using specific anubodics that
recognise only the active forms of p42/p44 MAPKs, first we were able to
demonstrate that active p42/p44 MAPKs translocate within five minutes to
the nucleus. Surprisingly, at times when we detect the maximal accumulation
of p42:p44 MAPKs in the nucleus (three hours), it is inactive. We therefore
propose thal long term p42/p44 MAPK nuclear retention is a mechanism of
signal termination.

Abstracts FEBS'99

(Mo/133/355)

Tyrosine Phosphorylation of IgB-c~ : a New Mechanism of

NF-gB Activation Involving LCK and ZAP-70 in T Cells
A. Livolsi, V. Busuttil, V. Imbert and J-F. Peyron

s259

(Mo/13.3/356)

INSERM CJF-9605, Facultd de Mddecme Pasteur, F-06107 Nice cedex 2

Sensitivity of Calcium-Dependent Protein Kinase

from maize seedlings to phospolipids
A. Liwosz, J. Szczegielniak, I. Jurkowski, G. Dobrowolska,
G Muszyfiska
lnstttute of Btochemtstry and Biophysics PAS, Warsaw, Poland

The transcription factor NF-~B regulates expression of many genes

participating in immune and inflammatory responses. NF-~B is activated by
removal of the inhibitory subunit of the I~B family (m iS, or ~,) that masks
its nuclear localization signal. In T lymphocytes upon stimulation, b:B-~ is
phosphorylated on serines 32 and 36 by a large multiprotein complex, the
I:<B kinase (IKK) signalsome, that leads to its polyubiquitination and
degradation by the proteasome [1]. We described an alternative mechanism
of NF-~cB activation through phosphorylation of IKB-c~ on tyrosine 42. In
this case, NF-~B nuclear translocation is observed without proteolytic
degradation of the inhibitor 12]. Using Jarkat mutants, we show here that
deficiency in the Protein Tyrosine Kinases (PTK) Lck or Zap-70 profondly
impaired pervanadate-induced both tyrosine pbosphorylation of I~B and
NF-~cB activation. The pharmacologic inhibitors PP1 and piceatannol, that
respectivcly affect Src or Zap/Syk PTK blocked activation of NF-gB by
pervanadate but not by TNFc. Reconstitution experiments in COS-7 cells
showcd that Lck and Zap-70 cooperate to induce tyrosine phosphorylation
of I~B-c~. These results demonstrate that the signaling pathway leading to
phosphotyrosine-dcpendent NF-~cB activation in T cells involves LCK and
ZAP-70 tyrosine kinases.
[1] Schcidereit, C. Nature 395. (1998) 225.
[2] lmbert. V. and al. ('ell 86. (1996) 787.

(Mo/13.3/357)

Peptide phosphorylation by Ca~-dependent protein

kinase from maize seedlings
M. Loog a,b, R. Toomik b K. Sak ~, G. Muszynska ~, P. Ek b,
J. Jarv"
a htstJtute of Chemical Physics, Tartu University, 2 Jakobi St., EE 5 I014
Tartu, Estoma
aDepartment of Medical Biochemistry and Mtcrobiology. Uppsala
Umversity, S-75 123 Uppsala, N~eden
lnsntute of Biochemistry caatBtk~physics, Pohsh Academy of Sciences, A.
PawmskJego 5a, 02-106 Warsaw, Poland

Ca2-dependent protein kinase (CDPK-I) was purified from maize seedlings

and substrate specificity of this enzyme was studied using a set of synthetic
peptides, derived from the phosphorylatable sequence RVLSRLHS(15)VRER
of maize sucrose synthase 2. The deca-peptide LARLHSVRER was found to be
a minimal substrate efficiently phospberylated by this enzyme, The same set of
peptides was found to be phosphorylated by mammalian protein kinase C[8
(PKC), but revealed low reactivity in the case of protein kinase A (PKA).
Proceeding from the sequence LARLHSVRER a series of SPOTs-attached
peptides of systematically modified structure was synthesized. These peptides
had hydrophobic (Ala, Leu) and ionic (Arg, Glu) amino acids subsequently in
each position. The phosphorylation of these substrates by CDPK-I was
measured and the substrate specificity of the maize protein kinase was
characterized by the consensus sequence motif
A/L.sX.4R.3X.IX_t SX+I R+2E+3R+4
This motif had a rather characteristic sequence element RER at positions from
+2 to +4 and resembled well the primary structure of the sucrose synthase
phosphorylation site. The sequence surrounding the phosphorylatable serine in
this consensus motif was close to the analogous sequence RXXSXR proposed
for mammalian PKC, but different from the consensus motif RRXSX for PKA.
This probably explains the different phosphorylation rate of the CDPK-1
peptide substrates by PKC and PKA and agrees with the hypothesis that by
using CDPKs for mediation of the Ca2+ -dependent cellular signals plants may
have surpassed the need for developing kinases homologous to PKC.

In plants, the presence of protein kinase activated by calcium and

phospholipids, resembling protein kinase C (PKC) is not clear, yet
Recently, we have dernonstrated that maize protein kinase is activated
by phospholipids
The m a i z e protein kinase, activated by calcium ions,
phosphatidyloserine and diolein was purified 500 times The
procedure of purification included ammonium sulfate fractionation,
hydrophobic chromatography (Octyl-Sepharose), ion exchange
chromatography (DEAE-52 and Mona Q - FPLC), and affinity
chromatography on immobilized substrate (histone -Sepharose). The
activity of partially purified enzyme was analyzed using ,,in solution
assay" and ,,in gel kinase assay" techniques with histone H1 and
Myelin Basic Protein (MBP) as substrates
Western blots experiments have shown that tile maize protein kinase,
sensitive to phospholipids, cross-reacts with antibodies directed
against the cahnodulin-like domain of Calcium-Dependent Protein
Kinase (CDPK) fiom soybean (the antibodies were a kind gil~ of Prof
Alice Harmon from Florida University). Tile enzyme activity was
COiTelated with the imnnmoreactivity of 54 kDa CDPK
The maize protein kinase was not inlnbited by the pept~de exhibiting
pseudosubstrate of PKC up to concentration 50 gM, whereas ttns
peptide was potent inhibitor of PKC from rat brain Contrary to that,
peptide from junctton domain of ('DPK mhibLts both protein kmases
with similar inhibttory constant of abater 4/.tM
The presented data suggests that the studied maize protetn kmase is a
member of Calclum-Dependent Protein Kmase l:annly and represents
a class of protein kmases which functionally could substitute PKC

(Mo/13.3/358)

KIS, a protein kinase with an RNA Recognition Motif.

A. Maucuer, V. Maneeau, S. Ozon, A. Sobel
INSERM U440, IFM, 17 rue du Fer h Mouhn, 75005 Paris, France

We are studying a novel kinase which possesses a characteristic RNA

Recognition Motif (RRM). We initially identified this kinase as a stathmin
partner in the two-hybrid system and thus called it KIS for "Kinase
Interagissant avec la Stathrnine" [1]. Stathrnin is a small cytoplasmic
ubiquitous phosphoprotein that is enriched in the nervous system. It is a
major phosphorylation substrate in response to diverse cell stimulations and
we proposed that it acts as a general regulator interacting with multiple
target proteins.
KIS is formed by the juxtaposition of an N-terminal kinase domain that is
not strongly related to any previously characterized one and a C-terminal
RRM containing domain [2]. This unique structure leads us to speculate that
KIS acts as a regulator of gene expression through phosphorylation and
regulation of RNA associated factors. Interestingly the RRM domain is
highly homologous (40% aa identity) with the C-terminal RRM of the
splicing factor U2af, which suggests a functional homology. Our analysis of
KIS expression indicates a particular implication in the developing as well
as the adult nervous system. Furthermore, when over-expressed in cultured
cells, KIS accumulates in the nucleus which might be relevant for its
proposed implication in controlling the metabolism of RNAs.
In order to analyse the catalytic activity of KIS we produced it in bacteria.
In vitro, recombinant KIS autophosphorylates and phosphorylates stathmin
on serine residues. It phosphorylates also other classical substrates such as
MBP and synapsin. A systematic approach for identifying K1S substrates in
brain fractions indicates that KIS phosphorylates a few specific substrates.
In vitro, we observe that KIS activity is repressed by histones and the Cterminal domain is necessary to have full autophosphorylation activity.
Phosphorylation might also participate in regulating KIS activity.
Identification of KIS substrates and regulations will shed light on its
implication in the control of protein expression.
[1] Maucuer at al., Proc. Natl. Acad. Sci. U.S.A., 92, (1995) 3100.
[2] Maucuer et al., J. Biol. Chem., 272, (1997) 23151.

s260

(Mo113.3/359)

Abstracts FEBS'99

New insight into RSK2 kinase activation.

K. M6rienne, Y. Lutz, M. Oulad-Abdelghani, S. Jacquot, S.
Pannetier, J.-L. Mandel, P. Sassone-Corsi, A. Hanauer.
IGBMC (CNRS/INSERM/ULP), BP 163, 67404 l]lkirch C U de
Strasbourz. France.

Ribosomal $6 kinase-2 is one member of a family of growth factorregulated serine/threonine protein kinases, that contain two heterologous
kinase domains linked together by a regulatory region. Substrate
phosphorylation is mediated by the N-terminal kinase domain of RSK2.
Full activation of the enzyme requires activation of both the C- and Nterminal kinase domains, and phosphorylation of serine and threonine
residues located in the linker region. Whereas extracellular signal-regulated
kinases (ERK) activate the C-domain upon mitogenic stimulation, the Ndomain is activated both by a constitutive-activated kinase, possibly
PDKI, and by the activated C-domain, suggesting PDK1 and ERK act in
a cooperative manner. However, the way the two kinase domains of
RSK2 cooperate according to their phosphorylation state is not yet fully
understood. The goal of this work is to study the subordination of one
domain to the other according to the stimulation state of the cells. For
this purpose, we have developed antibodies directed against PhosphoSer227RSK2 (activation loop of the N-domain) and PhosphoThr577RSK2 (activation loop of the C-domain). In addition, we have
used the commercially available anti-Phospho-Ser386RSK2 antibody
(linker region). Our first results indicate that in unstimulated cells, the Ndomain contributes to repress the activation of the C-domain. In this
conformational state, phosphorylations of Ser227 and Ser386 favor this
inhibition. In contrast, when cells are stimulated with growth factors, thc
inhibition is removed. This step allows the activation of the C-domain via
the ERK pathway and leads to an increase of the phosphorylation levels
of Ser227 and Ser386. As a consequence, the enzyme is fully activated.

(Mo/13.31361)

V E G F transcriptional induction by p42/p44 MAP

kinase (ERK) module is mediated by Spl and AP-2
J. Milanini, F, Vifials-Canals, J. Pouyss~gur and G. Pag+s.
UMR 6543-CNRS, Centre de Bzochtmw, Parc Vahvse. 06108 Nzce, France

Vascular Endothelial Growth Factor (VEGF) is a potent mitogen for

vascular endothelial cells and has been implicated in tumor
neovascularization. In hamster fibroblasts (CCL39 cells), VEGF mRNA is
expressed at low levels in serum deprived or exponentially growing cells
whereas it is rapidly induced after stimulation of quiescent cells with serum.
To analyse the contribution of ERK module in this induction, we used a
CCL39-derived cell line (Raf-I :ER) expressing a Raf-I chimera which can be
activated by estradiol. We clearly show a time and a dose-dependent upregulation of VEGF mRNA detectable between 1 and 2 hrs of stimulation
with estradiot.
Interestingly, hypoxia has an additive effect on VEGF induction in
CCL39 cells stimulated by serum or in Raf-I:ER cells stimulated by estradioh
We have identified a GC-rich region of the VEGF promoter between -88
and -66 base pairs which contains all the elements responsible of its upregulation by constitutive active Ras or MEK 1 By mutation of the putative
binding sites and electrophoretic mobility supershift experiments, we show
that the GC-rich region constitutively binds Spl and AP-2 transcription
factors. Furthermore, following exclusive activation of the ERK module, the
binding of Spl and AP-2 is increased in the complexes formed on this region
ofthe promoter. Raf-l-initiatedactivation of Spl and AP-2 binding activity
can be observed in 15 minutes, which corresponds to the peak of ERK
activation. In vitro, active ERK2 strongly phosphorylates the Zinc-finger
domain of Spl, which is implicated in its DNA binding, but not AP-2. We
are now looking for the ERK target site on Spl in vitro. We are also testing
whether this modification is sufficient for the enhancement of Spl DNA
binding.
This study demonstrates the importance of ERK pathway in the
mechanisms of angiogenesis via VEGF expression.

(Mo/13.3/360)

Role of G protein-coupled receptor kinases in

desensitization of human thyroid tissues
T Mrtayr, J-L Kraimps, J-M Goujon, P Babin and J Barbier
Groupe de Recherche en Endocrinologie F~pdrimemale et Chnique,
Hrpital Jean Bernard, BP 577. 86021 Pouters Cedex, France

Desensitization of a G protein-coupled receptor is a general phenomenon

which starts by phosphorylation of the receptor by G protein-coupled receptor
kinases (GRKs) or second messenger kinases (PKA or PKC), resulting in
receptor uncoupling from G protein. In thyroid, overexpression of GRK2,
GRK5 and GRK6 in cell lines suggested that these kinases were involved in
regulation of thyrotropin (TSH)-stimulated cAMP accumulation. The aim of
this study was to compare desensitization levels to GRK actwities or GRK2
expressions in different human thyroid tissues, to better prove the
physiological importance of GRKs in TSH signaling regulation.
Four human thyroid tissues, including a tissue from a patient with Graves'
disease, a toxic adenoma, a papillary carcinoma and its adjacent histologically
normal tissue were available for investigation in a same tissue, desensitization,
G R K activity and GRK2 expression. After 20 hrs in primary culture,
desensitization was studied on thyroid cells stimulated for 20 rnin or 2 hrs by 1
mU/ml TSH. Cells were washed and subsequently stimulated for 15 rain by 10
mU/ml TSH. Intracellular cAMP was assayed by means of radioimmunoassay,
G R K activities and GRK2 expressions were measured on tissue extracts
partially purified on SP Sepharose. GRK activity was assayed by studying
phosphorylation of light-activated rhodopsin contained in rod outer segments
of bovine retinas. GRK2 expression was assayed by immunoblot analysis.
A 20 min and 2 hrs preincubation of normal thyroid cells wtth 1 mU/ml
TSH decreased subsequent stimulation of cAMP production by 10 mU/ml
TSH, respectively by 32 and 78.6 %. In cells from the toxic adenoma, the
papillary carcinoma and the Graves' disease tissue, results for 20 min
desensitization were respectively, 68.6, 10.1 and 0 % and results for 2 hrs
desensitization were respectively, 89.3, 44.4 and 30.5 %. GRK activities
assayed m the normal tissue, the toxic adenoma, the papillary carcinoma and
the Graves' disease tissue, were respectively, 0.64, 0.67, 0.49 and 0.99 pmol
phosphate/mra/mg protein. A positive correlation was observed between GRK
activities and GRK2 expressions, suggesting that GRK2 was the main GRK
isoenzyme in human thyroid. A positive correlation was also observed
between GRK activities or GRK2 expressions and desensitization levels in the
normal tissue, the papillary carcinoma and the toxic adenoma, but not in the
Graves' disease tissue.
In conclusion, the results show the importance of GRKs, specially GRK2,
in desensitization of different human thyroid tissues. The therapeutic treatment
of the Graves' disease patient may explain the absence of GRK role in
desensitization of this tissue.

(Mo/13.3/362)

Selective increase of protein kinase CK2

during the cell cycle
F. Mir6, M. Plana, E. ltarte.
Dep. de BioqMmica i Biologia Molecular. Fac.de Ci~ncies. Universitat
Autbnoma de Barcelona. 08193 Cerdanyola. Spain.

The levels of protein kinase CK2 increased in the S I and $2 nuclear

subfractions during the transition from GO to GI/S of HepG2 cells. However the
levels of this protein kinase in cytosolic, MI and M2 fractions did not vary, A
specific subcellular increase of protein kinase CK2 was also observed in cells
arrested at G2/M, together with an increase in the p53 tumour suppressor
protein, an intracellular substrate of protein kinase CK2. No significant changes
were observed in the levels of elF-2 during the cell cycle but different isoforms
of elF-2 beta subunit, probably due to CK2 phosphorylation, were detected
when M2 and cytosolic subcellular fractions were compared. Another protein
kinase, ERK is a predominant cytosolic enzyme which levels per mg of protein
remain unchanged along the cell cycle. Nevertheless, an activated
phosphorylated form increased in the two nuclear subfractions at G I / S .

Abstracts FEBS'99

(Mo/13.3/363)

Augmentation O f Methylprednisolone-Indueed
Differentiation Of Myeioid Leukemia Cells By
Serine/Threonine Protein Phosphatase Inhibitors
S. B. Omaya S. Uzunoglub, R. Usluc, M. Tobu~, G. Saydama,
E.Terzioglua, F. Buyukkececi~,
' Ege Univ. School of Medicine, Dept of Hematology., bCelal
Bayar Univ., Fac. of Science and Art, Dept of Biology, Manisa,
~Ege Univ., Dept ofOncology., d Dept of Immunology.Izmir,
Turkey. omay@med.ege.edu.tr

Leukemogenesis is a complex phenomenon that is

characterized by uncoupling anomalies of proliferation and
differentiation resulting in maturation block and leukemic clonal
expansion. High-dose methylprednisolone treatment has been shown
to induce in vivo differentiation of myeloid leukemia cells to mature
granulocytes in patients with acute promyelocytic leukemia and other
subtypes of acute myeloblastic leukemia [1].
To elucidate
the roles of serine/tbxeonine protein
phosphatases type 1 and type 2A in methylprednisolone-induced
differentiation of HL60 cells into granulocytes and K562 cells into
monocytes, we examined the effect of serine/threonine protein
phosphatase inhibitors, okadaic acid and Calyculin-A on the
proliferation/differentiation of HL60 and K562 myeloid leukemia cell
lines[2,3].
Okadaic
acid
and
Calyculin-A
augmented
methylprednisolone induced granulocytic differentiation and cell
death of HL60 cells and monocytic differentiation and cell death of
K562 cells in different dose ranges, respectively. These data suggest
an important role of PPI and PP2A in the mechanism leading to
differentiation of leukemic cells.
REFERENCES
[1] Hicsonmez G., et al. Leuk Lymphoma 22, (1996):9L
[2] Morita K., et al. FEBS Lett. 321,(1992):224.
[3] Omay S.B, et al. Cancer Res. 55,(1995) :774.

(Mo/13.3/365)

Alpha-Kinases: Protein Kinases With A Novel Type Of

Catalytic Domain
K. S. Pavur, M. V. Dorovkov. A. G. Ryazanov
Dept q]"Pharmacolo~,,. UMDNJ-R. W .]ohnson Medwal School.
Piscatawav. N J. 08854. U.S A

Eukaryotic elongation factor-2 kinase (eEF-2 kinase) is a ubiquitous protein

kinase that regulates protein synthesis by phosphorylating and inactivating
elongation factor-2 (eEF-2). We cloned and sequenced eEF-2 kinase and found
tbat it belongs to a new class of protein kinases. ~hich also includes Dictyo.~tehum
myosin heavy chain kinases A and B. Members of this ne~ class of protein
kinases are structurally and evolutionarily distinct from conventional enkar3otic
protein kinases. In order to localize the functional domains of eEF-2 kinase, xxc
performed systematic in vztro deletion mutagenesis of the entire 725 amino acid
human eEF-2 kinase. The catalytic domain is located between amino acids 51335, and is completely different from the catalytic domains of the conventional
eukaryotic protein kinases. Ybe calmodulin-bmding domain is located between
amino acids 51-96. Mutations in the C-terminal portion of eEF-2 kinase between
amino acids 521-725 abolish the ability of eEF-2 kinase to phosphorylate eEF-2.
but do not affect its ability to undergo autophosphorylation, suggesting that this
region is involved in eEF-2 targeting. Deletion of amino acids 336-520 does not
affect eEF-2 kinase activity. This is the least-conserved region of eEF-2 kmasc.
and probably serves as a hinge between the catalytic domain and the eEF-2
targeting domain.
Existing evidence suggests that, in contrast to conventional protein kinases
that recognize primal' structure around the phosphorylation site. eEF-2 kinase
and the related kinases recognize secondary structure and phosphorylate amino
acids located within o~-helices. We identified a peptide that can serve as an
efficient substrate for eEF-2 kinase. C-terminal amidation of tlrls peptide
increases its o~-helical propensity, and also makes it a better substrate for eEF-2
kinase. This suggests an explanation as to ~vlly eEF-2 kinase and the related
protein kinases are so different from the conventional protein kinases: thetr
catalytic domains may be adapted to phospho~'late amino acids located ~vlthin c(helices. Therefore, we named this new class of protein kinases "'alpha-kinases".
We also identified and cloned cDNAs for three human proteins x~ith homologx to
the eEF-2 kinase catalytic domain, suggesting the widespread existence of this
no'~el class of protein kinases.

s261

(Mo/13.3/364)

The ERK and JNK MAP kinases activated by SF/HGF have

distinct effects on a Ras-dependent transcriptional response
in MDCK epithelial ceils. Paumelle R., Tulasne D., Kherrouche,
Z., Plaza S., Vandenbunder B., and Fafeur V. CNRS EP560, IBL/
iPL, BP447,59021 Lille, France.

Although different MAP kinases can be activated by a single

stimulus, and can target various transcriptional effectors, it is
important to address the question of how cells integrate these signals.
In MDCK epithelial cells, SF/HGF induce the phosphorylation
and activity of the MAP kinases ERK and JNK, but not of p38. In
particular, using transient transfections of epitope-tagged kinases, we
demonstrated that SF/HGF activates both ERK and JNK, an effect
that we evidenced by the specific phosphorylation of ETSI by ERK
and of JUN by JNK.
We next asked whether ERK and JNK activated by SF/HGF
play a role in mediating activation of a RAS-responsive promoter
element, constituted of combined EBS/AP1 binding sites for ETS and
FOS/JUN transcription factors, respectively. Surprisingly, activated
ERK allowed transactivation of the EBS/API-Lue reporter, while
activated JNK allowed its transrepression. The use of inactived forms
of ERK and JNK confirmed these results.
Furthermore, the opposite effects of these kinases were
modulated by cell density. Indeed, at high density, JNK mediated a
transrepressing effect, while at low density it had not effect. In
parallel, ERK mediated transactivation at all densities, but was more
efficient at low density; an effect which mimicked the one of SF/HGF
alone.
These results demonstrated that SF/HGF induces both the
activation of ERK and JNK, with ERK transmitting a RAS-dependent
transcriptional response, and JNK modulating this response
depending on the physiological status of the cells.

(Mo/13.3/366)

Phosphotyrosine phosphatase associated with band 3 in the

human red cell: inhibition by tbiol oxidation and by Ca 2+
A. Piade, Y Zipser, N S. Kosower
Tel~lvtv ~)Ttversz~:, Tel~tvzv 69978. Israel

The anion exchange band 3 protein is the main red blood cell (RBC)
protein phosphorylated by tyrosine kinase. The RBC also contains a
band 3-associated, Mg~'-activated phosphotyrosine phosphatase (PTP)
The PTP is PTP1B or related to it (as identified by an antibody to an
epitope in its catalytic domain and by molecular mass). We studied band
3 phosphotyrosine (PT) and phosphotyrosine phosphatase (PTP) in human
RBC. RBC band 3 is usually unphosphorylated in intact normal R B C ,
but is present as PT under conditions ofRBC thiol oxidation (by diamide)
and when RBC Ca'~ is increased (by ionophore) In both cases, the band
3 tyrosine phosphorylation is the result of PTP inhibition, allowing PT
formation by kinase activity In the case of red cell thiol oxidation, the
PTP (which has cysteine in the active site) is inhibited via PTP-band 3
mixed disulfides formation. The inhibition is reversible in the RBC upon
reducing RBC disulfides to thiols, dephosphorylation of band 3 PT is
also achieved in membranes isolated from the oxidized RBC, following
reduction of membrane disulfides. In contrast, we show here that the
inhibition of PTP by increased RBC Ca-~ is irreversible. No
dephosphorylation of band 3 PT occurred in these RBC or in membranes
isolated from them. The Ca>-induced inhibition of PTP in human RBC
is not due to PTP degradation by the Ca2'-activated protease calpain
(based on the use of cell penetrating calpain inhibitor); it may in part be
due to activation of PKC (as indicated by effects of PKC inhibitors),
probably via PTP serine phosphorylation. PTP inhibition may play a role
in tyrosine phosphorylation observed in other cells under conditions of
oxidative stress and increased Ca ~-*

s262

(Mo/13.3/367)

Abstracts FEBS'99

p42/p44 MAP kinases phosphorylate HIF-Io~ and enhance

its transcriptional activity.
D.E. Richard, E. Berra, E. Gothir, and J. Pouyssrgur

(Mo/13.3/368)

Centre de Blochmue, UMR CNRS 6543, Universitdde Nice, France

Isolation and characterization of a regulatory

subunit (CK2B1) of Zea m a y s protein kinase
CK2.
M. Riera, G. Peracchia, D. Kizis and M. Pag&s.
Departament de Genetica Molecular. Consell Superior de Investigacions
CientO'iques, Centre de Investigacid i Desenvolupament, Barcelona,
Spain.

Hypoxia-inducible factor la is a transcription factor that controls the

expression of a number of genes such as VEGF and EPO in low oxygen
conditions. However, molecular mechanisms underlying activation of HIF-Ia
are of yet unknown. Results showing that endogenous H I F - I a from
exponently growing cells migrated 12 kDa higher than in vitro translated
protein led us to evaluate the possible role of phosphorylation on this
phenomenon. We report here that HIF-Iec is strongly phosphorylated in vivo
and that this phosphorylation is responsible for the marked differences in the
migration pattern between in vitro translated and endogenous HIF-Ic~. We
also show that HIF-let is phosphorylated in vitro by p42/p44 MAPKs and not
by p38 MAPK or JNK and that this phosphorylation is able to reproduce the
SDS gel shift observed in vivo. More importantly, we demonstrate that in
quiescent CCL39 cells, strong p42/p44 MAPK activation induces the same
striking shift in HIF-lct's molecular weight. This shift is blocked by the
specific MAPK pathway inhibitor, PD98059. These results strongly suggest
that H1F-I ec is phosphorylated in vivo by p42/p44 MAPK. Finally, strong and
persistent activation of p42/p44 MAPK is sufficient to promote the
transcriptional activity of the HIF-I complex and induce the expression of
HIF-I dependent genes. This interaction between HIF-I~t and p42/p44 MAPK
suggest a cooperation between hypoxic and growth factor signals which
ultimately lead to the increase in HIF-I mediated gene expression.

Protein kinase CK2 has been described in many organisms as composed of

two catalitic (o~) subunits and two regulatory (13) subunits. However,until
now, no regulatory subunit has been discovered in maize. We have
previously eharacterizated the o.CK2 multigenic family [1] and we are
interested in knowing if the regulatory subunit also exists in maize. For this
reason we have screened a stress leaf maize eDNA library using a maize EST
as a probe and isolated a full-lengh cDNA, named CK2B1, which presents
high homology withArabidobsis thaliana C K 2 regulatory subunit.
Southern hybridization of total genomic DNA with CK2B 1 cDNA indicates
that probably this clon belongs to a multigenic family.
To test if CK2B1 has functional similarity to the others [ ~ K 2 subunits we
have transformed S.cerevisae mutant strain YDH8 with CK2B 1. YDH8
carries the ckal-A1 cka2-8 mutation, grows at 25C but not at 35.5(2, and
this temperature sensitivity can be compensated for by overexpressing I]CK2
subunits.We found that the regulatory subunit is able to compensate for the
temperature sensitive growth defect of YDH8, similar results has been
described for Arabidopsis and Drosophila ~CI,L2 subunits.
In addition, mating assays performed with ct and l] CK2 subunits constructs
confirm the interaction between o'[~ and 13/[~ subunits, as in all CK2
described to date.
In order to study the regulation of the enzime we are now screnning a stress
leaf maize eDNA library using the CK2B 1 as a bait.
[1]Peracchia G. et al. PlanLMol.Biol. In press

(Mo/13.3/369)

REDOX REGULATION OF PROTEIN PHOSPHORYLATION IN PLANT THYLAKOID MEMBRANES

E. Rintam~kt, P. Martmsuo, P. Mulo, S. Pursthelmo, E.M. Aro
Department of B+ology, Untverst(v of Turku. FIN-20014 Turku Finland

Photosystem I[ functions as a light-driven water-piastoquinone-oxidoreductase

in the thylakoid membranes of plant chloroplast. Four polypeptides of
Photosystem II core and three polypeptides of its light harvesting complex II
(LHCII) are reversibly phosphorylated. Phosphorylation of the thylakoid
proteins has been proposed to be involved in redox-signalling within the
chloroplast and between the chloroplast and the nucleus during biogenesis and
acclimation of chloroplasts. One of these phosphoproteins, the D1 reaction
centre protein undergoes rapid turnover in light and phosphorylatton has been
shown to control the degradation of the DI protein triggered to proteolysis [1].
We have investigated the regulation of thylakoid protein phosphorylation and
the expression of lhcb genes (encoding LHCII polypeptides) under various
light intensities and redox conditions of thylakoid membranes. The
photosystem I1 core phosphoproteins were maximally phosphorylated m vivo
already at moderate irradiances and this level was maintained even under high
light intensities. On the contrary, maximal phosphorylation of the polypeptides
of LHCII only occurred at low light intensity whereas a strong down
regulation of LHCII phosphorylation was observed at higher irradiances.
Prelirninary experiments indicated that down-regulation of LHCII
phosphorylation, induced by changes in the light intensity or by chemicals,
was concomitant to the decrease of lhcb gene expression in nucleus.
Phosphorylation ofLHCII polypeptides is proposed to be a positive feedback
regulatory mechanism enhancing lhcb gene expression. Moreover, we show
that besides the cytoehrome b/f complex-dependent activation of the LHCII
kinase, phosphorylation of LHCII polypeptides is additionally regulated by the
thiot-redox state of the chloroplast, e.g. via thmredoxin.
[1] Rmtam~ikt et al, J. Btol. Chem., 271 (1996) 14870.

(Mo/13.3/370)

PHOSPHORYLATION OF C-TERMINAL hrGRP94 BY

PROTEIN KINASE CK2.
N. Roher); M. PlanaJ; O-G. Issinger2; B. Boldyreff2;E. hartet.
i Dep Bioqufmica i Biologia Molecular, Fac. Ci~ncies. Univ. Autbnomade
Barcelona Barcelona.Spain
2 BiochemistryInsntut, OdenseUniversity.Odense, Denmark.

Grp94 is the counterpart of hsp90 in the endoplasmic reticulum. A

truncated form of grp94 was expressed as a fusion protein with a sixhistidine N-terminal tag in E. coli. The expressed protein was purified from
the soluble fraction of E. coli as a single band using one step
chromatography in Ni 2+ nitrilo-triacetic acid agarose (NiNTA agarose). The
yield was about 10 mg of purified protein from the soluble fraction of 400
ml ofE. coli culture.
The truncated form of grp94 corresponds to the C-terminal part of the
protein (AA470-756) that includes the phosphorylation site(s) for protein
kinase CK2 and the hypothetical dimerization region of grp94.
Dimerization, and also oligomerization of grp94, have been observed in
SDS/PAGE and is mediated by formation of disulfide bridges. C-terminal
hr-grp94 was phosphorylated by protein kinase CK2 in threonine residues.
When compared with [3-casein, C-terminal grp94 was a preferred substrate
with a low Km value (0.04 mg/ml). Dimerization of C-terminal grp94 was
not altered by CK2 pbosphorylation, and protein kinase CK2
phosphorylated preferentially the monomeric and oligorneric form of Cterminal grp94 over the dimeric form.

Abstracts FEBS'99

(Mo/13.3/371)

Identification of a new lgB-x partner, NICE.

Study of its potential role in NF-KB activation
F. Rossi, V. Bottero. A. Livolsi, V. Busuttil. J.-F. Peyron

s263

(Mo/13.3/372)

Induced hyperphosphorylation of ERK

E. Rovida, F. Marra*, P. D. Sbarba.

INSF.RM rDrF.9605. Facultg de Mdde'tne Pa.~teur, b 06107 NTce ceder 02

Department of Experimental Pathology and Oncology and *Department of
Internal Medicine; University of Florence; Firenze - Italy. E-mail:

In resting cells, the transcription factor NF-KB is sequestered in an inactive

form into the cytoplasm by the association with inhibitor), proteins temaed
IKB Ill. Two major NF-~B activating pathways have been described. They
both involve a specific pbosphorylation of the N-terminal moiety of h<B-~.
which leads to the dissociation of the IKB/NF-~B complex with 121 or
without 13l prior proteasome-mediated degradation of the inhibitor
molecule. Here we report the isolation of a new partner of I~B-~ obtained
by a yeast two-hybrid screening of a Jurkat library, using the N-terminus
(an 2-72) of IKB-~ as the bait. On the basis of the prelimina~" functio1~al
data we have collected since now, we named this new IKB-~ interactor
NICE (New IKB Complexed Element). NICE is a not yet characterized,
ubiquitously expressed cytosolic protein. The full-lenght NICE is able to
specifically interact with h<B-~ in the yeast system and in an independent in
vitro approach.When expressed in 293 cells, a myc-tagged variant of NICE
strongly activates ~<B-dependent transcription, both by itself and in synergy
with the combination of the phamaacological activators PMA/ionophore,
suggesting a possible role at some level of the NF-KB activation pathway.
Moreover. preliminary experiments show the ability of NICE to induce
phosphorylation of b<B-~ although is not yet dear if this effect is direct or
~ia interaction of NICE with kinases involved in NF-~B activation
(MEKKI. NIK, IKK,, IKKI~).
Goals of our future research will be a more extensive characterization of the
NICE protein and the study of its positionning in the kinase cascade that
regulates NF- ~B activation.
Ill Ghosh etaL Annu.Rev.lmmunol., 16, (1998) 225.
121Zandi etal. Cell, 91, (1997) 243
[3] lmbert etal. Cell. 86, (1996) 787

(Mo/13,3/373)

Up-regulation of tyrosine kinase c-Sre involves

the adaptor protein Shc
K.-L Sato, M. Kimoto, M. Kakumoto, Y, Hayakawa,
T. Iwasaki, A.A. Tokmakov, Y. Fukami
Laboratory of Molecular Biology, Biosignal Research Center, Kobe Umversir:',
Kobe 657-8501. Japan

Shc is an adaptor protein that exists in three isoforrns; P46, P52, and P66, in
mammalian fibroblasts. Shc has multiple protein-protein docking sites and is
believed to play an important role in cell proliferation and differentiation under the
control of activated tyrosine kinases. In the previous meeting, we have reported
that P52Shc, but not P46Shc, binds to the IDA (inter-DFG-APE) region of tyrosine
kinase c-Src that has been implicated in autoregulatory mechanism of the kinase
activity [1] and protein-protein interaction [2]. We have also reported that P52Shc
stimulates the kinase activity of c-Src several fold and the activated c-Src is in
association with P52Shc in mitotic NIH 3T3 cells.
In the present meeting, we have prepared NIH 3T3 cells expressing each of the
She isoforms as GFP (green fluorescent protein)-fusion protein and examined their
ability to stimulate endogenous c-Src activity. We have found that c-Src activity is
stimulated in GFP-P52-expressing cells but not in GFP-P46-expressing cells
irrespective of the serum stimulation of the cells. On the other hand, we could not
detect any significant difference in the subcellular localization of GFP-Shc proteins
in serum-starved, serum-stimulated, and nocodazole-treated mitotic cells. In rat
PC1.2 cells, it has been demonstrated that nerve growth factor-dependent tyrosine
phosphorylation of Shc is partially inhibited by tyrosine kinase inhibitors and by the
PI 3-kinase inhibitors such as wortmannin and LY294002 [3]. We have also found
that serum-dependent activation of c-Src in NIH 3T3 ceils is sensitive to the PI 3kinase inhibitors. We are now investigating the effect of the PI 3-kinase inhibitors
on the Sbc-induced up-regulation of c-Src. Present experiments have also been
designed to define a critical segment of P52Shc for its binding to the c-Src IDA
region and for the ability to stimulate the c-Src activity. A series of glutathione Stransferase (GST)-fusion proteins containing a part of P52Shc molecule were
prepared. By performing surface plasmon resonance analysis and in vitro kinase
assay, it was demonstrated that GST-fusion proteins containing amino acid residues
29~,9 of P52She, most of which is missing in P46Shc, are capable of interacting
with the e-Src IDA region and stimulating c-Src activity.
References
[I] Fukarni, Y. et al. J. Biol. Chem., 271, (1993) 13250.
[2] Sato, K.-I. et aI. Biochem. Biophys. Res. Commun., 210, (1995) 844.
[3] Sato, K.-I. et al. Biochem. Biophys. Res. Commun., 250, (1998) 223.

The cytoplasmic protein-tyrosine kinases of the fps/fes family, expressed

primarily in hematopoietic cells and in the vascular endothelium, are involved in
the response to several cytokines and believed to play a direct role in myeloid
differentiation and angiogenesis. The critical signaling pathways downstream fes
have not been fully characterized. To determine whether fes can mediate the
action of the Macrophage Colony-Stimulating Factor (MCSF), we previously
overexpressed c- and v-fes in the murine BAC1.2F5 macrophage cell line, that
depends on MCSF for survival and proliferation. We showed that the
overexpreasion of the product of fes could functionally substitut~ for the MCSF
requirement of macrophages for proliferation, and that, however, the fes tyrosine
ldnase acts largely independently of the MCSF receptor (MCSFR) tyrosine
kinase. It has been demonstrated that the activities of both ERK and JNK are
constitutively elevated in cells transformed with v-fes and an activated form of cfes (myr-fes), and that ras is the link between fes and ERK. On the other hand,
ERK is phosphorylated in tyrosine and threonine by llgand-activated MCSFR.
We show here, using an anti-phospho-ERK antibody, an increased
constitutive phosphorylation of both ERK-1 and ERK-2 in several v-fes- or cfes-overexpressing clones. In these clones, the MCSF stimulation determined a
marked decrease of ERK phosphorylation. This effect was paraleUed by a
decreased ability of raf to phosphorylate downstream substrates, as detected by
an in vitro kinase assay with rat" immunoprecipitated from MCSF-stirnulated
ceils. In c-fes- and v-fes-overexpressing clones, the levels of ERK and of
maximal ERK phosphorylation were identical to that of control ceils.
As the actions of both fes and ligand-activated MCSFR result in ERK
phosphorylation, the results obtained suggest that fes overexpression ensures the
maximal physiological levels of ERK phosphorylation, so that this
phospborylation cannot be increased by the activated MCSFR. On the contrary,
in fes-overexpressilag clones, the MCSF-dependent activation of tyrosinephosphatases active on ERK would be still possible, with the consequence of an
ERK de-phosphorylation.

(Mo/13.3/374)

Disseetion of Focal Adhesion Signaling Cascades Using

Phosphorylation Site-Speciflc Antibodies
E. Schaefer, L. Zorn, L. Lasovsky,C. Weinwurm,F. Burnsand J, Fishman
QCB/BtoSource lnternattonal. Hopkinton, ?via 01748, UNA

Cell surface reeeptor-ligand interactions involving soluble factors or

extracellular matrix activate a multitude of intracellular signaling molecules.
The focal adhesion kinase (FAK/pp125) is a cytoplasmic tyrosine kinase that
plays a key role in integrating signals initiated by a diverse group of stimuli.
For example, FAK signaling is regulated in response to bioactive peptides,
cross-linking antibodies, extracellular matrix, growth factors, mitogens and
cytokines/chemokines, thereby playing an important role in regulating
differentiation, apoptosis, cell morphology and motility [ 1]. A related enzyme,
the Ca+*-activared proline-rich tyrosine kinase 2 (PYK2, also known as CAK[3
CADTK or RAFTK) shares -60% identity with FAK in the catalytic domain,
however, these enzymes appear to be differentially regulated [2]. Both FAK
and PYK2 are phosphorylated on multiple tyrosine residues, which allows
coupling to downstream effectors. Interestingly, FAK but not PYK2, is also
phosphorylated on at least four highly conserved serine residues, resulting in
cell cycle-dependent patterns of phosphorylation [3]. To better understand the
role of specific phosphorylation events in focal adhesion signaling, we
generated a panel of site-directed phosphorylation-specific antibodies to FAK
(7 sites) and PYK2 (4 sites), as well as to other key proteins in related
pathways (i.e., c-raf, GSK31~, JAKs/STATs, MAPKs, PKB/AKT, p53 and
src). Western blotting results using a variety of cell types illustrates the utility
of these reagents in dissecting complex signal transduction events that are
activated in response to different stimuli. The ability to study site-specific
phospborylation provides considerably more information than standard
immunoblotting methods based on detection of total target phosphorylation
using generic anti-pTyr or pSer antibodies.
[I] Cary LA and Guan JL (1999) Focal adhesion kinase in integrin-medialed
signaling. Front Biosci 4:D 102
[2} Xiong WC et al. (1998) Expression and characterization of splice variants of
PYK2, a focal adhesion kinase-related protein. J Cell Sci 111 ( Pt 14):1981
[3] Yamakita Y el aL (1999) Dissociation of FAK/p130(CAS)/c-Src complex during
mitosis: role of mitosis-specific serine phosphorylation of FAK. J Cell Biol
144(2):315

s264

(Mo/13.3/375)

Abstracts FEBS'99

The Interaction of M A P kinase phosphatase 1 (MKP-1)

with the NF-r.B Activation Pathway

(Mo/13.3/376)

H.-C. Schneider, L. Scott, E. McKenna, S. Crowe, R. Jupp

Molecular Btology, Dagenham Research Centre, Rhone-Poulenc Rorer Ltd.
Rainham Road South, Dagenham, Essex, RMIO 7XS, United Kulgdom

The transcription factor NF-~cB is activated by a number of

proinflammatory factors, for example tumor necrosis factor-~ (TNF-oQ and
interleukin-I (IL-I). A number of kinases are involved in this signal
transduction pathway. Especially, NF-rd3 inducing kinase (NIK), MEK
kinase (MEKK), and the two Ir,.B-kinases IKK-o~ and IKK-[3 have been
shown to be key players an NF-rd3 activation. IKK-c~ and IKK-[3 are
subunits of a 700 kDa complex (IKK-complex) which can be coimmunoprecipitated with an antibody directed against the MAP kinase
phosphatase 1 (MKP-1). This interaction of MKP-1 with the IKK-complex
implicates the involvement of MKP-I in the regulation of NF-r,.B activity.
We are investigating the effect of MKP-1 on the activation of NF-rd3.
Using recombinant MKP-1 we have measured its effect on the activity of
NIK, MEKK-I, IKK-c~ and IKK-[3 in in vitro kinase assays. Also, data will
be presented on the impact of MKP-1 on NF-~B activatmn tested in
reporter gene assays in various cell lines. The possible target of MKP-I in
the NF-~B pathway and its sigmficance for the regulation of NF-r,B
activation will be discussed.
This project was sapported bv the EU Marie Curie Research Training
Grant ERBFMB1CT972584.

(Mo/13.3/377)

Mutational studies on phosphorylase kinase.

V.T Skamnaki', D.J. Owen b, M.E M Noble , E D. Lowe ~,

N.G Oikonomakos, ~ L.N. Johnson~.
~NHR~, Athens, Greece bA.IRCLaboratory of Molecular Btology, CambrMge,
L'h~ ~Laboratota, oMolacular Btol~hvsics. Umv.ofOxford. UA"

The reaction catalysed by protein kinases involves the transfer of the ?"
phosphate of ATP to a serine, threonine or tyrosine residue. It is likely that
all protein kinases utilise a common catalytic mechanism. Among the highly
conserved amino acids is an aspartate residue [1] Another key aspect of
regulation, is phosphorylation on a residue (or residues) located in the
activation segment at the centre of the kinase domain. The activation
segment in protein kinases refers to the amino acids between the conserved
DFG triplet to the APE triplet sequence and contains residue(s) that are
phosphorylated in many but not all protein kinases In this work, we address
the contributions of these residues to kinase catalysis using phosphorylase
kinase as a model system Phosphorylase kinase (PhK), the first protein
kinase to be discovered [2], catalyses the phosphorylation of a single serine
residue, S 14, in inactive glycogen phosphorylase b (GPb), thereby converting
phosphorylase to active glycogen phosphorylase a (GPa) and stimulating
glycogen degradation. The kinase domain of the 2" subunit which is the
catalytic one comprising residues 1-298 has been crystallised [3] in a ternary
complex with the non-hydrolysable ATP analogue, AMPPNP, and a peptide
substrate. The resulting structure showed the conformation of an active
kinase which is constitutively active without the need for any further
modification. In this case the role of the phosphorylated residue is played by
E182 In order to explore the contribution to catalysis of the conserved
D149 and the contributions of electrostatic interactions to the correct
localisation of the activation segment, we have carried out site-directed
mutagenesis studies on the catalytic DI49, as well as E182, RI48 and
surrounding interacting residue Y206 The kinetic parameters were measured
and the results suggest a mechanism in which the primary determinants for
catalysis come from the correct alignment of reacting groups coupled with
strong interactions that can stabilise the transition state.
[1] Taylor, S.S., et al., Structure 2 (1994), 345-355
[2]. Fischer, E H , et al, .Z Biol. Chem. 216, (1955) 121-132
[3] Lowe, E D., et al, E M B O ,I. 16, (1997) 6646-6658

Phosphatidylinositol 3-kinase contributes to the activation of the

MAP kinase cascade in the course of HGF-induced scattering of
HepG2 cells
Sz. Sipeki, L. Buday and A. Farag6

SOTE, Dept of Medical Chemistry, 9. Puskin St., Budapest, Hungary

MAP kinase cascade-dependent components of cellular responses

were investigated during the hepatocyte growth factor- (HGF) or phorbol
esterAnduced scattering of HopG2 hmnan hepatoma cells. Epidermal
growth factor (EGF) failed to stimulate HepG2 cells to scatter.
The inhibition of phospbatidylinositol 3-kinase (PI 3-kinase)
activity with LY294002 prevented completely the HGF- or phorbol esterinduced cell dispersion, while the inhibition of MAP kinase kinase (MEK)
activity with PD98059 prevented the development of the characteristic
morphological changes associated with cell migration.
HGF or phorbol ester treatment of the cells sustained the
phosphorylation of Erkl and Erk2 MAP kinases for several hours, while
EGF induced only a short-term activation of the MAP kinase cascade.
The inhibition o f P l 3-kinase activity with LY294002 reduced strongly the
HGF-induced Erkl and Erk2 phosphorylation, but it had no effect on the
short-term activation of the MAP kinase cascade induced by EGF, or on
the long-term MAP kinase cascade activation sustained by phorbol esteractivated protein kinase C.
The long-term activation of the MAP kinase cascade induced by
HGF or phorbol ester yielded the selective expression of a high-M r (>300
kDa) protein pair in HepG2 cells. The induced expression of these proteins
was inhibited by the PI 3-kinase inhibitor LY294002 in response to HGF
stimulation, but was not inhibited in phorbol ester-treated cells. The
inhibition of MEK activity with PD98059 prevented the expression of the
protein pair both in HGF- or phorbol ester-treated cells.
Our data indicate that PI 3-kinase contributes to the HGF-induced
activation of the MAP kinase cascade and this may lead to the selective
expression of certain proteins.

(Mo/13.3/378)

Unique structural and functional properties of the ATP

binding domain of atypical protein kinase C-t
M Spltaler ~, A Vtllmlger", S Kampfer", F Gantner b, H Grtmlcke" and ~ Uberall ~
aln~tuut fi~r AIed~zmtsche Chemze und Btochemte, Umversitat Inn~b~wk
Fntz-Pregl-Str 3 A-6020 bmsbruck Aust~Ta
Byk-(Julden Lomberg ( hemtsche ['abrtk GmbH, Konstanz Germam

Atypical protein kinase C-t (aPKC-t) plays an important role in mitogenic

signaling, actin cytoskeleton organization and cell survival. Apart from the
differences in the regulatory domain, the catalytic domain of aPKC-t differs
considerably from other known kinases, as it lacks the conserved glycine loop
motif (GXGXXG) that is found in the nucleotide fold of virtually all nucleotide
binding proteins including PKCs, Ras, adenylate kinase and the mitochondrial
F1-ATPase. We have used site directed mutagenesis and kinetic analysis to
investigate wether these sequence differences affect the nucleotide-binding
properties and catalytic activity of aPKC-t. When lysine 274, a residue essential
for ATP binding and activity in most protein kinases, was replaced by arginine
(K274R mutant), aPKC-1 retained its normal kinase activity This is in sharp
contrast to results published for any other PKC or even distantly related kinases
like phosphoinositide 3-kinase y (PI3Ky), where the same mutation aborted the
kinase activity completely Also the sensitivity of aPKCq for inhibition by
GF109203X, a substance acting on the ATP binding site, was not altered in the
K274R mutant. In contrast, replacement of Lys-274 by tryptophan (K274W)
completely abolished the kinase activity of PKCt. This defect could not be
reverted by cotransfection of lambda-interacting protein (LIP), a protein
activator of aPKC-t that is able to stimulate the in vitro activity of aPKC-t
extracted from cotransfected COS ceils In accordance with results obtained
with other kinase-defeetive PKC mutants, in cultured cells aPKC-t-K274W
acted in a dominant negative fashion on signal transduetion pathways involving
endogenous aPKC-t, while the effect of the catalytically active K274R mutant
was identical to the wild type enzyme. In summary, aPKC-t differs from
classical and novel PKCs also in the catalytic domain. The structural difference
is correlated with an unusual catalytic activity only known from a small number
of distantly related kinases like CDK-activating kinase (Cak) from yeast

Abstracts FEB S' 99

(M0/13.3/379)

s265

Expression of a Phosphatidylinositol 4-Kinase (PI4K230)

S. Suera, H. G01kan a, T. Gehrmann a, G. Vereb b, A. Balla b,
G.W. May:, F. Herberg a , L.M.G.Heilmeyer ~

(Mo/13.3/380) Serine 19 of Human 6-Pyruvoyl-tetrahydropterin Synthase

JPhyMol. Chem., RUB, Bochum. Germany,~Med. Chem., Debrecen,

lhmgao', ~Physiok Chem.. fhtiv. Hospital Eppendorf Hamburg, Germany

Department of Pedtatrics, Division of Clinical Chemistry and Biochemistry,

University of ZUrich, Steinviesstrasse 75, CH-8032ZUrich, 5hcitzerland

Polyphosphoinositides are involved in many signal tranduction pathways in

eukaryotic cells by which growth factors, hormones and neurotransminers can
exert their physiological effects. The first committed step is catalysed by
phosphatidylinositol 4-kinase leading to the formation of phosphatidohnositol
4-phosphat, which is phosphorylated consecutively at the 5"position by
phosphatidylinositol4P5-kinase. The resulting product, PtnIns(4,5)P,. is
cleaved hydrolytically by Phospholipasc C to produce two second messengers:
diacylglycerol and D-myo-inositol 1,4,5-trisphosphate.
The eDNA of a new human type 3 PtdIns4-kinase, named PI4K230, is
identified which encodes a protein of 2044 amino acids, Northern blot analysis
shows a hybridisation signal of 7.0 kb in many tissues [1]. The C-terminal part
of 260 amino acids represents the catalytic domain which is highly conserved
in all recently cloned PtdIns 4-kinases, similar to that of
(poly)pbosphoinositide 3-kinases and to that of more distantly related
lipid/protein kinases.
The coding region of PI4K230 and additionally two N-terminal
deletionmutants, AI-872 and A1-1427, both containing the catalytic domain
have been expressed in vitro using reticulocyte lysates. The expressed PI4K230
and AI-872 PI4K230 show a significant higher PtdIns4-kinase actiwty than
the negative control. A1-1427 PI4K230 exhibits no Ptdlns 4-kmase activity.
Polyclonal antibodies raised against the catalyhc C-termtal part of the enzyme
inhibit the human Ptdlns 4-kinase activity, while antibodies against the Nterminal part stimulate moderately. The coding region of PI4K230 has been
overexpresscd m an active and soluble form m Sf9 cells for further biochemical
charactefisation. The expressed protein with a MW of 200kDa was identified
from Sf9 cell lysate using antibodies against the polyHis-tag, the C- and Nterminal parts of the protein. The lipid kinasc activity of the expressed enzym
in the lysate was 20-fotd higher than the corresponding control The PI4K230
activity a~ayed in the presence of various concentrations of wortmannin was
inhibited with an IC5o of 20OHM and was not inhibited up to 2001aM adenosine.
Product analysis was performed by HPLC and by thin layer chromatography

is Phosphorylated by cGMP-Protein Kinase II

B. Th6ny, T. Scherer-Oppliger, W. Leimbacher, N. Blan

6-Pyruvoyl-tetrahydropterin

synthase

(PTPS)

participates

mutation. Argl6 is located in the protein surface exposed phosphorylation

motif RI6RISI9 , with Serl9 as the putative phosphorylation site for
serine/threonine protein kinases. Purification of recombinant PTPS-S19A
from bacterial cells resulted in an active enzyme ( k c J K M = 6.4 x 103 M -I st),
which was similar to wild-type PTPS (kcaJKM = 4.1 x 103 M "t s't). In assays
with purified enzymes, wild-type but not PTPS-S19A was a specific substrate
for the cGMP-dependent protein kinase (cGK) type I and II. Upon expression
in COS-1 cells, PTPS-S19A was stable but not phosphorylated and had a
reduced activity o f - 3 3 % in comparison to wild-type PTPS. Extracts from
several human cell lines, including brain, contained a kinase that bound to
and phosphorylated immobilized wild-type, but not mutant PTPS. Addition of
cGMP stimulated phosphotransferase activity two-fold. Extracts from
transfected COS-1 ceils over-expressing cGKII stimulated Serl9phosphorylation more than 100-fold, but only four-fold from cGKI overexpressing cells. Moreover, fibroblast extracts from mice lacking eGKII
exhibited significantly reduced phosphorylation of PTPS. These results
suggest that Serl9 of human PTPS may be a substrate for cGKII
phosphorylation also in vivo, a modification that is essential for normal
activity.

[21.
[11 Gehrmann T. et al.. Eur. J. Biochem. 253, 1998. 357
[2] Gehrmann T. et al.. Bioch]m. Biophys. Acta, in press
(Mo/13,3/381) Activation by EGF, T G F ~ and HGF of several members of
mitogen-activated protein kinases in hepatocytes
G. H. Thoresen, T. Christoffersen.
Department of Pharmacology, Universityof Oslo, N-0316 Oslo. Norwa)

We have investigated the effects of epidermal growth factor

(EGF), transforming growth factor x(TGF~), and hepatocyte growth
factor (HGF) on DNA synthesis and activity of mitogen activated
protein (MAP) kinases, p42/p44 or ERK (extracellular signal-regulated
kinase), p38, and JNK (c-jun N-terminal kinase), in cultured rat
hepatocytes. EGF and TGFet are ligands of the EGF receptor, while
HGF binds and activates another receptor tyrosine kinase, the HGF
receptor or c-Met. All three ligands increase the ERK MAP kinase
activity and stimulate the DNA synthesis in cultured hepatocytes [1-3].
Hepatocytes from adult male rats were cultured in a serum-free
medium in the presence of insulin and dexamethasone. Both p38 and
JNK t MAP kinase activity were stimulated by EGF, TGFa, or HGF.
Addition of the specific p38 MAP kinase inhibitor, SB 203580,
produced a marked, dose-dependent inhibition of EGF- and TGFotinduced, but not the HGF-induced, DNA synthesis. A single addition of
PD 098059, an inhibitor of MEK1, an enzyme in the ERK MAP kinase
pathway, gave little inhibition, but repeated additions every six hours
completely inhibited the EGF-induced DNA synthesis.
These results suggest that more than one of the MAP kinase
pathways may participate in mitogenic signals from tyrosine kinase
receptors. Further studies are in progress to elucidate the relative
importance of the different MAP kinase pathways in mitogen-induccd
proliferation.

References
[1] Peak Met al., Biochem Soc Trans 21(1993) 494S
[2] Adachi T et al., Hepatology 23 (1996) 1244.
[3] Thoresen GH et al., J Cell Physiol 175 (1998) 10-18.

in

tetrahydrobiopterin-cofactor biosynthesis. We previously identified in a

PTPS-deficient patient an inactive PTPS-aUele with an Axgl6 to Cys eodon

(Mo/13.3/382)

Activation of protein kinase B in v-src-transformed cells

Z. Tnhd~kovfi, E. ~loncovfi, V. Sovovd
Institute of Miolecular Genetics, Academy of Sciences, 166 Prague6,
Czech Republic

Two signalling pathways that are involved in the control of mRNA

translation have been found activated in hamster
flbroblasts
transformed by Rous sarcoma virus (RSV) [1]. While one of them, the
p70 $6 kinase pathway, is completely rapamycin-sensitive, regulation
of the activity of initiation factor eIF4E by phosphorylation of its
binding protein 4E-BPI is only partly sensitive to the inhibitory effects
of rapamycin in these cells. Protein kinase B (PKB) is also activated by
RSV-transformation in hamster fibroblasts.
The
increased
phosphorylation and activity of PKB is rapamycin-insensitive but
blocked by wortmannin, the inhibitor of phosphoinositol 3'-kinase
(PI3 '-K). Possible involvement of the PI3'-K/PKB signal transduction
pathway in the raparnycin-insensitive phosphorylation of 4E-BP1 in
RSV-transforrned fibroblasts was examined.
[ 1] Tuhfi~kovfi et al., Int. J. Cancer (in press)

s266

(Mo/13.3/383)

Abstracts FEBS'99

Regulation of adenylate cyclase

by lbrskolin in malignant cells
A.Vagonis
Laboratory of Boielectrochem&try and Btoenergettc, Insntute
~?fBIochemt.~03; Mok.~lhtinku 12, 2600 I'ihmts, Lithuama

We have previously shown [I] that in ovarian malignant cells

(OMC) the responsiveness of adenylate cyclase (AC) to the
stimulatory action of hormones is lost. A malfunctioning of AC in
malignant cells could be due to one or a combination of defects of
the AC complex compounds (hormone receptor, G-proteins, catalytic
subunit). G-proteins are molecular switches that regulate a host of
cellular processes including cell growth, division and malignancy.
Cholera toxin (CT) catalyses ADP-ribosylation of the et-subunits of
the stimulatory G-protein leading to intrinsic GYPase activity and
thereby causing constitutive stimulation of AC and elevated
intracellular cAMP levels. Here we have studied the effect of various
concentrations of forskolin alone and in combination with CT (10
~tg/ml) on the AC activity of membrane preparations obtained from
OMC, ovaries of OMC-bearing rats and ovaries of normal rats. We
have found that basal activity of AC of membrane preparations from
OMC was diminished. Forskolin, a direct activator of catalytic
subunit, caused a concentration dependent increase of AC activity in
all cells studied. CT stimulated activity of AC from ovaries of
normal rats (45 per cent) mad ovaries of OMC-bearing rats (20 per
cent) whereas in OMC CT caused a slight decrease of the AC
activity (8 per cent). When membrane preparations were
preincubated with CT the stimulatory effect of forskolin on AC
activity was either additive or synergistic in preparations from
normal ovaries and ovaries of OMC-bearing rats, whereas in OMC
forskolin caused a down-regulation effect. It is concluded that
malfunctioning of the adenylate cyclasc system in ovarian malignant
cciis is rclated to the occlusion o f a hormonai receptor.
[1] A. Vagonis, Biol. Zent. bl., 107. (1988) 97.

(Mo/13.3/385) Rapamyein blocks endothelial cell proliferation by

inhibiting protein synthesis

F. Vifials, J.C. Chambard & J. Pouyss6gur

Centre de Biochtmw-CNRS. Univel~ub de Nice, Pare Valrose, 06108
Ntce, f~)ance

In this work we have analysed the role of p42/p44MAPK and

P13K-p70S6K signalling cascades on the stimulation of endothelial
cell proliferation. We have found that inhibflors of p42/p44 MAPK
(PD98059) and of the PI3K-p70S6K pathways (rapamycin,
wortrnannin and Ly294002) all block thymidine incorporation
stimulated by fetal calf serum (FCS) in the mouse endothelial cell line
IGI 1. Rapamycin completely inhibits the mitogenic effect of FCS in
primary cultures (human umbilical vein endothelial cells, HUVEC)
and other established capillary endothelial cell lines (IBE and ECV304
cells), The inhibitory effect of rapamycin is only observed when the
inhibitor is added during the first hours of the GI phase, and it is lost
after eight hours of FCS stimulation. Moreover, rapamycin prevents
hyperphosphorylation of the retinoblastoma product (pRb) and BrdU
incorporation, indicating block of G1 phase. Rapamycin completely
blocks growth factor stimulation of protein synthesis. Moreover,
rapamycin IC50 is the same for both, inhibition of protein synthesis
and bock of cell proliferation. In accordance with its inhibitory effect
on protein synthesis, stimulation of cyclin DI and p21 proteins by
growth factors is blocked by preincubation with rapamycin.
Expression by retroviral infection of a p70 S6K form partially
resistant to rapamycin reverses the inhibitory effect of the drug,
indicating the participation of p70 S6K in the inhibitory effect of
rapamycin. Thus, in endothelial cells activation of protein synthesis by
p70 S6K via PI3K is essential for the stimulation of the cell cycle
progression by growth factors.

(Mo/13.3/384)

Potential strong regulation of guard cell phospho-

enolpyruvate carboxylase through phosphorylation

A Vavasseud V Cotelle ~ and J-N Pierre ~
~CEA Cadarache. DSV- DEVM. LEMS BPI F-I~I08. St Paul-lezDurancefIBP, Uul;' Pans-Sud. B~]I 630.91405 Orsa~,Cedex

In plants, guard cells are located at the end of the transpiration stream V,'lthin
the plant, and their control of stomatal aperture is crucial to manmise water loss
from the leaf tissues while balancing the requirement of CO_- exchange for
photosynthesis Water availability and CO, partial pressure modulate stomatal
aperture by modlf3mg the turgor of guard cells Phosphoenolpymvatc
carbox31ase (PEPCase) is a major enz3me in the pathwa? leading to malate
s.~nathesis which is. ~ith chloride, the mare countenon for potassium
accumulated m the guard cell xacuole during stomataI opening Whether
phosphor31ation of PEPCase could be a major event m guard cell regulation
was investigated Antibodies (APS-IgGs) raised to a synthetic pol3peptlde of 23
amino acids containing the phosphorylatmn site (ser-8) of the Sorghum
PEPCase recogmsed the guard cell PEPCase from Commehna communis L. at
110 and 120 kDa The in vitro phosphoBlatlon of the 110 kDa lsoform by PKA
~as 50% inhibited by APS-IgGs demonstrating that the regulator3
phosphorylation site x~as present and functional in the guard cell enzyane
Phospho~'lation b3 PKA resulted m a 50% increase in the Vmax of the enz3rne
(4 2 0 3 compared to 2 8 0 4 pmol h-I GCP-I. pH 7.3 and 200 ~tM PEP)
and a reduction in L-malate mb~bltlon (64% compared to 82% mhibman by" 1
mM L-malate) In the presence of 1 mM L-malate (pH 7 3) phosphor31ationof
the enzyme by PKA resulted in a 3-fold increase in the Vmax. Binding of APSIgGs to the phosphorylatlon site of the enz3me led to the highest activity (10 9
2.6 pmol h- l per guard cell protoplast) and to an absence of inhibition by 1 mM
L-malate at pH 7 3 and 8.0. These changes in the kinetic properties of the
enzyme after phosphorylaUon should have important consequences in terms of
stomatal regulation

(Mo/13.3/386) Ligand-dependent Phosphorylation of Serine 118 of Human

Estrogen Receptor c~ does not involve MAP Kinase
E. washbrook, P. Pace, V. Thirunavukkarasu,R. Epstein, R. Coombes, S. Ali
CRC Laboratories, Dept. of Cancer Medicine, Imperial College School of
Medicine, Charing Cross Campus, St Dunstan 's Road, London W6 8RP, UK

Previous studies have shown that human estrogen receptor (hERa) ts

phosphorylated on multiple sites in a ligand-dependent manner. We
previously identified serine 118 (S 118) of hERo~ as a major ligand-inducible
phosphorylation site, the mutation of which significantly reduced
transcription activation by the receptor. Using SDS-PAGE and 2-D
phosphopeptide mapping we now show that Sl18 is phosphorylated by
mitogen-activated protein kinase (MAPK) in vitro. We also show that in
vivo, the MAPK activators epidermal growth factor (EGF) and phorbol 12myristate 13-acetate (PMA) increase phosphorylation of Sl18.
Phosphopeptide mapping further shows that h E R s is phosphorylated in
vivo on S102, S104 and/or 106 and in vitro by MAPK, although Sl18
appears to be the major MAPK substrate in vivo. Using an approach we
developed to detect functionally distinct protein isoforms using
phosphorylation-specific antibodies [1] we show that Sl18 is efficiently
phosphorylated in response to inducers of MAPK activity. In the case of
the wild-type receptor, 171~-estradiol (E2), the partial agonist 4hydroxytamoxifen (OHT) but not the complete antagonist ICI 182,780, as
well as EGF and PMA induce S118 phosphorylation. However, the E2- and
OHT-mediated increase in Sl18 phosphorylation is not inhibited by
PD98059, indicative of the involvement of (an)other signal transduction
pathway(s) in the ligand-dependent phosphorylation of S 118.
[1] Epstein RJ et al., Proc Natl Acad Sci USA 89, (1992) 10435.

Abstracts FEBS'99

(Mo/13.3/387)

GSK 3 in human semen

B. Willipinski and A.K.Mukhopadhyay
Institutefor Hormoneand FertilityResearch at the Universityof Hamburg.
Hamburg, Germany

The presence of GSK 3 m human sperm and a correlation between

bovine cauda and caput sperm motility and GSK 3-activity have been
reported. In addition, it is well known, that cAMP is involved in
regulating sperm motility. In this study the aim was to determine if a
correlation between cAMP-stimulated GSK 3-activity and motility in
human sperm exists. Human semen were collected from healthy
donors with a sperm motility greater than 50 percent and were
processed by swimup procedure. The swimup (motile) and bottom
(immotile) fractions and also the sperm-free seminal fluid were
treated with varying concentrations of cAMP for different durations.
Following this treatment the proteins were dissolved in sample buffer
and were subjected to SDS-PAGE. GSK 3 was visualized by Western
Blotting procedure using a specific antibody and ECL-detection
system. We report that GSK 3 is not only present in human sperm but
also in human seminal flied. This was true for several individual
specimen. In addition, we could show differential effects of treatment
with cAMP in the bottom versus the swimup fraction and in the
seminal fluid. Thus we propose that cAMP-mediated modulation in
GSK 3 could be relevant for sperm motility.

s267

(M0/13.3/388) Osmotic stress activation of p38-SAPK2 in astrocytes

induces the phosphorylation o f PEA-15 through a PKCdependent pathway
L
D. Zvalova ~,2, J. Cordier I, M. Kubes2 , J. Glowinski . and H.
Chneiweiss* I
I INS'ERM Ull4/Coll~ge de France, Paris, France. 2Institute
of virology, Slovak academy of science, Bratislava, Slovakia.
PEA-15 (Phosphoprotein Enriched in Astrocytes, Mr= 15,000) is
an acidic serine-phosphorylated protein highly expressed in the
central nervous system where it might play a protective role against
cytokine-induced apoptosis. PEA-15 was characterized on the basis of
its high level of expression and degree of phosphorylation in
astrocytes. This protein is phosphorylated on two different seryl
residues, Serl04 being identified as a site of regulation for protein
kinase C (PKC) whereas Ser116 is regulated by Calcium/calmodulin
kinase II (CaMKII).
In an attempt to analyze the role of PEA-15 phosphorylation in
its anti-apoptotic effect, we investigated wether the protein could be a
target for stress kinase pathways. Indeed, astrocytes responded to a
sorbitol-induced hyperosmotic treatment by a robust, long lasting and
reversible, activation of p38-SAPK2. Maximal activation of the
kinase was already observed after 10 min. of exposure to 500mM of
sorbitol whereas its inactivation required 60 rain. after renewal of a
fresh normal medium. Astrocyte exposure to sorbito[ resulted in a 3fold increase in PEA-15 phosphorylation, an effect inhibited by
SB203580, a selective inhibitor of p38-SAPK2. Interestingly, several
PKC inhibitors also prevented sorbitol-triggered PEA-15
phosphorylation but did not affect the p38-SAPK2 activation.
Treatment ofastrocytes with a high concentration of the phorbol ester
PMA (1 [tM, 24h) resulted in the desensitization of classical and new
PKCs. Sorbitol still induced p38-SAPK2 phosphorylation in these
conditions, but no further phosphorylation of either PEA-15 or
MARCKS could be detected. This suggests that a PKC, belonging to
the family of diaeyglycerol and/or calcium-dependent kinases, is
among the downstream effectors of p38-SAPK2 dependent
phosphorylations.

s268

Abstracts FEBS'99

19.1 Molecular mechanisms in neuronal and muscular diseases

(Mo/19.1/389)

Ape E and ACE Genotypes in Alzheimer's Disease

B.Agaqhanr,H YllmazI,M Aydm~,lKara~ T Isbir ~

(Mo/19.1/390)

tUniversiO' o f ]stanbnL Institute f o r Experimental Medical

Research DepLMoleeular Medicine.ZDept, Neuro.~cience. Istanhul,
l'urke3,

Apolipoprotein E (Ape E) e4 has been associated with both early and

late-onset Alzheimer's disease (AD).Angiotensin Converting Enzyme
(ACE) Deletion (D) polymorphism have been associated with a high
risk for coronaw heart disease and may effect longevity/survival into old
age We investigated possible relations between ACE I/D and ape E
gene polymorphism and Alzheimer's disease in 35 subjects. PCR
(Polymerase Chain Reaction), RFLP (Restriction Fragment Length
Polymorphism), and agarose gel electrophoresis techniques were used to
determine lhe ACE and ape E genotypes Here we report that the ACE D
and Ape E ~4 allele are more frequent in patients with Alzheimer's
disease than in control
Table 1, Comparison o f a p o c4 and ACE D alleles presence

Apo c4 allele
~:4(-I
c41~ )
ACE D ~dlele
Dl-)
Dr+)

}
~
[
[
]

Alzheimer(%}
Frequenc),

Control,
Frequenc
)' s,(%)

28
7

80
20

28
1

96
-~4

4
31

114
886

8
21

276
724

v4:-, . lh~eace q f apo ~t atle/e . zd : ~ Pre.wpwe ~?/'ape c4 allele. ~ 3 97 p 0 (/5:

I)(-) , ll~.wngc o/ape 1) MMe: l) : : Pre~em e el ape D allele, Z" 2. ~ I. p 0 09

I
2
3

I%osoncIIO. 'FalaslacllUS.. Lchto',ma M. HelnoIlCllO. Hehsalnn S . Manllennaa

A" Relanon of coronar3 atherosclerosls and apohpoprolln E gllol)'~:s in
Al/hemler patients Stroke. 26. 743-748. 1995.
SchaehterF. Faure-Delanef L Guenot F, Rouser H. Frogul P. Lcsueur-Gmot.
Cohell D GeneueassoemUons".~.ilhhtllllarlIonge~il) at the APOE alld ACE 10el.
Nature Gcnetics. 6.29-~2. 1994
Strlnmattcr W.J. Roses A D. ApohpoprotemE arid Al:hemler disease l~'oc Nail
Acad ScJ USA. 92.4725-4727 1995

(Mo/19.1/391)

Phoeetn, a newly discovered neuronal protein

abundant in some axon bundles.
G. Baillat, A. Moqrich, F. Castets and A. Monneron.
LNCF, CNRS, 21 chemin Joseph AIguter, 13420 Marseille Cedex, France

Antioxidant status of penthylentetrazolium induced

epileptic r a t s
H. Akbas, A. Yegifl, S. Aktas, T. ~)zben
Akdeniz University, School o f Medicine,
Departments o f Biochemistry, Antalya, Turkey

This study investigates the effects o f pentylentetrazolium (PTZ)

induced-epilepsy on erythrocyte and liver tissue antioxidant enzyme
systems. Adult Wistar female rats (n=24) were assigned into two
groups. The first group was controls and the second one was the
epileptic group. Epileptic seizure was induced with a single injection of
PTZ (50 m s / k s i.p.). Blood samples and liver tissues were obtained
under ether anesthesia 24 hours after PTZ injection. Oxidative injury
was evaluated in erythrocyte and liver tissue by measuring the
glutathione (GSH) and malondialdehide (MDA) levels and glucose-6phosphate dehydrogenase (G-6-PD), selenium-dependent glutathione
peroxidase (GSH-Px), catalase (Cat) and Cu-Zn superoxide dismutase
(Cu-Zn SOD) activities. The results were compared between controls
and epileptic group. Erythrocyte and liver tissue Cu-Zn SOD activities
were significantly decreased in epileptic group (p<0.001). Erythrocyte
catalase activity was found to be significantly decreased in epileptic
group, where as it was not significantly different from control values in
liver tissue. In erythrocyte and liver MDA levels were significantly
increased in epileptic group (p<0.001). The GSH levels of control
group were found to be significantly higher than epileptic groups in
erythrocyte and liver tissue (p<0.001). No statistically significant
difference was found in erythrocyte G-6-PD and GSH-Px activities
between controls and epileptic group. G-6-PD and GSH-Px activities in
liver tissue were significantly increased in epileptic group (p<0.001),

(Mo/19.1/392)

l n t e r f e r o n s Induce E x p r e s s i o n o |
SMN and SMNc Genes and Restore SMNc Protein
Level in SMA. S. Baron-Delag(, A. Abadie ~, J. Melki~ and
L. Beretta'. INSERM, Utlitd 365. hlstitut Curie. Paris and 'IGBMC.
INSERM/CNRS/ULP. Srtasbottr~. France.

To identify partners of striatin, a neuronal, post-synaptic protein with a

signalling function (Castets et al., 1996, J. Cell. Biol. 134, 1051; Barloli
et al., 1999, J. Neurobiol. in press), interaction screening of a rat brain

axons are strongly stained. The distribution of phocein being more

Spinal muscular atrophy (SMA) is a cotmnon recessive disorder,

characterized by degeneration of motor nem'ons of the spinal cord,
Deletions, conversions or mutations of the survival motor neuron gene
(SMN) are responsible for SMA [i]. A highly homologous centromeric
copy of the SMN gene (SMNc) remains intact in SMA patients, However,
there is an inverse COiTelation between the amount of the SMNc gene
product and the clinical severity of the disease. An understanding of SMN
and SMNc gene regulation is therefore an important step towards therapy
for SMA. We identified a candidate Interferon-Stimulated Response
Element (ISRE), overlapping with an Interferon Regulatory Factors
binding motif (IRF-E) in the promoter region of SMN and SMNc genes.
Both ISRE and IRF-E motifs are involved in mediating transcriptional
induction of inte,'feron-stimulated gene expression. We therefore
investigated whether SMN and SMNc genes eae regulated by interferons
(IFN). Here we show that both IFN-13 and IFN-y rapidly induced SMN
and SMNc mRNA and protein expression in various cell lines. The
transcription factor IRF-I bound to the candidate ISRE/IRF-E sequence of
SMN and SMNc genes in vitro and overexpression of IRF-I induced
expression of both genes in transfection assays. IRF-I is therefore at least
in part responsible for the induchon of SMN and SMNc by IFNs. In
prinlary culture of fibroblasts from SMA patients, IFN-t3 and IFN-'?
induced SMNc gene expression and restored protein defect.

widespread than that of striatin, it follows that striatin is but one of several

i. Lefebvre. S. et al., Cell. 60. (1995) 155.

eDNA library by the yeast two-hybrid method has been achieved. A major
partner was found, phocein, a hitherto unknown protein of 26 kDa which
appears to be well conserved during evolution. Validation of the mteraction
of phocein with endogenous brain striatin was achieved by in vitro assays.
Incubation of the fusion protein GST-phocein with various brain
subfraetions resulted in the coprecipitation of striatin. Endogenous striatin
and phocein were also found to colmmunoprecipitate, as shown by using
their respective antibodies. The interaction of stnatin and phocein is thus
established. Phocem transcripts are mostly present in the central nervous
system, cerebellum, brain and spinal cord, and in the adrenal glands.
Phocein is detected by lmmunocytochemistry in the soma and processes of
a majority of neurons in adult rat brain. Axon bundles heavily labeled by
anti-phocein antibodies are scattered all over the brain, without any
obvious systematisation; m the cerebellum, m particular, the basket cells

partners of phocem, the search of which is actively pursued.

Abstracts FEBS'99

(Mo/19.1/393)

Amyloid precursor protein - lipid interactions

S. Coillet-Matillon, L. de La Fourni~re-Bessueille, R. Buchet

s269

(Mo/19.1/394)

Laboratoite de physicochimie blologique CNRS UPRESA 5013. UCB L3ml 1,

43 bd du I I novembre 1918, 69622 Villeurbanne Codex. France

The amyloid precursor protein (APP) is one of the major protein involved in
Alzheimer's disease. Soluble and membrane-bound isoforms of 13-amyloid
protein precursor (APPs and APPm) of Alzheimer's disease were extracted
and purified from porcine brains [1]. APPm is an integral type-I membrane
glycoprotein that spans the lipidic bilayer once.
A lipid-APP binding assay was developed to demonstrate the direct
interaction of APP w~th various lipids. Solid phase ELISA for APP binding
to phospholipid was performed. Microwell plates were coated with different
lipids and subsequently incubated with APP. Control binding of APP to the
microtiter plates was performed without lipid. It was found that APPm binds
to all lipids tested with a preference for phosphatidyl serine, sphingomyelin
and lactocerebrosides. The binding of APPm to sphingomyelin was
approximately 35% higher than the observed binding without lipids.
To better understand the binding of APP in lipid membranes, and eventually
to precise how it is cleaved to give 13-amyloid peptides, incorporation of
APPm into biomimetic membranes was performed. Liposomes were
obtained by dialysis of a mixture of phospholipid and octylglucoside.
Incorporation of APPm was carried out by preparing the Liposomes
suspensions with octylglucoside, addition of APPm and removal of detergent
by dialysis. The resulting vesicles were separated by sucrose density
gradient. The distribution of APPm into the liposomes was determined by an
ELISA test using polyclonal antibodies. It is possible to incorporate about 40
pmoles of APPm into the vesicles. Studies of factors controlling the binding,
e.g. lipid composition, pH, molar lipid/APPm ratio are in progress.
[1] L. de La Fourni6re-Bessueille et al. Eur. J. Biochem. (1997) 250 : 705.

(Mo/19.1f395)

A new signaling protein family.

F. Castets', T. Rakitina', S. Galliard ~, M.-G. Mattei b and
A. Monneron~.
a LNCF, CNRS, 2l chemin Joseph Aiguier, 13420 Marseille Cedex, France
blNSERM U491, Bd Jean Moulin, 13385 Marseille Cedex 5, France

Striatin is a protein specifically expressed in the somatodendritic

compartment of a few subsets of neurons, mostly pertaining to the
locomotor system [I]. A minor protein, it belongs to the WD repeat
family, binds Ca2*/ealmodulin and is most probably involved in
transduction phenomena [2]. Human and mouse EST data banks allowed
us to identify two proteins closely related to striatin, one known and called
SG2NA [3] and a "new" one that we called zinedin, These three proteins
share almost identical domains and the same general topology from the N
to the C terminus: a caveolin binding domain, a coiled-coil structure, a
CaZ+/calmodulin binding domain and 8 WD repeats suggesting similar
functions. However, we shown here, that the expression patterns of these
three proteins are very different: striatin is expressed in the CNS, SG2NA
is expressed in skeletal muscle and heart, zinedin being ubiquitous. We
report here the cloning and sequencing of human zinedin eDNA. We also
produced recombinant fusion proteins corresponding to these three human
eDNA and raised specific antibody for each of them. The three human
genes are distributed on different chromosomes: 2p22-p21 for striatin,
14q13-q21 for SG2NA and 19q13.2 for zinedin.
[1] Castets et al., J. Cell Biol., 134, (1996) 105l.
[2] Bartoli et al., J. Neurobiol., (1999) in press.
[3] Muro etal., Biochem Biophys Res Commun, 207, (1995) 1029.

Preconditioning against hippocampal neuronal death using a

sublethal global ischemia or kainate-lnduced seizures
N. Blondeau, H. Plamondon, C. Heurteaux & M. Lazdunski
IPMC - CNRS - 660, route des Lucloles, 06560 Sophm Antzpolis. France

Preconditioning with sublethal ischemia improves the brain resistance to a

subsequent prolonged ischemic insult. The mechanisms underlying this
neuroprotection remain largely unknown. The main objective of this work is
to analyze in vivo the potential cross-tolerance between different neuronal
death inducing treatments such as kainate administration that induces
seizures and global ~schemia and characterize common endogenous
mechanisms in these different models of tolerance. Global ischemia was
obtained by four vessel occlusion while kainic acid (KA) was used to induce
limbic epileptic seizures. Three preconditioning models were developped in
which ischemic and KA-induced epileptic episodes were combined in the
following manner. Group 1 was injected with 5 mg/kg KA prior to a 6 min
global ischemia (KA5 - I6). Group 2 received a 3 rnin global ischemia prior
to 7.5 mg/kg KA (I3 - KA7.5). Group 3 was injected with a 5 mg/kg dose of
KA prior to a 7.5 mg/kg KA injection (KA5 - KA7.5). The interval between
treatments was of three days. Groups of rats were sacrificed at 3 and 7 days
following the last treatment. Neuronal degeneration, assessed using the
silver impregnation method was strongly reduced in rats preconditioned
with a sublethal ischemia or a 5 mg/kg KA treatment (KA5 I6 >- KA5 KA7.5 > I3 - KA7.5). Apoptosis was drastically reduced in all
preconditioned animals as compared to ischemic or epileptic controls. The
present work supports the existence of cross-tolerance between KA
excitotoxicity and global ischemia in terms of neuronal death and suggests
the involvement of adenosine At receptors and sulfonylurea-sensitive ATPsensitive K" channels in this protective mechanism.

(Mo/19.1/396)

Effects of lipid peroxidation products on human

adenylosuecinate lyase
C. Crif6, C. Salerno, W. Stems, M. T. Mungo
Dept.of Biochemwtry, Umv Roma La Sapienza. Roma, Italy

Adenylosuecinate lyase (ASL; EC 4.3.2.2) catalyses two distinct

reactions in the purine synthesis, both involving the removal of fumarate~ the
conversion of SAICAR in AICAR along the de novo pathway, and the
formation of AMP from adenylosuccinate in the conversion of IMP into
adenine nucleotides. Decreased levels of ASL activity have been reported in
approximately 40 patients showing psychomotor retardation [1, 2]. A
remarkable feature of the disease is the high incidence of autistic symptoms.
It has been reported that ASL deficiency in a subset of patients is more
severe in liver, kidney, and lymphocytes than in skeletal muscle and
erythrocytes. Since the available data seem to exclude the presence of tissuespecific isozymes [3], this may imply that yet undefined regulatory
mechanisms play a role in controlling adenylosuccinase level within certain
cell types.
We studied the effect of the lipid peroxidation product, trans-4hydroxy-2-nonenal, on the wild type human ASL and on the enzyme from a
patient compound-heterozygous for two missense mutations (P75A/D397Y)
[3]. Both the enzymes were inhibited by 10-50 ~tM trans-4-hydroxy-2nonenal in a concentration-dependent manner by means of a mixed type cooperative mechanism. A significantly stronger inhibition was noticed in the
presence of the defective enzyme. Nonanal and trans-2,3-nonenal inhibited
the enzymes to a less extent and at about ten-times higher concentrations.
Hydroxylamine reversed the inhibition by trans-4-hydroxy-2-nonenal, trans2,3-nonenal or nonanal in the case of the wild type enzyme, but it was
ineffective to reverse the inhibition by trans-4-hydroxy-2-nonenal on the
defective enzyme. Dithiothreitol slightly decreased the inhibition exerted by
trans-4-hydroxy-2-nonenal on both the wild-type and the defective ASL,
while it did not produce practically any change in the presence of trans-2,3nonenal or nonanal.
[1] J. Jaeken & G. Van den Berghe Lancet 2, (1984) 1058.
[2] C Salerno el at. J. Inher. Metab. Dis. 18, (1995) 602
[3] D. Verginelli et al. Biochim Biophys. Aeta 1406, (1998) 81.

s270

(Mo/19.1/397)

Abstracts FEBS'99

Post-transcriptional modification patterns of mitochondrial

wild-type and mutant human tRNALYs and tRNA Leu(UUR)
C. Florentz 1,2, M. Helm 1,2, A. Chomyn l, and G. Attardi 1

(Mo/19.1/398)

1Diviston of Biology, Catech Pasadena 91125, USA and 2UPR 9002 du

CNRS 1BMC, 15, rue RenO Descartes, 67084 Strasbourg

Post-transcriptional modifications are characteristic features of tRNAs and have

been shown in a number of cases to influence both their structural and
functional properties, including structure stabilization, aminoacylation and
codon-recognition. We have developped an approach which allows the
investigation of the post-transcriptional modification patterns of human
mitochondrial wild-type and mutant tRNAs at both the qualitative and the
quantitative levels. Specific tRNA species are long-term labeled in vivo with
~-'P-orthophosphate, isolated in a highly selective way, enzymatically digested
to mononucleotides, and then subjected to two-dimensional thin layer
chromatographic analysis. The wild-type tRNALy~ and the corresponding
tRNALy~ carrying the A8344G mutation associated with the MERRF
(Myoclonic Epilepsy with Ragged Red Fibers) syndrome exhibit the same
modified nucleotides at the same molar concentrations. By contrast, a
quantitatively different modification pattern was observed between the wildtype tRNA e~u(t:t3R) and its counterpart carrying the A3243G mutation
associated with the MELAS (Mitochondrial Myopathy, Encephalopathy with
Lactic Acidosis and Stroke-lake episodes) syndrome, the latter exhibiting a 50%
decrease in rn~-G content. Complementary sequencing of tRNA~(uUa)has
allowed the localization of this modification at position 10 within the D-stem of
the tRNA. The decreased level of this modification may have important
implications for understanding the molecular mechanism underlying the
MELAS-associated nfitochondrial dysfunction.
Hehn et al., Nucleic Acids Res, 27 (1999) 756.

(Mo/19.1/399)

Effects of various stress and inflammatory stimuli on

cultured rat astrocyte cyclooxygenase activity
B KalmarL E Madarfisz~. t3 Kiss~. S Farkas"
" Gedeon Rtchter Ltd .Budape.st. lhmgarl : ~h~stttute qf
Expermtenta131edtcme qf the lhmgmTan Academy ,fSclences

Prostanoids are commonly known as regulators of immune and

inflammatory processes. They are also present in the CNS where they play
important roles in the regulation of diverse CNS functions, such as
cerebral blood flow, temperature regulation Although concentrations of
prostaglandins are low in the normal brain, their levels increase under
pathological conditions such as ischemia, seizures and inflammation
While shear stress is known to act on vascular endothelial cells, its
effect is not clarified on astroglial cells in vitro Astroglia may play in
vivo an important mechanical sensory function in brain homeostasis. The
prostaglandin E2 (PGEz) production by cultured rat astroglial cells was
investigated in response to various stimuli, e g , shear stress, caused by
turbulent flow of culture medium covering the cells, lipopolysaccharide
(LPS, 10 ~tg/ml) and IL-lf3 (10 ng/ml) treatment.
Under our experimental conditions PGE2 production by astroglial cells
was increased by shear stress, LPS and 1L-I[3 LPS and IL-I[3 induced
PGE2 production could be inhibited by the non-selective cyclooxygenase
inhibitor indomethacin

Neuroprotective role of cyclooxygenase (COX)

inhibitors in ischemic and traumatic brain damage
A Gere. fi~KIs-Varga. M Par6czal. B Kalmar. G3. Dom~iny.
J Tofie3. B Kiss and E K~trpati
Gedeon Rtchter Lid. Budapest. Hungary

Recent data indicate that inflammatory processes may contribute

to ischemic and traumatic brain damage [1, 2]. The aim of this study was
to investigate the effect of COX inhibitors, i.e indomethacin, diclofenac,
ketoprofen, celecoxib, meloxicam and nimesulid against pathological
changes in brain ischemia or head injury
Methods" The infarced area was measured in focal ischemia model [3]
(permanent occlusion of MCA where mice were treated with drugs in a
dose of 10 mg/kg i p 30 rain before and 1 h after injury In the sublethal
head trauma model [4} drugs were given 5 rain after injury (1 mg/kg, i.v.)
and the neurological status of mice was evaluated 1, 24 and 48 h later.
Results Pretreatment with COX-1 inhibitors (indomethacin, diclofenac
and ketoprofen) was without any effect on infarct size (3 1%, 8.7% and
7 1%, resp ) whereas COX-2 inhibitors (celecoxib, meloxicam and
nimesulid) decreased the area of infarct (30.3%, 4 3% and 43 3%, resp ).
Posttreatment with COX-I inhibitors (indomethacin, diclofenac) was also
beneficial (326% and 173%, resp.). COX-2 inhibitor celecoxib
significantly reduced the infarct area by 40 0% and the effect of nimesulid
was also beneficial (24.8%). In the sublethal head trauma model,
admimstration of single i.v.dose of COX inhibitors produced significant
improvement. The neurological score was increased by the treatment of
indomethacin, diclofenac, ketoprofen, celecoxib, meloxicam and nimesulid
24 h after injury (score +196, +117, +152, +96, +209 and +293, resp.).
The beneficial action of indomethacin, celecoxib, meloxicam and
nimesulid remained until 48 h (score +240, +163, +204 and +311, resp )
Conclusion:
This study demonstrates that administration of COX
inhibitors may ameliorate the symptoms and/or consequences of ischemic
and traumatic brain injury
References'
[1 ] S Nogawaet al J Neurosci 15" (1997) 2746
[2] L TDunn Exp Opin Invest Drugs: 6 (1997) 1511
[3] C Backhan[3 e t a l ' J Pharmacol Methods: 27' (1992) 27
[4.] E D Hall et al/J. Neurosurg 68 (1988) 456
(Mo/19.1/400)

Effect of estradiol on calcium flux induced by amyloid

peptide AB 25-35in SK-N-SH human neuroblastoma cells
A. Lebeau, A. Gompel, W. Rost~ne, A. Lombet
INSERM U339, HOpital St-Antoine, 75012 Paris, France

Many studies have described the neuroprotective effects of estmdiol

on amyloid peptide AB ,-5-35 neurotoxicity in many different cellular
models such as SK-N-SH cell line. Variations of intmcellular free calcium
levels seem to play an important role during the first steps of amyloid
peptide insult. The present study provides a dynamic analysis of the effect
of 171Lestradiol on such calcium levels during amyloid peptide insult,
using a dynamic analysis station of Fura-2-detected intracellular free
calcium levels (Quanticell 700, Applied Imaging Ltd).
AB :5-~s (1/aM) induces both an accute and a delayed neurotoxicity
on the SK-N-SH cells. The accute necrotic form of cell death (about 25%
of the monitored cells) is characterized by a massive and sustained
intracellular free calcium level elevation, followed a few minutes later by
an extinction of the fluorescent signal caused by the loose of cellular
membrane integrity, The delayed apoptotic form of neurotoxicity
(concerning about 75% of the monitored cells) is characterized by (i) an
initial slight and transient elevation of intracellular free calcium level and
(ii) after several hours by a massive intracellular free calcium level raise
just before generation of membrane blebbs that are typical features of
apoptosis. In the same way, the cellular viability decreases by 40% after
24 hours incubation with A8 25-35 (l~lM) as determined by MT'F viability
test.
The initial intracelIular free calcium elevation registred at the
beginning of the apoptic form of cell death implicates both an entry of
extracellular calcium (blocked by 2raM EGTA) and an intracellular
calcium stocks release (blocked by 1/aM Ryanodine). In these conditions,
17B-estradiol (1/aM) induces a small calcium transient and prevents any
Af~ 25-35-induced free intracellular calcium levels elevation that normally
cause neurotoxicity.
These study provides additional imaging results in order to explain
the mechanims of neumprotection affored by 17B-estradiol that are still
controversial.

Abstracts FEBS'99

(Mo/19.1/401)

CLONING OF GENES UP- AND DOWNREGULATED

IN SKELETAL MUSCLE DISUSE
N Cros, L Leclerc l., A. Chopard I, J-F Marini 1 , and C A
Dechesne2 1NSI'3~,MU 300, Montpelher, France. i anti,present

s271

(Mo/19.1/402)

address- ('NRS U.~47~6548, Ntce, France. E-mall' dechesne(db~umcefr

Situations such as limb immobilization, bed rest, and space flights imply
reduction or abolition of skeletal muscle activity. This muscle disuse
leads to progressive muscle atrophy. Regulation of the atrophy process is
poorly understood but has already been shown to take place at the
transcription level for several genes
In order to draw a more global picture of the molecular mechanisms
induced by muscle disuse, we developed a strategy to find genes with an
altered expression in the model of rat soleus muscle unloaded by
hindlimb suspension. This strategy is based on the differential screening
of subtracted libraries made with the recently described suppression
subtractive hybridization technology. More than 30 genes have been
found up- or downregulated in our model. Approximately one third of
them represent novel genes without match in public available databases
Here is presented a preliminary characterization of these novel genes in
terms of RNA messenger sizes, full length cloning, sequence analysis
and tissue specificity
This work indicates that i) a wide spectrum of cellular functions is
involved in the response of muscle to disuse, ii) muscle activity
participates to the regulation of gene expression, and iii) suppression
subtractive hybridization technology is an efficient approach to detect
alteration of gene expression and to clone novel moderately expressed
genes

(Mo/19.1/403)

A cellular model that recapitulates major pathogenic

steps of Huntington's disease
A. Lunkes, Y. Trottier, D Devys, G. Yvert, C. Weber
and JL Mandel
lnstitut de Gdndtique et de Btologie Moldculatre et Cellulaire (1GBMC),
CNRS/1NSERM/ULP. 67404 lllkwch Strasbourg, France

To gain insight into the pathogenic mechanisms of Huntington's disease we

have developed a stable cellular model, using a neuroblastoma cell line in
which the expression of full length or truncated forms of wildtype and
mutant huntingtin can be induced. While the wildtype forms have the
expected cytoplasmic localization, the expression of mutant proteins leads to
the formation of cytoplasmic and nuclear inclusions in a time and
polyglutamine length dependent manner. The inclusions appear more rapidly
in cells expressing truncated forms of mutant hunfingtin and react with
antibodies against ubiquifin and the proteasome.
In lines expressing mutant full length huntingtin major characteristics present
in Huntington's patients could be modeled. Selective processing of the
mutant, but not the wildtype full length huntingtin was observed at late time
points, with appearance of a breakdown product corresponding to a
predicted caspase-3 cleavage product. A more truncated amino-terminal
fragment of huntingtin containing the polyglutamine expansion is also
produced, that appears involved in building up cytoplasmic inclusions at
early time points, and later on also nuclear inclusions. This fits with the
finding that inclusions in the brain of HD patients are detected only using
antibodies directed against epitopes very close to the polyglutamine stretch.
We localized the cleavage site for the short truncated amino-terminal fragment
in a domain of about 60 amino acids. A fine mapping and mutagenesis is being
performed in order to evaluate the involvement of the potential cleavage site
in the release of a toxic polyglutamine-containing fragment leading to the
formation of nuclear inclusions.

The evolutionary conserved RNA-binding properties of

SMN, the Spinal Muscular Atrophy gene product
S. Lcfebvre, S. Bertrandy, P. Burlet, O. Clermont, C. Huber, D.
Thierry-Mieg and A. Murmich.
INSERMU393, InstttutNecker-IFREM,75743 Pariscedex 15, France.

Spinal Muscular Atrophy (SMA) is an autosomal recessive disorder

characterized by degeneration of spinal motoneurons and muscular atrophy.
SMA results from alterations of the Survival Motor Neuron (SMN) gene
encoding a protein of unclear function that interacts with RNA and RNAbinding proteins involved in mRNA processing. SMN protein is detected in
the cytoplasm and in novel nuclear bodies termed gems for gemini of coiled
bodies. SMN gene is located in chromosome 5ql 3 and a highly homologous
copy (SMNc) maps proximal to the disease gene. A tight correlation between
clinical severity and SMNc protein level is demonstrated in all tissues,
including spinal cord and muscles, derived from SMA patients. Subcellular
fractionation reveals that SMN is associated with heavy sedimenting
complexes. A better knowledge of conserved domains should provide further
insight into functional/structural requirements of SMN. We have isolated the
zebrafish Danio rerto and nematode Caenorhabditts elegans orthologue
cDNA clones and have found that the RNA-binding capacity is conserved
across species. Purified recombinant SMN proteins from both species showed
preference to poly(G) homopolymer RNA in vitro, similar to the human
protein. Deletions of the zebrafish SMN protein allowed to define a RNAbinding element at the N-terminal end of SMN in a conserved domain. Our
deletion analyses also favor the view that the RNA capacity is modulated by
domains located C-terminal to this element, in exons 2b and 3. Finally, the
deleted zebrafish SM'N protein mimicking a SMA frameshift mutation
showed a dramatic change in the in vitro RNA-protein interaction.
Recombinant SMN proteins should allow the isolation of sequence-specific
RNA ligands and help identify putative aberrant RNA maturation in SMA.
Hopefully, these studies should contribute to a better understanding of the
patbogenesis of SMA.

(Mo/19.1/404)

Calcium signalling and homeostasis in dystrophin deficient

Sol8 myotubes
E Marchand, B Constantin, C Vandebrouck, G Raymond,
C Cognard
Laboratmre de l'hysloh~gte o~endrale. ~'~lR 6558 86022 Pmtwrs. France

'Duchenne muscular dystrophy (DMD) is an X-linked recessive lethal

neuromuscular disease characterised by a progressive degeneration of human
skeletal muscle The mutated gene product in DMD named dystrophin, a major
constituent of the cell cytoskeleton localised at the inner face of skeletal muscle
cell membrane, is absent in dystrophic muscle cells Although the precise
degenerative mechanisms causing cell necrosis are still unknown, it has been
proposed early on that the subsarcolemmal dystrophin may play a simple
structural role in protecting the sarcolemma from contraction-induced injury
This hypothesis has failed to explain all the pathological sequences that
produce muscle degeneration, and dystrophin is thought to normally play an
active role in regulating cell signalling systems A possible cause of cell
damage has been suggested to be a sustained elevation of cytoplasmic free
calcium concentration ([Ca2-]i) exceeding the buffering capacity of cells which
could be the primer of cellular necrosis In the aim to investigate the role of the
different calcium signalling systems potentially implied in the pathogenesis of
DMD, we propose the So18 muscle cell line as possible cellular model. We
have shown by Western blotting and lmmunostaining that differentiated So18
myotubes do not express dystrophin A ratiometric fluorescence method using
the calcium probe lndo-1 under Xenon lamp illumination have shown that
differentiated myotubes present intracellular calcium transients following both
membrane depolarisatton induced by 10"~ M acetylcholine and high potassium
stimulation (100 mM KCI) or sarcoplasmic retieulum calcium release
consequently to a pharmacological stimulation of ryanodine receptors induced
by caffeine Interestingly, resting calcium level increases with the age of
cultured So18 myotubes and they rapidly undergo cell death in 4 or 5 days in
our culture conditions They present chromatin condensation, membrane blebs
and a characteristic shrunken morphology, which is reminiscent of an apoptotic
pattern Simultaneously, we have observed an elevation in the occurrence of
dead cells shown with a propidium iodide staining The description of the
calcium homeostasis and calcium transients in sol8 myotubes might be useful
to analyse further the consequences of the re-expression of dystrophin The
obvious hope is that critical examination of the cellular function of dystrophin
will help to understand the fine process of Duehenne muscle dystrophy

s272

(Mo/19.1/405)

Abstracts FEBS'99

Coproporphyrinogen oxidase gent defects in hereditary copropor~hyria

P. Mart,'isek~. R. Rosipal~. J. Laraoril~'.J. Z,eman~, J.C Dcybach
~Dept of Pedtatmcs, Facul~ of A4edscmeL 12109Prague, Czech Repubhc
bUentreFrangats des Porpt~vrtes. 1VSERAI(.'409. 92701 Colombes, France

Hereditary coproporphyria (HC) is classified in the group of acute

hepatic porphyrias
HC is caused by defect in the enzyme
coproporphyrinogen Ill oxidase (CPX) which converts coproporphyrinogen
III (coprogen) to protoporphyrinogen IX. Coprogen is made mainly in the
liver and is exctreted predominantly in the feces. HC is inherited in an
autosomal dominant mode We recently reported molecular cloning of a
cDNA complementary to human CPX (i) We also have used a human
cDNA probe for cloning of CPX gene (2). The human genome contains a
single CPX gene, which is localized to chromozome 3q12, spans about 14 kb,
and consists of seven exons and six introns (2). The probability of a lifethreatening porphyric attack in HC is a significant personal burden, and
create a challenge for counseling and medical management. Availability of
screening for all HC families using melecular biological techniques will
ensure the correct diagnoses in all gene carriers. Therefore, the seven exons,
the exon/intron boundaries and part of 3'noncoding sequence of the CPX
gene were systematically analyzed by an exon-by-exon denaturating gradient
gel electrophoresis (DGGE) followed by direct sequencing in four unrelated
heterozygous patients from Czech Republic
Mutations founded in the CPX gene"
Locahzation
Mutatton
Sequence modification
Exon 2
G589T
Gly197Trp
662 delAAAT
Frameshifi (stop codon +8)
Exon 5
1168 delGGA
390 del Gly
Exon 6
GI277A
Exon 6-skipping
Procaryotic expression of mutated cDNAs from patients with the amino acid
substitution Gly197Trp and amino acid deletion 390 del Gly were performed
and residual activity of 22% of control and less than 1% were found,
respectively. Five itragenic dimorphisms were also characterized (promoter
region -142C/G, Exon 4 814 A/C and 880 G/A, Exon 5:990 G/A, Exon 7
delATTCTT)
1. Martzisek P. et al., Proc Natl Acad. Sci. USA, 91, (1994), 3024
2. Delfau-Larue M.H et aL, Hum. Mol. Genet. 3, (1994), 1325
Supported by Grant Arc). 302"99.0651 o f GA("R.
(Mo/19.1/407) Control Of Adenine Nucleotides In lschaemic Myocardium
A. Meneses, J Mowbray
Gower St, Dept o f Biochemistry and Molecular Biology,
Umversity College London, London W('IE 6BT, ~LK

Measurements of adenine nucleotides in ischaemic and reperfused rat

hearts have shown that free adenine nucleotides appear to be
augmented by up to 30% from some unidentified source separate from
the highly phosphorylated adenosine and glyceric acid polymer,
Purinogen, found in mitochondrial intermembrane space [1]. The
endothelial cell barrier makes direct radiolabelling of myocyte purine
nucleotides virtually impossible in intact hearts.
A new system for perifusing isolated rat myocytes has been developed.
It has a small interior space, rapidly responds to substrate changes,
requires no mechanical agitation of the cells, and provides adequate
reoxygenation and reliable sampling of the cells Using this system,
adenine nucleotide contents and radioincoporation experiments have
been repeated and shown to mirror the findings of earlier myocyte
incubations [2] Subjecting cardiomyocytes in our perifusion system to
ischaemic insult we have confirmed the observation that a putative
storage pool able to supply adenine nucleotide in ischaemia appears to
exist. Using 8- J4 C adenosine labelling in ischaemia and reperfusion
experiments we expect to identify and characterise this store.

(Mo/19.1/406)

Effect of oxidative stress on stability and structure of

neurofilaments. S Gdinas, D. Germain, J-C Guimond,
I Gosselin, L Cossette, C Chapados, M-G Martinoli.
Dept Biochemistry,Umversitd du Oudbec11Trois-Rivtbres.Oc,Canado

Neurofilament proteins (NFs) are highly phosphorylated molecules in the

axonal compartment of the adult nervous system The phosphorylation of
NFs is considered an important determinant of filament caliber, plasticity
and stability. The structure of neuronal cytoskeleton could be altered by
oxidative stress and NFs represent an important potential target for free
radical damage because of their abundance and their long biological halflife. We report the structural analysis of NFs after oxidative damage.
Electron microscopy, SDS page analysis, immunoblots and Fourier
Transform Infrared (FT-IR) spectroscopy were used to investigate the
relative sensitivity of NFs to oxidative stress and to identify the molecular
perturbation that follows.
A Fe3+/ascorbate buffer system as well as catechols were used to generate
free radicals on a substrate of native and dephosphorylated NFs By FT-IR
spectroscopic analysis, we have established that free radical damage on
native and dephosphorylated NTs gives rise to a new band at 1720 cm "t
that we assigned to the formation of new carbonyl groups on the amino
acid side-chains. Moreover, modifications in the profile of the Amide I
and 11 bands were seen after exposure of native and dephosphorylated NFs
to oxidative injury. This suggests the formation of a ~3-sheet structure,
which may be an indication of pathological disorders. However, more
drastic injuries were detected on dephosphorylated NFs where peptidebond cleavage was observed in addition to structural changes, suggesting
that a cytoplasmic pool of dephosphorylated NFs might represent an
important potential target for free radical activity. Our studies provide
strong evidence that oxidative stress can modify the primary structure of
NF proteins and may thus contribute to creating pathological
abnormalities of the neural cytoskeleton that are characteristic of a variety
of neurodegenerative diseases.

(Mo/19.1/408)

Striatin, a novel signaling protein

in the central nervous system
A. Moqrich, A. Monneron, M. Djabali
LNCF, CNRS, 21 CherainJoseph Alguier, 13420 Marsedle, France

Striatin, a recently discovered signaling protein, Is present in dendrites

and sprees of certain neurons mostly involved in motor (Castets et al.,
J. Cell Biol., 1996; Mcqrich et col., Genomics, 1998),). Its molecular
features are those of a signaling protein, endowed with several proteinprotein interaction domains: a WD-repeat domain, a CaZ*-calmodulin
binding domain (Bartoli et al., J. Biol. Chem., 1998), a coiled-coil
structure and a caveolin-binding domain. We have just shown, by an
antisense strategy, that striatin is involved in: 1) the growth of neurites
in cultured motoneurons; 2) the locomotor behavior of adult rats
(Bartoli, 1999, J. Neurobiol., in press). The effect of the transient
inhibition of striatin synthesis lead us to achieve the knock-oat of the
striatin gene. This gene has been cloned and its structure determined. A
construct deleting a 14kb fragment coding for the first six WD-repeats
of the striatin gene has been realized. It allowed us to obtain
recombinant ES cells by homologous recombination. Chimeras have
been obtained by injection of recomhinants ES cells. Their offsprings

References.

are studied. In parallel, the expression profile of striatin during

development is studied by means of in situ hybridization on whole

[1] J Mowbray et al., Eur. J. Biochem, 254, (1998), 75

[2] P. G.Stanley et al., J. Mol. Cell Cardiol. 27, (1995), 1797.

mounts embryos (from day 8 1/2 to 10 I/2) and on sections. Striatin is

localized in the forebrain, heart and limb buds. Immunocytochemistry
on sections confirm the expression of the protein within brain and
heart.

Abstracts FEBS'99

(Mo/19.1/409)

Persyn in development, neurodegeneration and

malignancy
N. Ninkina,.b.V. Buchman",b

s273

(Mo/19.1/410)

Lipid Peroxidation in Stroke: Effects of Glutamate Release

Inhibition, Glutamate Receptor Antagonist and L-NAME
T.Ozben a, E.Balkan b, S Balkan c, M.Serteser a, S.Giimia~lO~

"School of Biomedical Sciences, Univeestty of St. Andrews, Fife, UK

blnstitute of Gene Biology, Russian Academy of Sciences, Moscow, Russia

Departments of Biochetmstry, bOtorhinolaryngology, 'Neurology, Akdeniz

University, School of Medwme, Antalya. Turkey

The synucleins are a unique family of small intracellular proteins expressed

in the nervous system and localised in synaptic terminals, u-synuclein has
been implicated in at least one form of learning and memory and is believed
to be involved in the pathogenesis of Alzheimer's disease and familial
Parkinson's disease. However, the functions of synucleins remain elusive.
We cloned mouse and human genes encoding persyn, a new member of the
synuclein family [1, 2]. Although amino acid sequence and some physicochemical properties of persyn and other synucleins are very similar there are
substantial differences in their expression patterns [1]. Persyn displays
distinctive patterns of expression in subsets of neurons and their axons in
both the developing and ageing nervous system. Furthermore it was shown
that persyn is concentrated in cortical axonal lesions seen in
neurodegenerative conditions. Overexpression of persyn in cultured sensory
neurons by the microinj ection of an expression plasmid into nuclei of these
cells and showed that persyn is involved in regulating the integrity of the
neurofilament protein network [3]. These findings suggest that persyn plays
a role in modulating axonal architecture in the developing and mature
nervous system and implicate persyn in the axonal pathology of
degenerative conditions. Substantial level of persyn expression was also
found in particular layer of skin epidermis (stratum granulosum) of
postnatal mice and in several infiltrating breast tumours. In both types of
tissues severe changes of keratin intermediate filaments take place,
suggesting the involvement of persyn in modulating not only neurofilament,
but also keratin network. We also studied the expression and intracelhilar
disuibution of abnormal isoforms of the persyn protein. 16 kD persyn
protein is cytosolic but in some tumour cell lines, a "heavy" persyn isoform
has a nuclear loealisafion. Further functional studies of persyn involves
generation of mice with targeted deletion of persyn gene.

Stroke is the third common cause of death in industrialized countries.The

mechanisms of ischemic injury is complex and involves more than a single
pathophysiologic pathway. Excessive activation of the NMDA subtype of
glutamate receptor has been implicated in the sequence of neurochemical
events that results in irreversible neuronal damage in cerebral ischemia. Focal
cerebral ischemia was produced by the permanent occlusion of right MCA in
urethane anesthesized rats. Nitrite and cGMP as indicators of NO production
and malondialdehyde(MDA) and lipid conjugated dienes as indicators of lipid
peroxidation were measured in both cortex and cerebellum at 0,10,60 minutes
of ischemia. The same parameters were measured in rats treated with
glutamate receptor antagonist MK-801 (0.5 mg/kg, i.p.) glutamate release
inhibitor Lamotrigine (20 mg/kg, i.p.) and Nitric Oxide Synthase inhibitor LNAME (10 mg/kg, i.p.) 30 minutes before or just after the occlusion of the
right MCA. lpsilateral cerebral cortical nitrite levels were increased relative to
contralateral cortex after MCAO. No significant changes were observed in
ipsilateral or contralateral cerebellum. There was also a marked increase in
CGMP levels in ipsilateral cortex and contralateral cerebellum at 10 and 60
minutes when compared with opposite cortex and cerebellum. In ipsilateral
cortex, MDA values at 60 minutes and conjugated diene levels at 0, 10 and 60
minutes of ischemia were found higher than those in contralateral cortex. On
the other hand, contralateral cerebellar MDA and conjugated diene levels were
found higher than those in ipsilateral cerebellum. Our results showed that preand postischemic administration of MK-801, Lamotrigine and L-NAME
significantly decreased the Nitrite, cGMP, MDA and conjugated diene
production.
Key Words: stroke, nilric oxide, lipid peroxidation
[1]Balkan S, Ozben T, Balkan E, Oguz N, Serteser M, GtimO~lti S. Effects of
lamotrigine on brain nitrite and cGMP levels during focal cerebral ischemia in
rats. Acta Neurologica Scandinavica 95: 140-146, 1997;
[2]Free Radicals, Oxidative Stress and Antioxidants. Pathological and
Physiological Significance. Edited by T. Ozben Plenum Press New York and
London ISBN 0-306-45813-6, 1998.

[1] Buchman et at., J. Neurosci., lg, (1998) 9335

[2] Ninkina et at., Hum. Mol. G-enet., 7, (1998) 1417
[3] Buchman et at., Nat. Neurosei., 1, (1998) 101
(Mo/19.1/411)

The importance of apolipoprotein E e2 allele in stroke:

A case-control study
M. Serteser a, S. Visvikisb, T.Ozben a, B. Herbeth b, S. Ataus c,
M. Kasapoglua, S. Balkan , G. Siestb
Akdeniz University School of Medicine, ~Dept of Biochemistry and
~Neurology, 07070 Antalya, Turkey & bCentre de Mddecine Prdventive,
2, Avenue du Doyen Jacques Parisot, 54501 Vandoeuvre-Lks-Nanoy, France

The eventual effect of apolipoprotein E polymorphism in the development of

ischemic cerebrovascular disease has not been investigated sufficiently and
contraversial results were obtained from the few existing studies. Therefore we
aimed to find out the possible role of apolipoprotein E polymorphism in a well
characterized stroke patients. Genotyping of apolipoprotein E carried out in 79
patients ( 26 thrombotic, 20 embolic, 26 lacunar and 7 miscellaneous) and in
126 age and sex matched controls free of eerebrovascular disease. In addition,
serum apolipoprotein E, A I, C III and B and lipoprotein (a) levels were also
determined. The prevalences of well-known vascular risk factors were
significantly higher in patients. Epsilon 2 allele was found significantly higher in
controls ( 8.34% ) than in patients ( 3.16% ) (X2= 4.37, p<0.05). Patients with
large-vessel stroke or lacunar stroke had higher triglyceride and lower HDL
levels. In all stroke subtypes, apolipoprotein A I levels were lower than in
controls and the ratio of apolipoprotein B to A I was higher. A stepwise logistic
regression showed that; prescence of vascular stroke was related with epsilon 4
allele, diabetes, high systolic blood pressure, high apolipoprotein E serum levels,
but inversely related with epsilon 2 allele and apolipoprotein A I levels. Epsilon
2 may protect individuals against stroke even if the epsilon 2 carrying patients
had higher apo E levels and epsilon 4 may be a genetic risk factor together with
other well-known vascular risk factors. Large population based studies are
needed to clarify the exact relationship between stroke and lipid metabolism.

(M0/19.1/412)

NMDA receptor mediated PARP activity in

hippocampus of adult and aged brain. Effect of amyloid.
R.P. Strosznajder, H J~sko and J. Strosznajder
Aledtcal Rewarch Center. l'oh.~h .tcademv of Sciences.
PawtJ~sktego 5 ?,'treet. Warsaw 02-106. Polaml

Poly(ADP-ribose)polymerase (PARP) is a nuclear enzyme which is involved

m DNA repair, cell prohferation, differentiation and transformation
However, overstlmulation of PARP activity may lead to ATP depletion and
cell death In these studies NMDA receptor stimulation on Poly(ADPnbose)polymerase activity m hlppocampus slices from adult and aged brain
was investigated. Additionally, the effect of A13(25-35) the neurotoxlc
fragment of lull length anayloid beta peptide, on basal and NMDA receptor
regulated PARP activity was evaluated The studies were carried out using
slices of hippocampus from 4 and 27 months old rats. PARP activaty was
determined under optimal expermlental condition in the presence of [14C]
NAD. It was tbund that in hlppocampus shces from 4 months old rats NMDA
at 100 I.tM concentration significantly stimulated PARP actixaty and this effect
was abohshed by noncompetitive antagonist of NMDA receptor, MK-801
(101.tM). Amyloid beta peptlde (25-35) at 25 I.tM had also significant
st~mulatory effect on PARP actwity and this effect was significantly decreased
by inhibitor of mtric oxide synthase, N-Nitro-L-argmine (NNLA) at 100 ~.tM
concentration Concomitantly, it was found that A[?, activated free radical
evoked lipid perox~datlon These results suggest that NO and other free
radicals may be responsible for AI3 evoked sumulatlon of PARP activity In
hlppocampus slices from 27 months old rats, NMDA receptor stimulation and
amylmd beta peptlde had neghgible effect on PARP actiwty
Summansmg, our data indicates that alteratmns of PARP activity evoked by
NMDA-receptor and amyloid beta peptides, m hippocampus are age
dependent

s274

(Mo/19.1/413)

Abstracts FEBS'99

Role of the W W Domain in Signaling & Disease

M. Sudol, S. Rentschler, K. Scheinerman & X. Espanel.

(Mo/19.1/414)

Mechanism Leading to the Formation of Fibrils.

Dept of Biochem. & Mol. BioL, Mt Sinai School

of Medicine, New York, New York 10029, USA

Similar to SH2 and SH3 modules, the WW domain mediates proteinprotein interaction[l]. It is present in more than 100 structural and signaling
molecules in Eucaryotes, including ubiquitin ligases, dystrophin and adaptor
proteins [2]. The WW domain binds proline-rich ligands with the core
consensus Pro:Pro:Xxx:Tyr or 6xPro. Phosphorylation of Tyr abolishes the
binding. Recently a subset of WW domains was shown to bind to phosphoserine and phospho-threonine containing ligands in the phosphorylationdependent manner. The WW domains have been implicated in several genetic
diseases including Liddle Syndrome (hypertension), Muscular Dystrophy,
Alzheimer and Huntington Diseases [1,2]. We focused on Muscular Dystrophy
by identifying a protein ligand that binds to the WW domain of human
dystrophin. In addition, we decided to study ligands to the WW domain of
FE65 adaptor protein.
FE65 interacts with the internalization signal of
Alzheimer's beta-amyloid precursor protein and was proposed to regulate its
processing. We elucidated the role of the WW domain of dystrophin and
surrounding sequence in binding t3 -dystroglycan. We showed that the WW
domain of dystrophin along with the EF-hand motifs is sufficient for binding to
the carboxy-terminus of 13-dystroglycan. Through site-specific mutagenesis and
in vitro binding assays, we demonstrated that binding of dystrophin to the
carboxy-terminus of 13-dystroglycan occurs via a ILdystroglycan
Pro:Pro:Xxx:Tyr core motif [3]. Precise mapping of this interaction could aid in
therapeutic design. To understand the function of the FE65 adapter protein we
identified its binding partners.
Proline-rich sequences sharing a
Pro:Pro:Leu:Pro core motif were recovered by screening expression libraries
for ligands of the FE65 WW domain. We identified two FE65WW ligands as
the 80 and 140 kDa isoforms of Mena, the mammalian homolog of the
Drosophila Enabled gene [4]. Finally, we demonstrated that Mena binds to
FE65 in vivo by coimmunoprecipitation assay from COS cell extracts. Further
characterization of the Mena-FE65 complex should identify a physiological role
for these proteins in amyloid precursor protein biogenesis.
Study of WW domain of dystrophin and FE65 adapter protein should
illuminate molecular mechanisms that underlie Muscular Dystrophy and
Alzheimer's Disease.

Biophysical Characterization of Litheetathine: Evidences for

a Polymeric Structure at Physiological pH and an Autolysis

C. Cedni, V. Peyrot, L. Duplan, S. Veesler~, J.P. Le Caer, J.P.

Bernard, H. Bouteille, 1L Michel, A. Vazi, B. Michel, Y.
Berland, J.M. Verdier.

Lithostathine, an inflammatory protein, that we have described as a

calcium carbonate crystal habit modifier (1), is found precipitated under the
form of fibrils in various diseases like chronic calcifying pancreatifis,
Alzheimer disease or Down syndrome. In order to gain better insight into
the nature and the formation of fibrils, we have expressed and purified
recombinant lithostathine using a mammalian expression system. Analytical
ultracentrifugation and quasi-elastic light scattering techniques were
employed to demonstrate that lithostathine remains essentially monomerie at
acidic pH whereas it is highly aggregated at physiological pH. Analysis of
these aggregated forms by electron microscopy showed an apparently
unorganized structure of numerous monomers which tend to precipitate
forming regular unbranched fibrils. Aggregated forms always preceded the
apparition of fibrils. In addition, we demonstrated that these fibrils resulted
from an autolysis mechanism due to a specific cleavage of the Argl 1-11el2
peptide bond. It is deduced that the N-terminal undecapeptide of
lithostathine normally impedes fiber formation but not aggregation. We
therefore propose that lithostathine, whose major function is unknown,
defines a new class of molecules which spontaneously undergoes autolysis
and is not involved in cytoskeleton nor intermediate filament functions. As
lithostathine is expressed in very early stages in Alzheimer's disease (JMV,
unpublished), we propose a theoritical model explaining the formation of
amyloid plaques in neurodegenerative diseases starting from lithostathine.
(1) Geider, S. et al., J. Biol. Chem. 271, 26302-26306 (1996)

1. Sudol, M. Trends in Biochem. Sci. 21, (1996), 162. 2. Sudol, M. Prog.

in Biophys. a n d M o l . Biol. 65, (1996) 113. 3. Rentschler, S., et al.,. Biol.
C h e m . (1999), April Issue. 4. Ermekova, K.S., et al., Z Biol. Chem. 272,
(1997), 32869.

(Mo/19.1/415)

Influence of H.E.L.P. therapy on serum homocysteine

and neopterin in atherosclerosis

K. Vrecko ~, M. Walzl b, E. Tafeit ~, G. Reibnegger~

" ~ u t e ~r ~
C ~ r ~ , F-,F~r/~s/y, 80"t0C~z; Aus~a,
~D~. ot'Cormm~S~/~, State/-/~o.~N~ora., 8o61GrazAus~
Elevated serum homocysteine (HC) is regarded as k'deperdera risk factor for
~ s c k ~ o ~ (1). ~
s~am neol~rin shows i n v o l v ~ of the cellar romaine
system ha atherosclerosis (2). Worldwide trials are made to lower serum HC by
~tion
of fo]ic acid. heserdy no longtime studies ~xe available showing whether
this can corarbate to decrease incktence of stroke and cardiac hfarctionAs cholesterol, ~
LDL and fibrinogen can be precipitated and washed out
extracorporany from serum of at_rm-oscleroticpafiems by I-I.E.L.P. (Hepam-induced
Extmcoq~ral LDL ~ o n ) t ~ by the Phsmat secure (B.Bram~ FRG) (3), we
investigated the poss~qity of decreasing H C insermn and the~
of this mahod on
the cellularin~ame system
We measured HC and noopta~ concenwationsin serum of 67 patients with untreated
atha'osclerosis ( ~ v a s c u l a r i n s ~ ,
hyper~;~ar~ coroma'y heart disease,
p ~
occlusivevesselsdisease)before and after each H.E.L.P. thaapy in a long-term
study up to 12 therapies within 200 days. We found increased HC levels in all patients
(15.80pmoH serum, SD=7.89), whereby it should be mardoned that HC inaease over
12~moH doubles the risk of atheroga'~c diseases. We found a highly sigafram HC
reduction ~er H_E.L.P. t t ~
(13.4571xmol/1, SD=6.474; 19<0.001, t-test for
samples). Neopterin corr.ea'~rationswere also hcrezsed in all patients (9.18 nmol/l,
SD=3.23) c o n ~
to age matched healthy corgxols(5.34nmol/I,SD=2.70), and were
s i ~
lowered after H.E.L.P. therapy (8.22raaxfl/1, SD=3.22; p=0.002, t-test).
Long-term neopt~in concentrations from the first up to the 12~ H.E.L.P. session
deaxasal s~gnificalxly,in cormtst to HC which stayed stable.
We conclude that the decrease of neop~erin after long-term t r e a ~ t reflects an

improven-~ of chronicm

stimda~n ard thus, k~..ate clinicalbene~ whereby

HC carmotbe decreased penmnenfly, but the decrease al~r each H.E.L.P. sessionr t ~ a
be able to supIress a timtgr ~aease ard consequam'yprevent from further deterioration
of the disease.
(1) Malk~w MR. et aL CI~_Chem_40, (1996) 857.
(2) Weiss G. et al., Atherosclemsis106, (1994) 263.
(3) WalzlM. et al., Slroke 24, (1993) 1447.

(Mo/19.1/416)

ROLE OF CALPAINS DURING MYOGENESIS AND

SKELETAL MUSCLE REGENERATION
M.Zimowska, M.Sobolewska, I.Martelly*, J.Moraczewski
Dept.Cytolog3; Warsaw, ~hm,ersl~,, Poland; gostaz a.eco l.bloLuw.edu pl
*Lah.CRRET~ ~bnverstty Parts Xll, France

Calpains are calcium-dependent non-lysosomal cysteine endopeptidases,

which are actwe at neutral pH The physiological functions of calpain are
still unknown, meanwhile this protease has been implicated in a wide variety
of cellular processes including myogenic dlfferenttatlon. They are involved
for instance m s~gnal transductmn by membrane-associated down-regulation
of PKC, receptors processing and cytoskeletal protein proteolysis.
We investigated the activity and localisation of the two ubiquitous
lsoenzymes: mill- and micro- calpains in primary cultures of the myoblasts
isolated from adult rat muscles called satellite cells. Striking differences
were observed in calpains cellular distribution during differentiation of
satellite cells. In myoblasts mili-calpain was present in cytoplasm,
concentrated near the nuclear envelope, micro-calpain was detected in the
nuclei. In differentiated myotubes, both were in cytoplasm showing a
sarcomeric-like distribution.
Since satelhte cells are the myoblasts involved in muscle repair and
regeneration, we analysed calpain activity in muscle fibers of intact and
regenerating muscles. We purified active ~- and mill- calpams from
sarcoplasmic reticulum, myofibrils, cytosol and cytoskeleton of adult muscle
fibres and we shown that their act~vlties were different in the isolated
organelles of adult muscles. Using a crush-induced muscle regeneration
model, we established the variations of calpain activity during regeneration
of the fast-twitch (EDL) and the slow-twitch (Soleus) muscles We further
investigated the influence of two dextran derivates called RGTAs
(ReGeneraTingAgent) on calpain activity during adult rat muscle
regeneration. These molecules are heparan sulfate functional analogues,
which present different amount of sulfate. They were injected in the muscles
just after the crush Compared to untreated muscles, both used RGTA
accelerated the regeneration of EDL and Soleus muscles.
Taken together, these results support the hypothesis that two calpains
play different role in myogenesis Our results further illustrate future
potential pharmacological use of RGTA m neuromuscular diseases.
This work was supported by funds from grant .,Polonium".

Abstracts FEBS'99

s275

2.2 DNA recombination, repair and plasticity

(We/2.2/001)

AP site detemlinanls for Fpg spedfie ~ecognifion

B. C,~~aga, N. Hervotm~, J.-L Fourr@,S. ~ a x l C
Zelw~a
aUPR4.JOI,CNRS.450710d~ca~O2 bUPR2301.CNRS.
Gif'YvetteCUMR217,CNRS-CEA,Fcn~o~3.Au.xRoses(Fm~e)

Fpg proteitt is one of the enzymes that initiate base excision repair
(BER). It removes 8-oxoguanine (8-oxoG) and irnidazole ringopened purines (Fapy) from oxidatively damaged DNA. Fpg
catalyses the hydrolysis of the base-sugar bond (N-glycnsylase
activity) and then, the DNA chain cleavages (,/3- and &elimination) at
3' and 5' side of the resulting abasic site (AP lyase activity) (1,2).
Using electrophoretic mobility shift assays, we compare the
specific binding of E. cull and L. lactis Fpg proteins to DNA duplexes
containing various cyclic and non cyclic AP site analogs. This
approach allows to show that:
(i) the ring-opened form of the AP site is the active form of
the substrate for Fpg AP lyase activity (the reduced AP site is the best
substrate analog for both enzymes as compared with the other analogs
tested in this study).
(ii) the 1,3-propanediol is a minimal AP site structure
allowing a Fpg specific DNA recognition,
(iii) the newly described cyclopentanol (a cyclic AP site
analog) is better recognized than tetrahydrofuran suggesting a
hydrogen bond between the C4'-OH of the sugar and a Fpg residue.
Footprinting experiments show that Fpg binds to six nucleotides
on the damaged strand and contacts only the base opposite the lesion
on the complementary strand.
This comparative study (3) and limited proteo~ysis experiments of
the free or liganded protein provide new structural and/or functional
reformations about frpg specific DNA recognition.
1. Boiteux, S. et al, EMBO J., 6, (1987) 3177,
2. Castaing, B. et al. Nucleic Acids Res., 21, (1993) 2899.
3. Castaing, B. et al, Nucleic Acids Res., 7. (1999) 608.

(Wed2.2/003) Activation of DNA-hound PARP: A Novel Target for Signal

Transduetion Mechanisms.
M. Cohen-Armour" S. Homburg ~, F. Dantzer ~, N. Moran 3,
L. Visochek 1, N. Dekel J, E. Priel 4, D. Schwartz 5, V. Rotter ~ .

~l"eI-Aviv University, TeI-Aviv, Israel. eLouis Pasteur University,

Strasbourg, Franc~3Hebrew University of Jerusalem, ~BenGurion University, Beer-Sheva , and ~The Weizmann lnstltute o f
Science, Rehavot, Israel.

We present the fwst evidence for activation of polyADP-ribosepolymerase (PARP) by signals evoked in the eel] membrane (eg.
membrane depolarization~ nerve growth factors). PARP is an
abundant nuclear protein in eukaryotes that Catalyzes
polyADP-ribusylation of DNA-binding proteins, and thereby
modulates their activity. PolyADP-ribosylation is involved in
regulation of DNA repair, transcription and replication. The
presented evidence indicate that membrane depolarization
induces within minutes PARP activation in rat cortical neurons
and cardiomyocytes. In cortical neurons, signal-induced polyADP-ribosylation is mediated by activation of phospbolipase C
and inositol 1,4,5,-trisphosphate (IPj)-dependent Ca*2-release. It
is independent of the activity of phosphoinosltide 3-kinase.
Signal-induced activation of PARP is also independet of DNAnicks formation. These findings present a novel targert for
signal transduction from membrane to nucleus, which may
regulate the activity of proteins involved in DNA repair,
transcription and replication. They also predict a vital influence
of membrane depolarization on the viability of cortical neurons.

(We/2.2/002)

Regulated Formation of Extrachromosomal Circular

DNA Molecules During development in Xenopus laevis
S. Cohen, S. Menut and M. M~chali
Institut de G~n~tique Humaine. CNRS, 141 rue de la Cardonille 34396
Montpelher Cedex 5, France.

Extrachromosomal circular DNA molecules of chromosomal origin

have been detected in many organisms, and are thought to reflect genomic
plasticity in eukaryotic cells. They are also associated with genomic
instability which characterise cancerous processes and aging,
Here we report a developmentally-regulated formation of
extrachromosomal circular DNA that occurs preferentially in pre-blastula
Xenopus embryos, This specific population is not detected in the male and
female germ cells and is dramatically reduced in later developmental stages
and in adult tissues. The activity responsible for the de novo production of
extrachr~mosomal circles is maternally inherited, stored in the unfertilised
egg, and requires genomic DNA as a template, The formation of circular
molecules does not require genomie DNA replication but both processes can
occur simultaneously in the early development.
The production ofextrachromosoma[ circular DNA does not proceed
at random as multimers of the tandemly repeated sequence satellite I are
over-represented in the circle population while other sequences (such as
rDNA and the dispersed repetitive JCC31 element) are not detected.
Telomeric repeats were also detected in the circular DNA population
from early embryos, and comprised up to 1~3 % of the cellular telomere
content. Like other circular molecules, their amount is decreased in later
developmental stages. These circles are resistant to cleavage hy frequent
cutters indicating that they consist exclusively of telomeric repeats, Their
size is heterogeneous and they can be larger than 15 kbp.
This phenomenon reveals an unexpected plasticity of the embryonic
genuine which is restricted to the early development stage. We are currently
investigating the mechanisms involved in circle formation and the possible
role of circular DNA in chromosomal re-organisation during development.

(We/2.2/004)

Repair of oxidised guanine in plant DNA.

A.L Dany~, T. Doukib, J. Muchemble&, J Cadet b, and C
Triantaphylid~s' ~CF~Cadarache, Saint Paul Lez Durance, France.
~CENG, Grenoble. France

Base oxidation is one of the main types of DNA damage Among these
lesions, 8-oxo-7,g-dihydro-2'-deoxyguanosine (8-oxodGtm) is thought the
most abundant, and is highly mutagenic because of its propensity to mispair
with adenine[l] In Escherlchia coli, 8-oxodGuo is repaired by the enzyme
Fpg. Recently, a gene encoding an homolog ofFpg, AtM?cIH, was cloned in
the higher plant Arabidopsis thaliana [2] Like Fpg, its product seems to be
able to specifically excise 8-oxodGuo from double stranded DNA. Aider
oxidative treatments of Arabtdopsts thaliana cells, we measured the 8oxodGuo amount, and we examined the induction of rnRNA coding for
AtMMH (AtMMH-mRNA).
We observe that ~' radiation (up to 3 kGy), mostly inducing OH formation,
generates few 8-oxodGuo in plant DNA (15 8-oxodGuo/10~ Gua/Gy). In
parallel, y rays (100 Gy) do not increase the expression level of AtMMttmRNA over a 4 hours period. We notice that in cells grown in the dark
(cgd), the basal level of 8-oxodGuo is lower than in cells grown under light
(cgl) However, the basal level of AtMMtt-mRNA is the same either in cgd
or in cgl. We show that photosensitization by methylene blue, involving ~O2
formation, is responsible for an increase of 8-oxodGuo amount in DNA of
cgl. Its level increases up to 30 minutes of light stress, and then stabilizes
However, an exposure to ~O2 (up to 1 hour) seems to have no effect on the
AtMMIq-mRNA expression either in cgd and in cgl.
We hypothesise that OH induced DNA damage should be more rapidly
repaired than tO2 We think that the AtMMtt activity may be constitutive,
and that it may not be correlated with the level of AtMMH-mRNA. Plant
DNA oxidative lesions may also be repaired by other enzymes like an
hypothetic homolog of Oggl, the eucaryotic 8-oxodGoo repair protein
Analysis of 8-oxoGuo DNA excision activities are presently under
investigation
{1] Wood et aL, Biochem, 29, (1990), 7024.
[2] Ohtsubo et al., Mol Gen Genet ~259, (1998), 577

s276

(We/2.2/OOS)

Abstracts FEBS'99

Poly(ADP-ribose)Polymerase gene expression is induced

by ionizing radiations in Arabidopsis thaliana.
G. Doucet-Chabeauda, G. de Murciaband M. gazmaiera.

(We/2.2/006)

Laboratoire d'Oncologle Moldculaire, CentreJeaJl Perrm,

58, rue Montalembert. B P392. 63011 Clermont-Ferra~cede.~, France

aLRV/DEVM/DSV/CEA Cad, 13108 St Paul-tds-Durance, France.

bUPR CNRS 9003, ESBS, 67400 lllkirch, France.

Differential Display RT-PCR was used to screen for genes induced

by ionizing radiation (IR) in Arabidopsis thaliana. We have isolated a 3,2
kb cDNA which encodes a protein of 980 amino acids (ll0kDa),
homologue to the mammalian 116 kDa Poly(ADP-ribose)Polymerase
(PARP), due to the extensive amino acid sequence conservation in all
functional domains. PARP is a nuclear protein detector of DNA strand
breaks, that catalyses the formation of polymers of poly(ADP-ribose) on
numerous nuclear proteins such as histories and also PARP itself, required
for recovery from DNA damage [l]. As the mammalian PAP,P, the 110 kDa
A.th. PARP is composed of three functional modules [1] : the N-terminal
DNA binding domain with two Zinc Finger motifs and a bipartite nuclear
location signal; the central portion with ADP-ribosylation sites and a BRCT
module involved in the contact protein-protein [2] ; and the C-terminal
catalytic domain with NAD ~ binding sites. The A.th PARP 110 kDa protein
is recognized by antibody against human PARP, furthermore, this A.th
PARP protein binds to damaged DNA and is able to synthesized poly(ADPribose) on itself. We report for the first time that DNA damage,
experimentally introduced by 1R treatment, triggers PARP gone expression
at the RNA level : IR induce a rapid accumulation of 110 kDa A.th. PAR/'
mRNA. The 110 kDa A.th PARP protein is present only after gamma
irradiation in tissues containing actively dividing ceils (flowers, meristems
and roots). Our results suggest a high homology in structure and in function
between the animals 116 kDa PARP and the 110 kDa A.th. PARP, and is
likely that they play the same role in DNA repair pathway but control
mechanisms leading to PARP activation after the DNA damage are different
in plants.
[1] : de Murcia et al., TiBs, 19, 1994, p172.
[2] : Masson etal., Mol. Cell. Biol., 18, 1998, p3563.

(We/2.2/007)

Induction of the expression of Saccharomyces cerevisiae

UMP1 gene in the response to DNA damage
A. Galuszewska, P. Mieczkowski, W. Dajewski, Z. Ciesla,
E. Sledziewska-Gojska

BRCAI quantification with real-time quantitative PCR

D. Favy, S. Lafarge.C Vissac,P. Rio, Y-J. Bignon,D. Bernard-Gallon.

Germline B R C A I alterations predispose women to early onset familial

breast cancer and/or ovarian cancer. Although mutations of the
coding sequence of B R C A 1 were not detected in sporadic human
breast cancer, several lines of evidence have correlated the level of
B R C A 1 expression and malignancy.
Numerous experiments based on hybridization methods such as
northern blotting have been carried out to study the expression of
B R C A 1 in cell lines and turnour samples. They have yielded relative
values and because of sensitivity of the method were limited by the
very low abundance of transcripts. Until recently, competitive
reverse-transcription PCR (RT-PCR) was an alternative way to
quantify the level of some rare mRNAs.
Real-time quantitative PCR (RTQ-PCR), based on the TaqMan
methodology, represents a new powerful tool to quantify gene
expression. RTQ-PCR is a very sensitive method and allows faster
analysis and higher sample throughput. Moreover no post-PCR
man]pulation is required, thus cross-contamination is minimalized
We designed TaqMan probes to evaluate the copy number of
B R C A 1 (both full length cDNA and one of its most important splice
variants tacking exon ll) in several turnout (MCF-7, MDA-MB-231,
T-47D, SKOV-3, OAW28, LnCAP, DLD-1, MOLT-4) and normal
(MCF10a, HBLI00) cell lines. A reference gene (18S rRNA) was used
as an internal control.
Priliminary results confimq that B R C A 1 mRNA level was higher in
notmal cells than in tumoral cells and that the expression level of
B R C A 1 splice variant, relative to the full length B R C A 1 , was cell line
dependant.

(We/2.2/008)
CRYSTAL STRUCTURE OF A THERMOSTABLE TYPE B DNA POLYMERASE FROM
THERMOCOCCUS GORGON,4RIUS
Hopfner, K.-P *~, Eichinger, E. ~, Engh, R A. ~'2, Laue, F t, Ankenbauer, W. 2, Huber, R.~, Angerer, B.z

lnst#ute of Biochemistry and Biophysics PAS, Poland

TAbtellung Strukturforscllung, Max-Planck-lnst~tut fur Biochemie,/3-82152 Martinsried, Germany
2 Roche Diagnostics, D-82372 Penzberg, Germany

In response to DNA damage the cells of Saccharomyces

cerevisiae induce a set of physiological responses which facilitate
DNA repair processes. Among these responses are: cell cycle arrest in
so called checkpoints and increased transcription of genes encoding
proteins involved mainly in the metabolism of DNA, especially in
DNA repair.
We have constructed the series ofS. cerevisiae strains carrying
random fusions of the genomic DNA with reporter lacZ gone. These
constructs allowed us to identify several genes, expression of which
was elevated in cells treated with DNA damaging agent MMS (methyl
methan'esulfonate). Among of these MMS inducible genes we
identified UA#PI, which encodes the protein engaged in maturation of
20S proteasome complex.
We have shown that induction of UMP1 by M M s occurs on
transcriptional level. Expression of UMPI is inducible also in
response to UV or HU, so U M P I can be classified to the family of
D I N (Damage INducible) genes. Results of our experiments indicate
that M e c l - L Rad53-21 orDun21:TRP1 regulatory mutations have no
effect on the level of MMS induced fl-galactosidase activity in strains
carrying U M P I - l a c Z fusion.
To analyze the possible role of Ump I p in DNA repair processes
we investigated the sensitivity ofS. cerevtsiae cells, carrying
disruption of the UMP1 gone, to DNA damaging agents. It has been
found that cerevisiae cells carrying disruption of UMPI are
substantially more sensitive to UV radiation then wildtype cells.We
have not observed increased sensitivity to MMS.

Most known archaeal DNA polymerases belong to the type 8 family, which also includes the DNA
replication polymerases of eukaryotes, but maintain high fidelity at extreme conditions. We describe
here the first crystal structure of an archaeal DNA polymerase, at 2.5 A resolution, from the
hypertherophilic Archaea Thermococcua gorgonanus and identify structural features of the fold and
the active site that are hkely responsible for its thermostable function Comparison w~th the
mesophilic B type ONA polymerase gp43 from the bacteriophage RB6g hightlghts thermophl~ic
adaptabons, which include the presence of two disulfide bonds and an enhanced electrostatic
complementarity at the DNA-protein ~nterface. in contrast to gp43, several loops in the exonuclease
and thumb domains are more closely packed This apparently blocks primer bmding to the
exonucleaae active site. A physiological role for this 'closed' conformation is unknown but may
represent a polymerase mode, in contrast to an editing mode with an open exonuclease site. This
archaea[ B DNA pclymerase structure provides a starting point for structure-based design of
poIymerases or ligands with applications in biotechnology and the development of antJvlral or
antlcanoer agents
References:
Hopfner KP, Eichinger E, Engh RA, Laue F, Ankenbauer W, Huber R, Angerer 8 (1999) Prec. Nat/.
Acad. Sci. USA, in press.

Abstracts FEBS'99

(wa2.2t009)

Sequential analysis of junctions between two copies of

prokaryotic T-DNA integrated in tobacco chromosome
L. Krizkova and M. Hrouda*

s277

(We/2.2/010)

P. Koprowski, M. O. Fikus, P. A. Mieczkowski, J. gytka,

E. Sledziewska-G6jska, Z. Ciefila

Institute of Experimental Botany, Academy of Sctences of the Czech

Republic, Rozvojovd 135, 165 02 Prague 6, Czech Republic

Integration of prokaryotic DNA ("T-DNA", transferred DNA) into

plant chromosome is a process of illegitimate recombination naturally
occuring in plants as a result of infection by Agrobacterium. This mechanism
is also widely used in genetic transformation of plants. After transformation
of plants via Agrobacterium the T-DNA copies are frequently integrated into
the plant chromosome as direct repeats or inverted repeats.
We have constructed Ti plasmid-derived transformation vectors
enabling direct selection for transformants carrying tandemly and invertedly
repeated copies of T-DNA. After cocultivation of tobacco protoplasts with
Agrobacterium and subsequent regeneration we have obtained transformed
plants out of which as much as 30% carried multiple T-DNA copies. The
junction regions between two T-DNAs were amplified, cloned and
sequenced. The involvement of the border sequences of vectors in the
junctions was determined as well as the origin of "filler" sequences detected
in junction regions of some clones. The implications for a mode of T-DNA
transfer and integration are discussed.
"present address: Organon Teknika CS, Radimova 36, 162 O0 Prague 6, Czech Republic

The product of the DNA damage-inducible gene of

S.cerevisiae, DIN7, specifically affects stability of mtDNA.

Institute of Biochemistry and Biophysics, Warsaw. Poland

We have previously reported the cloning and sequence analysis of a

novel DNA damage-inducible gene orS. cerevisiae, DIN7 [1]. By comparison
of the predicted Din7p amino acid sequence with those in databases we
found that Din7p belongs to a family of proteins which are involved in DNA
repair and replication. This family includes Rad2p and its
Schizosaccharomyces pombe and human homologs Radl3p and XPGC;
Rad27p and its human homolog FEN-l, and Exolp. All these proteins are
endowed with exonuclease activity. Din7p is remarkably homologous to
Exolp, which is involved in mismatch correction. However, overproduction
of Din7p fails to suppress exol mutator phenotype, suggesting that the
physiological role of Din7p is different from that of ExoIp. We have found
that an increased cellular concentration of Din7p, e.g. in the dunI strains,
specifically enhances the frequency of the mitochondrial petite mutants,
which result from large deletions of mitochondrial DNA. On the other hand,
this elevated level o f petite production in dunl is partially supressed by the
din7 mutation. We have also found that overproduction of Din7p results in
the increased frequency of the mitochondrial erythromycin resistant mutants.
We have fused Din7p to the green fluorescent protein (GFP) of Aequorea
victoria and with the use of this hybrid protein we have found that Din7p is
specifically located in mitochondria. Our results indicate that Din7p
specifically affects stability of mitochondrial D N A
References:
[I] P. Mieczkowski etal. Mol. Gen. Genet. Vol. 253, 1997, pp. 655

(We/2.2/011)

Preliminary study of the human TBP complexed with a

cisplatin-damaged DNA
V. Lamoura, F. Legendre ~, J-P. Renaud ~, A. Poterszman '~,
J-C.Thierry ~, J-M. Egly a, J-C. Chottard b and D.Moras a.
"IGBMC. Illktrch, FRANCE and t'CNRS URA400 Paris. FRANCE.

The understanding of the mechanisms of the transcription of class II

genes is one of the research orientations of the laboratory. A connection
between transcription and DNA repair was demonstrated previously
through the characterization of TFIIH. Recently, it was shown that the
TBP is also implied in the DNA repair by the recognitton of the lesions
induced by UV radiation or cisplatin, a compound used in chemotherapy
of cancers [1 ]. The crystallographic structure of a DNA duplex covalently
bound by cisplatin [2] presents a bend similar to the one adopted by the
TATA box m its complex with TBP [3]. It thus appears that various
antitumour agents could turn G-C rich sequences into potenttal s~tes for
TBP, reducing transcription m target cells.
We present here the preliminary study of a complex between the
human TBP and a platinated DNA duplex.

[1] Vlchi P. et al., EMBO J., 24, (1997) p.7444.

[2] Takahara P.M. et ah, Nature, 377, (1995) p.649.
[3] Juo Z.S. et al., J. Mol. Biol., 261, (1996) p.239.

(We/2.2/012)

Expression of the Kin protein in Vertebrate brains

N. Mermet a, J. Angulo b, J. Repdrant c, S. Araneda"
aNeurobtologte des Etats de Sommeds et d'Eveil, INSERM U480, Say
RockeJeller. 69373 Lyon. bCEA-FAR, 92265 Fontenay-auv-Roses. 3MNHN
55 rue Buffon. 75012 Parts France

Kin is a nuclear protein presenting cross-immunoreactivity with the

bacterial RecA protein and binds efficiently to curved DNA, a genomic
interaction associated with replication, repair, illegitimate recombination
and also with transcription. By using antibodies directed against the
bacterial RecA protein, we study the Kin protein distribution in the brain
of representatives belonging to four major vertebrate classes: amphibians
(Frog, Rana esculenta), reptiles (Turtle, Trachemys scripta elegans), aves
(Quail, Coturnix coturnix ) and mammals (Mouse, Mus musculus). We
also detect the K i n l 7 mARN in the different brains by ISH, using either a
specific Kin176o ~ cDNA probe or a forty base oligonucleotide probe. A
striking overlap was observed between the pattern of distribution of the
Kin protein and the one corresponding to Kinl 7 mRNA : immunoreactive
cell nuclei were ubiquitously identified in all major neuroanatomic
subdivisions of the four species brains, with a preferential localization in
cortical structures of the telencephalon, the hypothalamus and the preoptic
area in the diencephalon, the tectum opticum in the mesencephalon, the
locus coeruleus, the raphe nuclei and most of the motor and sensory nerve
nuclei in the rhombencephalon as well as cerebellar nuclei. A strong Kinimmunoreactivity was also observed in specific cells such as the mitral and
Purkinje cells in all brains. These findings provide then evidence for a
strong expression of the Kin protein in cerebral structures mainly
conservative in their evolution and homologous between species. In
addition, the detection of the Kinl 7 gene accounts for its encoding of the
Kin protein. Our results support the hypothesis that mechanisms
underlying DNA replication-recombination and repair are fairly
conservative through the brain evolution.
References :
[1] Mazin et al., Mol.Gen.Genet., 244, (1994) 244.
[2] Araneda et al, Brain Res.. 762, (1997) 103.
[3] Mermet et al., Neurosc.Letters, 243. (1998) 97.

s278

(We/2.2/013)

Abstracts FEBS'99

Domain analysis of Holliday junction binding protein RuvA

Tatsuya Nishino and Kosuke Morikawa
D~7~L o] Stru~ ntrtd Htolo~

BERI, 6-2 3 Ftmlelhtt, Sutta. hqmn 56%0874

Three gene products, RuvA. RuvB, and RuvC, are revolved in the late
stages of homologous recombination which process a branching intermediate into
mature recombinant DNA duplexes. The RuvA-RuvB complcx facilitates the
ATP dependent migration of the Holliday junction created primarily by the
function of RecA protein. RuvA specifically brads to the four-way junction and
recruits RuvB helicase.
RuvA consists of three distinct domains termed domain 1. II, and 1II.
We have construcled an overproducing strain, which allows a large scale
purification of the NH2 fragment, containing domain I and I1 alone. The crystal
structure of the NH2 fragment determined at atomic resolution showed that its
backbone structure is identical to that in the full length protein, suggesting that
domain Ill makes no contribution to the formation of the RuvA tetrameric
structure. Biochemical characterization indicated that the NH2 fragment solely is
responsible for the junction DNA binding but domain II1 is not.
The function of isolated recombinant Domain Ill was examined using
the wild type and mutant molecules, which impair the ATP dependent branch
migration activity. The wild type domain Ill retained the secondary structure
comparable to that of the wdd type, while the mutant domain Ill reduced it. The
addition of the excessive amount of domain 11I in vitro inhibited the RuvAB
mediated branch migration, possibly because domain III competes with the intact
RuvA in RuvB binding. The excessive amount of wild type domain Ill or intact
RuvA inhibited the spontaneous ATPase activity of RuvB but its mutant did not.
In the presence of DNA, RuvA greatly stimulated the ATPase activity of RuvB,
while domain Ill still acted in a inhibitory manner. These results suggest that
domain Ill could modulate the RuvB activity. In the presence of DNA, the
stimulation of the RuvB ATPase activity by domain Ill wtthm RuvA may be due
to the maximization of the activdy in the formation of the complete RuvAB
complex.
To examine the solution structure of RuvA, small angel X-ray scattering
analyses were applied for full length RuvA and the NH2 fragment. Distance
distribution of the NH2 fragment m solution was in good agreement with that in
crystal structure, although the full length RuvA in solution showed a more
extended shape than the crystal structure. We assume that this difference should
be explained by the mobility of domain Ill, which is weakly bound to its
netghbormg subunit in the crystal.
UV sensitivity assay tn vtvo showed that neither of the NH2 fragment
(domain 1 and II) and domain III could not complement the ruvA deficient cells.
When they were expressed in the wild type strain, both of them increased the
sensitivity of the cells toward UV . Taken together, these findings imply that,
independently of domain I and It, mobile domain Ill parucipates in the regulation
of ATP dependent branch migration through interaction with RuvB.

(We/2.2/015)

(We/2.2/014)

S u b n u c l e a r distribution of p o l y ( A D P - r i b o s e ) and
its target proteins in differentiating rat g e r m cells
P. Quesada, M. Malanga, L. Atorino, F. Tramontano, A.
Petrella & B. Farina
Dipt. Chimica Organica e Biologica - Universtth "Federico I1"- Napoli

Poly(ADP-ribose) is a heterogeneously sized h o m o p o l y m e r of

ADP-ribose units synthesized from N A D + by the nuclear D N A binding
enzyme poly(ADP-ribose) polymerase on a protein acceptor [1]. Besides
modulating the function of the covalently modified proteins, poly(ADPribose) may participate to the regulation of cellular functions via noncovalent interaction with specific proteins (histones, the tumor suppressor
protein p53, M A R C K S proteins) [2].
We have previously shown that the poly(ADP-ribosyl)ation system
is highly active in murine testis where it may play a role in meiotic
recombination and in the remodelling of the chromatin structure that occurs
during germ cell differentiation [3]. The present study was aimed at: i)
identifying potential target proteins for poly(ADP-ribose) binding in
different chromatin subfractions from rat testis nuclei; ii) defining the
poly(ADP-ribose) size classes involved in such interactions; iii) analyzing
the intranuclear distribution of differently sized ADP-ribose polymers.
Our results show that among nuclear proteins only a few are able to
establish noncovalent interactions with poly(ADP-ribose). These include,
besides the core histones, nuclear matrix proteins and individual histone
H 1 variants. Such binding is salt-resistant and is not competed out by an
excess of DNA over poly(ADP-ribose). Interestingly, the testis-specific
H1 variant, H i t , which has the lowest D N A binding capacity among the
Hlsubtypes, appears to bind poly(ADP-ribose) with the highest affinity.
We also found that specific classes of ADP-ribose polymers, i.e. long and
branched molecules, are preferentially involved in the binding to the H1
variants; such polymers are produced both in vivo and in vitro in rat testis,
and are non-randomly distributed among subnuclear fractions.
Our results further corroborate the concept that non-covalent
interacttons of poly(ADP-ribose) with nuclear proteins may constitute an
important mechanism to modulate chromatin structure. The identification
of nuclear matrix proteins as specific targets for poly(ADP-ribose) binding
deserves further consideration due to the important role that the nuclear
matrix plays both in chromatin archttecture and nuclear functions.
References
1) Oei S.Li et al., Rev.Physiol.Biochem.Pharmacol.,131, (1997) 3
2) Malanga M. et al., J.Biol.Chem., 273, (1998) 11839
3) Quesada P. et al., Exp.Cell Res., 226, (1996) 183

Double strand break End-Joining is affected

in Fanconi anemia cells.
D. Papadopoulo, C. Baldeyron and J. Smith
UMR218 du CNRS, lnstitut Curie-Recherche, Paris, France;

Fanconi anemia cells display marked chromosomal instability, which is

reflected at the gene level by an excessive production of deletions. Knowing
that improperly repaired DNA double strand breaks (DSB) are at the origin
o f deletion mutagenesis and that DSB are produced during fundamental
cellular processes (replication, oxygen metabolism, repair intermediates), the
question arose as to whether the high frequency of deletions in FA originates
from error-prone DSB processing. We set up a <<host cell recombination
assay >> to define the efficiency and the fidelity o f DSB end-joining
processes. We analysed the fate o f model DSB, restriction endonucleseintroduced within extrachromosomal substrates transiently replicated in
normal and FA cells. DSB with different termini (blunt, cohesive, 5' or 3'protruding) as well as gaps with dissimilar termini, have been examined.
The overall rejoining efficiency o f all examined DSB is similar in
normal and in FA cells. However, the error-free processing o f blunt-ended
DSB is markedly decreased in FA cells from all complementation groups
examined (A-D). The fidelity o f processing o f blunt-ended DSB was
completely restored in FACC cells (FA-C cells complemented with wildtype F A N C C gene). Sequence analysis of recombinants indicates that errorfree <<Nonhomologous end-joining >>(NHEJ) o f plasmids carrying a DSB
with a blunt-end and various partner termini, is also reduced in FA cells. In
contrast, cohesive-ended DSB, with four base pair homologies at the
breakpoints, are recircularized with the same accuracy in normal and FA
cells, suggesting that the <~Repeat driven end-joining >> pathway is not
affected in FA.
We propose that the deficiency in FA genes specifically affects the
fidelity o f NHEJ resulting in a higher proportion o f deletions with a larger
deletion size. The ability of the wt F A N C C gene to correct abnormalities in
end-joining in FA provides strong evidence for a direct role of F A N C C and
perhaps all FA genes for the observed phenotype.

(We/2.2/016)

Human meioszs-specificMSH4 protein

(MutS Homolog4) interacts wuh the MutL homolog MLHI.
S. Santucci, F. Lespinasse, A. Sauni&es, C. Desnuene and
V. Paquis-Flucklinger.
UMR UNSA/CNRS 6.$49, Facultd de Mddecine. Nice.

The E. coli MutHLS system has been highly conserved throughout

evolution. The eucaryotic pathway results m a specialization of MutS and
MutL homologs that have evolved to play crucial roles in both DNA
mismatch repair and meiotic recombination. In Saccharomyces cerevisiae
MSH4 (MutS Homolog 4) is a meiosis-specific protein that is required
for reciprocal recombination and proper segregation of homologous
chromosomes at meiosis I. In order to determine the role of MSH
proteins in mammalian meiosis, we have cloned the human homolog of
the yeast MSH4 gene [1], and analyzed the expression of this novel
human gene (hMSH4) [2]. We have found that the expression of h M S H 4 encoded proteins is restricted to meiotic cells, as seen in yeast [2]. As a
first step toward understanding biological function of hMSH4 protein
during meiosis, we decided to determine whether this protein interacts
with factors implicated in meiotic recombination. MLH1 (MutL
Homolog 1) protein presents different features leading us to search for an
interaction between this later and hMSH4. First, MLH1 interacts with
other MSH proteins in the DNA repair pathway. Second, MLH I has been
shown to participate also to meiotic recombination mechanisms [3]. To
test physical interaction between h M S H 4 and M L H I , we have
constructed vectors allowing the overexpression of these proteins in
bacteria. Then, immunoprecipitation and Western blot analysis were
performed. The results presented here show that hMSH4 and M L H 1 can
be co-immunoprecipitated, s u g g e s t i n g that these proteins form a
heterodimer. The co-immunoprecipitation is observed regardless the
presence of DNA or ATP in the reactions. This result shows that the
interaction between hMSH4 and M L H I does not require the binding of
MSH4 to DNA. This situation is different from the one observed during
DNA repair pathway. In this case, MLHI interacts with MutS homologs
already bound to DNA. The identification of hMSH4 domains involved
in interaction with M L H I is in progress. To our knowledge, our results
represent the first characterization of an interaction between a meiosisspecific MutS homolog and a MutL homolog. Further identification of
h M S H 4 and M L H I partners should provide new insights into
fundamental meiotic recombination mechanisms in mammals.
[ 1] V. Paquis-Flucklinger et al., Gcnomics, 44 (1997), 188.
[2] S. Santucci-Darmanin et aI., accepted in Mammalian Genome.
[3] W. Edehnann et al., Cell, 85 (1996), 1125.

Abstracts FEBS'99

(We/2.2/017)

Photocrosslinking identifies a contact site for hRPA on the

damaged strand of eisplatin modified DNA
U.Schweizer, T.Hey, G.Lipps and G.Krauss
Department of Biochemistty, University of Bayreuth, Germany

Human replication protein A (hRPA) is a heterotrimeric protein that plays a

role in DNA replication, recombination, and repair, hRPA has been
proposed to be involved in initial steps of nucleotide excision repair (NER).
It is known that RPA acts in concert with the XPA protein which is affected
in many cases of the genetic disorder xeroderma pigmentosum (XP).
Patients suffering from XP show a high incidence in skin cancer that is
caused by an inability to remove UV-indueed photoproducts from their
DNA. It is an open question whether RPA and XPA are responsible for the
primary recognition of DNA lesions and where they bind with respect to the
site of damage.
To address this question we performed photocrosslinking experiments with
a DNA substrate carrying a single 1,3-d(GTG)-cisplatin modification. The
photoreactive base analogue 5-iodo-2'-deoxyuridine (5-IdU) was incorporated in our model DNA at specific positions to probe for close proteinDNA contacts.
The 70 kDa subunit of RPA very efficiently underwent crosslinking with the
damaged strand. Crosslinking is dependent on the presence of the DNA
lesion. Examination of the crosslinking efficiency in dependence of the
position of 5-IdU indicates preferential and specific positioning of hRPA 5'
of the site of platination.
The presence of XPA increases the rate of crosslinking of hRPA to the
model DNA. XPA, however, is not crosslinked significantly under these
conditions.
By electrophoretic mobility shift experiments we could show the formation
of a stable complex of heterotrimeric hRPA, XPA and singly modified
DNA. A more than tenfold increase in the affinity of hRPA for damaged
DNA is observed in the presence of XPA.
Our data suggest that the hRPA-XPA complex could function as an
important element of primary damage recognition in NER.

(We/2.2/019)

C R Y S T A L STRUCTURE OF A SYNAPTIC CRE

R E C O M B I N A S E - LOXP C O M P L E X
D. Suck, F. Buchholz, H. Dreher, F. Stewart, J.E.W. Meyer
EMBL, Meyerhofstr. 1, D-69117 Heidelberg, Germany

The structure of a synaptic Cre recombinase - DNA complex has been

determined at 3.2A resolution from orthorhombie crystals containing four Cre
monomers bound to two synapsed wild-type loxP sites in the asymmetric
unit. The structure suggests that a Cre-induced kink in the spacer region
initiates first-strand cleavage during site-specific recombination.
The 38.5 kD Cre protein from phage P1 belongs to the ~. integrase family of
site-specific recombinases which proceed through the formation of a Holliday
junction and a covalent phosphotyrosine protein-DNA intermediate. Cre
recognizes a 34bp target site ("loxP site") consisting of two 13bp repeats
separated by an non-symmetric 8bp spacer.
The structure was solved by molecular replacement (AMoRe) using the
coordinates of a complex containing Cre bound to a symmetrized lox-site
formed from a 16mer and 19met oligonucleotide [ 1]. Applying tight geometrk
and B-factor restraints, and adding independent phase information from an
EMTS derivative (SHARP), the Cre-loxP structure was refined to an R-factor
of 21.8% (Rrrc = 27.0%) using the program Refmac.
The tetrameric Cre-loxP complex shows local 2-fold symmetry with an
arrangement of subunits closely resembling that seen in the structure solved
by van Duyne and coworkers [1]. There are however slight, but significan~
changes in quaternary structure involvingrigid body movements of domains
and furthermore, while two of the subunits are closely similar in structure to
the Cre-loxA [1] complex, the other two show distinct differences.
particularly around the active site and the linker region between N- and Cterminal domains.
A striking feature of the synaptic Cre-loxP structure is a kink in the spacer
region of the DNA adjacent to the first strand cleavagesite associated with a
45 degree major groove-directed roll and complete unstacking of bases at this
step. This finding may provide an explanation for the observed order of strand
exchange in Cre-inducedrecombination.
1. F. Guo, D.N. Gopaul & G.D. Van Duyne (1997). Nature, 389, 40-46.

s279

(We/2.2/018)

D N A end-joining: are all ends repaired equal?

J. Smith, C. Baldeyron and D. Papadopoulo
UMR 218 du CNRS, Institut Curie-Recherche, Paris, 75231 France

In eukaryotes, two major pathways have evolved for the repair of

DNA double-strand breaks (DSB), homologous recombination and DNA
end-joining. In vivo, the process of DNA end-joining has been studied at a
mechanistic level in several systems, most notably in the yeast
Saccharomyces cerevisiae. However, little is known about the process in
human cells. Thus, we have used a plasmid rejoining assay to examine the
ability of human cell lines to join different DNA ends in vivo. To dissect
finely this process, repair of complementary 5' and 3' extensions as well as
several types of non-homologous termini were examined (i.e. blunt, blunt / 5'
extension, 5' extension / 3' extension, non-complementary 5' extensions).
Our results indicate that in human cells the repair efficiency of all DSB
examined was approximately the same, although the accuracy of the joining
event varied dramatically. Interestingly, DSB with complementary and blunt
termini as well as blunt / 5' extension were repaired frequently without loss of
nucleotides. Thus, in contrast to S. cerevisiae, human cells have the capability
to repair homologous and non-homologous termini with the same fidelity.
However, in human cells, accurate joining of non-homologous ends appears
to be limited to blunt ends, as the fidelity of repair of termim with 5'
extension / 3' extension or non-complementary 5' extensions was
significantly reduced. Sequence analysis of repair events associated with
nucleotide loss also revealed a difference between the repair of blunt ends and
that of other non-homologous termini. These results indicate that in human
cells there are differences in the repair of non-homologous DSB as a function
of the type of termini.

(We/2.2/020)

Detection of OGG1 transcripts and 8-oxoG DNA

glycosylase activity in the rat CNS
T. Verjat ~, P. Ra&cella,
"
b S. Araneda a oNeurobtologw
.
. des
des etats
Sommeds et d'Eveil, lnserm U480, , 69373 Lyon 6CEA, 92265 Fontenayclux-Ro~es P"

Central neurons of the adult rats are post-mitotic cells. They cannot
divide but present a high metabolic activity. Oggl protein, a functional
homologue of the bacterial Fpg protein, is a DNA glycosylase which excises
8-oxoguanine (8-oxoG) and Fapy from damaged DNA [1,2]. These
metabolites are formed in living cells by actions of the reactive oxygen
species which are normally generated by physiological process. In this
work, we studied the presence of the OGG1 transcript and the Oggl
enzymatic activity in the CNS of rats. The OGG1 cDNA sequence (1.2kb)
and forty base oligonucleotide, that spans the region for catalytic site [3],
were radiolabelled using ramdom priming or terminal transferase methods
respectively. Coronal sections of 3% paraformaldehyde fixed brains were in
situ hybridised and treated for autoradiography. Radiolabelings by using
cDNA or oligoprobe were observed in the cytoplasm of a great number of
nervous cells. High levels of OGG1 transcript were observed in the
superficial cortical layers, hyppocampal formation, the supraoptic area, the
paraventricular and arquate nuclei, the dorsal and median raphe, the
cochlear nucleus, the motor nucleus of the brainstem, and the granular and
Purkinje cells of the cerebellum. Control experiment using 100-fold excess
of cold oligonucleotide showed a dissapearence of the label. In addition, a
low Oggl enzymatic activity was detected in brain homogenates from
punchs containing raphe, hypothalamus and cerebellum by using a
radiolabeled 34 mer oligonucleotide carrying a single 8-oxoG as specific
substrate. In conclusion, Oggl glycosylase activity and OGG1 trancript are
present in the rat CNS supporting the existence of DNA repair pathways in
neurons.
1) Thomas et al, Mol Gen Gent, 254, 1997, 171.
2) Radicella et al. PNAS, 94, 1997, 8010.
3) Prieto Alamo et al, Nuc. Acids Res, 26, 1998, 5199

s280

Abstracts FEBS'99

(Wed2.2/021) RecBC(D) enzyme in UV-irradiated E. coil interferes with

site-specific and general recombination of ~. prophage
K. Vlahovi~, M. Petranovi~, D Zahradka, D. PetranoviC
DeFt. of A.folecular Genettcs. Rutller Bo[ckovt,2Institute. Zagreb. Croatia

RecBCD enzyme is involved in the radiation-induced process known as

prophage inactivation [I]. The process leads to the inability of )~ prophage
to excise itself from the E. coli chromosome via site-specific
recombination [2]. In this work we wanted to fimher characterize the role
of RecBCD enzyme in this process, ha addition, we examined the ability of
irradiated prophage to recombine with the infecting homologous phage.
We used several E. coli mutants differentially altered in the RecBCD's
activities. The results showed that in the mutants carrying either recB2109
or reeD1903, which do not exhibit considerable nuclease activities, the
prophage progressively loses its capacity for both site-specific and general
recombination. In the recB268 null mutant, however, prophage
recombinogenicity remained preserved. We also showed that the prophage
unable to recombine remained transcribable and that the recombination
frequencies in phage x phage crosses were not affected by postirradiation
incubation. Our results suggest that the helicase activity of RecBCD is
responsible for the loss of prophage recombinogenicity. This loss is most
probably a consequence of the unsuccessful RecBC(D)-dependent
recombinational repair of damaged cell chromosome, which may produce
intermediates unsuitable for further recombination reactions. Such
intermediates, when having reached the prophage, may affect its
recombinogenicity.
[1] Petranovi~ et al., Mol. Gen. Genet. 196, (1984) 167.
[2] Medi6-Petranovi~ et al., Int. J. Radiat. Biol. 32, (1977) 103.

(We/2.2/022)

Chromosome segregation and cell morphology in

recBC s b c B C ruvC mutants of Escherichia coil
D. Zahradka, K. Vlahovi~, M. Petranovi~, D. PetranoviC
DeFt. of Molecular Genetics. Rudler Bo,6kovi~ ]nstttute. Zagreb. Croatia

RuvC protein is important for DNA recombination and recombinational

repedr in E. colL Together with guvA and guvB proteins, it is involved in
the final, postsynaptic stage of recombination, in which Holliday
intermediates are processed into mature recombinant products. In this
work we have been focused on cytological effects provoked by ruvC
mutation in both wild type and recBC sbcBC backgrounds. By the usage
of phase-contrast and fluorescence microscopy we have found that ruvC
mutation causes chromosome segregation and cell division defects in
exponentially growing cells. These defects were extremely pronounced in
recBC sbeBC ruvC mutants, in which about 50 % of population consisted
of long filaments with unsegregated chromosomes. A small proportion of
filaments displayed unusual shape irregularities including bulges and lateral
appendages. Both chromosome segregation and cell morphology were
significantly improved when additional recA mutation was introduced into
ruvC mutants. From these results we infer that chromosome segregation
and cell division defects result from the abortive recombination initiated by
RecA protein. We hypothesize that unresolved chromosomes exert a
negative effect on cell division process and that they affect cell wall
structure.

Abstracts FEBS'99

s281

4.1 Structural motifs, dynamics and function in proteins

(We/4.1/023)

Site directed mutagenesis on archaeal elongation factor lc~

P. Areari, P, Cantiello, M. Masullo, O. Fattoruso, V. Bocchini

(We/4.1/024)

Dipartimento di Biochimica e Biotecnologte Mediche Unn'ersltgt di Napoli

Federlco IL Via Sergio Pansim 5, 1-80131 Napoli, Italy.

The elongation factor 1~xfrom the archaeon Su(/'olobus sol[ataricus (SsEF-l a)

belongs to the family of the GTP-binding protein. As such, it binds GTP and
GDP and shows an intrinsic GTPase activity stimulated by NaCI at high
concentration [1 ]. To investigate on the role of specific amino acid residues,
single mutations in SsEF-le~ have been designed in the region of the first
nucleotide-binding consensus sequence [2] which in SsEF-I ot corresponds to
the segment GI3HVDHGK. The mutations GI3A and V114K were chosen. In
the 3D structure of EF-Tu from T h e r m u s t h e r m o p h i l u s these residues
correspond to G 18 and V 105 which face each other in a ~3-strand region [31.
S s E F - I a gene, cloned in the pT7-7 expression vector [4], was mutated in the
desired position by PCR. Purification of the recornbinant protein was achieved
by heat treatment of the S100 cell extract tbllowed by FPLC on MonoS.
The biochemical properties of the two mutants were compared with those of
native SsEF-let. Both G I 3 A and V l l 4 K bound GDP and GTP with an
affinity similar to that displayed by SsEF-lot. The V114K mutant showed an
intrinsic GTPase activity similar to that of SsEF-Ic~ whereas the G I 3 A had a
significantly reduced catalytic efficiency; this last finding was opposite to that
produced on the elongation factor 2 by the A26G mutation. More dramatic
was the effect of the mutation on the temperature tbr half inactivation that for
the V114K mutant was 79C whereas for SsEF-lo~ and G I 3 A was 15C and
12C higher, respectively. Even though in the absence of the 3D structure of
S s E F - [ a only inferences can be made, these results might indicate that in
SsEF-lct. G13 plays a role in the GTPase activity, whereas the V114 seems to
be important tbr the thermostability of the entire molecule.
Work supported by M U R S T (PRLV 97) and ('NR (Rome)

[I]
[2]
[3]
[4]

Masullo et al.. J. Biol. Chem. 269, (1994). 20376

Dever et al.. Proc. Natl. Aca. Sci. USA 84. (1987), 1814
Berth[old et al., Nature 365, (1993), 126
lanniciello e| al.. Bmtecnol. Appl. Biochem. 23, (1996), 41

(We/4.1/025)

Micellar Activation of the pancreatic lipase:

orientation of eolipase plays a critical role
L Ayvazian,I Crenon,J Hermoso, D Plgnol", C Chapusand B Kerfelec
BIP, UPR 9036 CNRS. 13009 Alarsetlle, France, LCCP, CEA-CY,RS,
38027 Grenoble, France,*lnsmuto Qutmtca-Fistca "Rocasolano" CSIC,
28006 Madrid, Spaol

The pancreatic lipase-colipase complex fulfills a key function in dletar3

fat digestion by converting insoluble long chain triacy[glycerols into more
polar products able to cross the bmsh border membrane of enterocytes As
confirmed by structural data, pancreatic lipase possesses a taro-domain
organization, the N-terminal domain bearing the active site and the Cteiminal domain devoted to colipase binding. The enzyme has been shown to
exhibit two conformations, an mactive closed conformation in solution, and
an active open conformation resulting from the motion of the flap and the
exposure of the active site. It is supposed that this conformational change
requires the presence of oil-in-water droplets and this phenomenon has been
called interracial activation
Recently, we have shown that detergent micelles together with colipase are
able to induce the opening of the flap in the absence of any substrate
interface. This finding was further supported by the observation of a terna~'
complex associating pancreatic lipase in its open conformation, colipase and
a well characterized detergent mieelle [1]. We found that a disk-shaped
micelle interacts extensively with the concave face of colipase and to a lesser
extend to the distal tip of the C-terminal domain of lipase away from the
active site of the enzyme Therefore, we conclude that lipase activation is
not interracial but is mediated, tn solution, by a pre-formed lipase / colipase /
micelle ternary complex [2].
To better understand the activation pathway of lipase, we have investigated,
by site directed mutagenesis, the importance of the sole ion pair (Lys400 /
Glu45) existing between lipase and colipase. As expected, impeding the
formation of this interaction affects the apparent affinity between the
mutant protein and its native partner. This effect depends on the form of
presentation of the bile salts Moreover, the resulting lipase/colipase
complexes are unable to perform an efficient catalysis, notably m the
presence of bile salts micelles. Inhibition experiments suggest a poor
stabilization of the flap These results indicate that the ion pair Lys400/Glu45
plays a critical role in the active conformation of the lipase/colipase/mlcelle
complex by contributing to a correct orientation of colipase relative to lipase
[31.
References
11] Hermoso et al, J.Blol.Chem., 271, (1996). 18007
[2] Hermoso et at, EMBO, 18, (1997). 5531
[3] Ayvazian ct at, J.BiolChem, 273, (1998). 33604

Experimental based spatial

refinement of m e m b r a n e protein structures.
Arkin IT., Hensen RH., Kukol A., StevensJS. and Tortes J.
Cambridge Umvers~ty, Department of BtochemtstpT, 80 Tenms
Court Road. Cambridge CB2 IGA, UK.

Structures of membrane proteins are notoriously difficult to solve

using standard tools such as X-ray crystallography and NMR.
Where structural based methods fail, molecular modelling
procedures come into their own. While molecular modelling
procedures are somewhat useful on their own, it is obvious that
experimental methods are needed to ensure model reliability.
Herein we describe a novel experimental based method to refine
models of transmembrane or-helical bundles using orientational
data obtained from site directed infrared dichroism. The method is
based on the phenomena of site directed dichroism in which
dichroism at unique site (in the helix) will be dependent on the
rotational pitch angle at the site, the angle between the helix axis
and the laboratory z axis and the sample fractional order. When
measuring dichroism at two or more sites (1-UC introduced during
synthesis) all of the above parameters can be analytically obtained.
This is due to the fact that the rotational pitch angle difference
between the two sites is known, due to helical periodicity. The
results are incorporated into orientational energy functions in the
program CNS, constraining the helix tilt and the angle between the
J3C=O bond and the z axis. Global searching is then undertaken
with the experimental data utilised as an unbiased refinement tool.
We present results for several different systems of
symmetrical helical bundles including the M2 t f channel; from
i n f l u e n z a A, vpu from HIV and Human phospholamban. The
unique structures obtained correlate exceptionally well with all
additional experimental data. thereby reflecting the reliability and
accuracy of this procedure.

(We/4.1/026)

The 3-D structure of the bovine PEBP and the study

of other h o m o l o g o u s proteins
H. B6n6detti, L. Serre, N. Bureaud, B. ValiSe, F. Schoentgen,
and C. Zelwer
C B.M. UPR 4301, CNRS, Rue Charles Sadron, 45071 Or[clans, Cedex 2~

Phosphatidylethanolamine-binding proteins (PEBP) are found in numerous

tissues of mammalian species. Homologs of these proteins are found in a
variety of organisms including D r o s o p h i l a , plants ( A n t i r r h i n u m ,
Arabidopsis, tomato), parasites, and Saccharomyces cerevisiae.
The precise mode of action of these proteins in their respective organism is
unknown at present but some of them appear to be implicated in important
cellular processes.
Mammalian PEBP which have been shown to have affinity for nucleotides
(GTP) and phospholipids (phosphatidylethanolamine) might play a role in
tissue development and cellular morphogenesis. Indeed, in rat, PEBP is
found in oligodendrocytes of developping brains and in elongated
spermatides during rat spermatogenesis.
In plants, PEBP homologs are clearly involved in inflorescence development.
In yeast, one of the two PEBP homologs (TFSI) might be involved in
cellular growth and cell cycle.
To obtain new insights on the function of proteins of the PEBP family, two
directions are being explored.
First, the 3-D structure of the bovine brain PEBP has been determined by Xray cristallography. The structure displays a ]3 fold and exhibits one
nonprolyl cis peptide bond in a particularly conserved region of this protein
family. Furthermore, it defines a novel structural family of phospholipidbinding proteins. A small cavity close to the protein surface presents affinity
for anions such as phosphate, acetate, and phosphory[ethanolamine
s u g g e s t i n g that it may c o n s t i t u t e the b i n d i n g s i t e of
phosphatidylethanolamine.
Second, PEBP homologs from Saccharomyces cerevisiae are being studied.
The structure of the bovine brain PEBP will he presented together with the
first results on S.cerevisiae PEBP homologs.

s282

(We/4.1/027)

Abstracts FEBS'99

Conformational ion-dependent changes of alkaline

phosphatase from bovine intestinal mucosa

Distinct regions of cyclophilin B involved in the recognition

of functional receptor and glvcosaminoglyeans on T cells

M. Bortolato. F. Besson, B. R.oux

M Carpentier", F. AIlain~, B. Haendlerb, A. Den,vs~. G. Spik"

Laboratoire de Phrsico-CTlimie Btologique CNRS UPRESA 5013, UCBLyon I, 43 bd du ] I novembre 1918, 69622 Villeurbanne Cedex, France

" UMR N/II du CNRS, USTL, 59635 Vtlleneuve d'Aseq, France

Research Laboratories of Sehering AG, 13342 Berhn, Germato~

Alkaline phosphatase (EC 3.1.3.1) from bovine intestinal mucosa (BIAP)

is an homodimeric meta}loenzyme, containing one Mg 2+ and two Zn 2+ ions
in each active site. The metal-depleted BIAP (apoBIAP), prepared by using
ion-chelating agents, exhibited a dramatic decrease of its hydrolase activity,
concomittant to conformational changes in the quaternary structure of the
protein, Evidenced by rate-zonal eentrifugation and electrophoresis, we
demonstrated, for the first time, that the lost of divalent ions leads to some
monomerization process for metal-depleted alkaline phosphatase.
Divalent ions are not only necessary for the stabilisation of the quaternary
structure of BIAP but they are also involved in its secondary and tertiary
structures. Metal-depletion induced more exposure of some Trp residues and
hydrophobic regions to the solvent molecules (as evidenced by intrinsic and
ANS fluorescences). These changes might correspond to the disappearance
of a-helices and/or turns with a concomittant appearance of unordered
structures and B-sheets (as probed by FTIR spectroscopy).
Concerning the influence of temperature on the conformation of BIAP,
three steps were evidenced: (i) at 45-60C, the lost of BIAP activity,
correlated to some modifications of strength of the ion-binding with Asp
and/or Glu COO- groups and to the deuteration of Arg residues near the
metal binding sites, (ii) at 65C, the thermal unfolding transition of BIAP
probed by intrinsic fluorescence with exposure of hydrophobic regions,
corresponding to the heat-induced monomerization and (iii) at 70C, the
formation of intermolecular g-sheets with a concomittant lost of some ahelices, reflecting the temperature-induced aggregation of BIAP, On the
other hand, only one step of temperature-induced conformational changes
was evidenced for apoBIAP at 55C.
Metal depletion might lead to modifications in the subunit structure that
finally allow to monomer formation. Addition of metal ions to apoBIAP did
not conduct to appreciable enzymatic activity recovery. The apoBIAP
monomerization is either irreversible or the dimer Ibrmed ~ould be
catalytically different on account of new structural arrangements in the
secondary and tertiary structures.
(We/4.1/029)

(We/4.1/028)

Modeling and site-directed mutagenesis

of protein kinase CK2 catalytic ~ subunits from Y. lipolytiea and S, pomb

D ChallloQT Chardot~, K Niefindh, D Schomburgh. J -C Meunier

l.aNl~tolre de Chlmle Blologlquc /NRA INA-PG, CI~AI. 78R50 ~I]ll~crxal-nngn~m.France
b lnstltut Ihr HlOChemle,UlllX~rMtalgU Koeln. Zu]plch~[ Stf 47, 50674 Koeln, Allemagn

Protein kmase CK2 is an ubiquitous serine-threonine protein kinase found in

all eukaryotic organisms examined. The CK2 catalytic ct subunit (CK2ct) is
able to phosphorylate numerous substrates and can use both ATP and GTP as
phosphate donors. This ability to use the both NTP is a property of CK2et
among all protein kinases. Moreover, among kinases, it was shown that
phosphofructokinases can use ATP (ATP-PFK) or pyrophosphate (PPi-PFK)
as phosphate donor depending on the organism that produces it Therefore,
our purpose is to mutate CK2a, so that it uses pyrophosphate instead of ATP.
One problem is to choose the amino acid residues that should be mutated by
site-directed mutagenesis. First, we determined few residues from sequence
comparisons between protein kinases and phosphofructokinases (ATP-PFK
and PPi-PFK) sequences. These comparison enabled us to choose just two
residues. The first one, Gly48, belongs to the glycine-rich loop, typically
found in protein kinases The second residue, Gly177, belongs to the triplet
Asp-Trp-Gly that represents the most hfghly conserved short stretch in the
catalytic domains. To choose other residues, we modeled the structure of
Yurrowta hpolytwa and Schlcosaccharomyces pombe CK2a. There is a very
high degree identity between these two last CK2c~ amino acid sequences and
the Zea maize one. So we made the models from the crystal structure of Zea
maize CK2ct using the AMBER software. The CK2ot structural model
comprises two lobes, linked by a hinge. The C-terminal lobe mainly contains
co-helix structures and the N-terminal mainly 13-strands The results obtained
for the first studied amino acids (Gly48 and Gly177) confirmed the predicted
structural model for ~ hpolytwa. Other Y. hpolyt~ca and 5: pombe CK2a
mutants were constructed but, to date, none of them seems to use
preferentially the pyrophosphate. Molecular dynamics studies showed that, as
those of cAMP-dependent protein kmase, the CK2ct two lobes could move
between open and closed conformanon. The closed conformation would
correspond to the active structure where ATP is fitted in a pocket formed at
the interface between the two lobes. We particularly study the residues
located within that cavJty and especmlly those of the flexible glycine-rich
loop which could act as a cap

Cyclophilin B is a cyclosporin A-binding protein exhibiting peptidylprolyl cis/trans isomerase activity. We have previously shown that it
interacts with two types of binding sites on T lymphocytes. The type I
sites correspond to specific functiona/receptors and the type I1 sites to
sulphated glycosaminoglycans. The interactions of cyclophilin B with
type I and type lI sites are reduced in the presence of cyclosporin A and
of a synthetic peptide mimicking the N-terminal part of eyclophilin B
respectively, suggesting that the protein possesses two distinct binding
regions. In the present study, we intended to characterize the areas of
cyclophilin B involved in the interactions with binding sites present on
Jurkat cells. The use of eyclophilin B mutants modified in the Nterminal region demonstrated that the 3KKK5 and ~4yFD~6clusters are
probably solely required for the interactions with the type II sites. We
further engineered mutants of the conserved central core of cyclophilin
B which bears the catalytic and the cyclosporin A-binding sites as an
approach to localize the binding regions for the type I sites. The
enzymatic activity of cyclophilin B was dramatically reduced after
substitution of the R 62 and F ~7 residues, while the cyclosporin Abinding activity was destroyed by mutation of the W ~28 residue and
strongly decreased after modification of the F 67 residue. Only the
substitution of the W ~zs residue reduced the binding of the resulting
cyclophilin B mutant to type I binding sites. The catalytic site of
cyclophilin B did therefore not seem to be essential for cellular binding
and the cyclosporin A -binding site appeared to be partially involved in
the binding to type I sites.

(We/4.1/O30)

Possible existence of isozymes of tbioredoxin reductase

in different tissues within the same species
P.Y. Cheung, L. Xtt and K. S. Lee

The Ho*lgKo~gPolytechmc University. HollgKongSAR, PR (Tnna

Thioredoxin reductase is a ubiqmtous protein that exists in nearly

all species and tissues. It is a multifunctional enzyme that is well
characterized in E. colt, but not in mammalian tissue especially the brain
tissue.
We have developed the protocol |br the purification of porcine brain
thioredoxin reductase and studied its physiochemical characteristic. Partial
sequence of the enzyme was obtained by using tryptie digest Ibllowed by
HPLC and sequencing techniques. The partial sequence of the enzyme has
around 80% positivity when compared to that obtained from the human
placenta thioredoxin reductase. The sequence of porcine brain thioredoxin
reductase has high homology when compared to the partial sequence of
purified porcine red cell thioredoxm reductase, but yet not identical.
Potyclonal antibody against porcine red cell thioredoxin reductase
has been raised. The antibody was then added onto the brain thioredoxin
reductase and results shown that the polyclonal antibody has affinity for the
porcine brain thloredoxm reductase, but the affinity was much lower when
compared to that of porcine red cell thioredoxm reductase.
All these evidence indicate that thioredoxm reductase is present in
both tissues, but there are structural differences. We propose here, the
presence of isozymes of thioredoxin reduetase m different tissues within
the same species.

Abstracts FEBS'99

(We/4.1/031)

Unfolding of mammal alkaline phosphatase.

/3. Couturier and B. Roux

s283

(We/4.1/032)

Laboratoire de Physico-Chimiebiologique UniversitJ C. Bernard l~voJ~

UPRESA CNRS5013 - 43 boulevarddu II novembre 1918

Alkaline phosphatases (APs) are ubiquitous enzymes found in

different mammalian tissues. They are hydrolases which catalyse the
hydrolysis of ester bonds in phosphomonoesters. APs which belong to Zncontaining enzymes, are dimeric proteins. They interact with plasnw
membranes via a glycosylphosphatidyl inositol (GPI) anchor. Soluble lbrm
may be obtained by action of specific phosphatidylinositol phospholipasc.
In this work, equilibrium unfolding of the bovine intestinal alkzfi;
phosphatase(B1AP) has been monitored by activity measuremcol,
fluorescence, and exposition of cysteinyl residues. The monomer contains 5
Cys and 4 Trp. The locations of disulfide bonds, if they exist, are unknown,
We describe here the results obtained on the soluble form.
The guanidine hydrochloride (Gua,HCI) unfolding curve shows two
transitions at about 0,35M and around 2M depending on concentration of
BIAP. This indicates the presence of at least one stable intermediate lbrm
between 0,5-1,2M Gua,HCI.
In the native dimeric form, neither free Cys nor disulfide bonds can
be evidenced using Ellman's and Tannhauser's reactions. The wavelength of
the maximum fluorescence intensity is 325nm, for an excitation at 295nm.
In the intermediate form, which is always active, only one disulfide
bond is determined. The wavelength of the fluorescence maximum does not
change in these conditions. Sulfitolysis of the exposed disufide bond
inactivate the enzyme.
In presence of higher Gua,HCI, the denatured monomeric form is
obtained with one free thiol and two disufide bridges. The wavelength of
the maximum fluorescence emission is shifted to 351nm.
Therefore the Gua,HCI induced denaturation of the soluble lbrm of
BIAP follows a three states model. The intermediate state is an active dimer
resulting of a conformational change which exposes a disulfide bridge
probably involved in the stability of the active site.
The same study on the GP[ form is in progress.
(Wed4.1/033)

Structure-function relationships of a catalytic antibody

with a beta-laetamase activity.
H. Ddbat, B. Avalle, C-O. Sarde, A. Friboulet, D. Thomas
UPRES A 6022 du CNRS, Centre de recherche de RoyaUieu.
BP20529, 60205 CompibgneC~dex, France

We have developed an original approach to generate a catalytic antibody

(abzyme) by basing a strategy on the mimicry of an enzymatic active site
[1]. The first step consists in producing a monoclonal antibody Abl
against the active site of an enzyme, 13-lactamase, thus exhibiting inhibition
properties. This Abl is then used to elicit a second generation of
antibodies Ab2, directed against the hypervariable specific sequence of
Abl, and thus potentially mimicking the enzyme active site [2]. This
abzyme is an antibody in which the hypervariable domain sequence was
designed to specially recognize the antigen, but also to mimic the catalytic
activity of the enzyme. In a further s~ep, molecular cloning allowed us to
characterize and express Ab2 light and heavy chain in E. coil as a scFv
fragment. Data generated by sequencing where considered in a molecular
modeling approach and allowed us, when cnmpared to these available
residues responsible for the 13-1actamase activity, to discuss molecular
parameters involved in fi-lactam recognition and in 13-1actamase activity,
These studies are important to improve the knowledge of structurefunction relationships in abzymatic mechanism.
The relevance of this abzyme was, on the one hand, to demonstrate the
ability to generate a new kind of catalysts by the idiotypic mimicry
concept and, on the other hand, to enforce the possibility of new
promissing therapeutic perspectives such as ADAPT (Antibody-Directed
Abzyme prodrug Therapy).
[1] Izadyar et al., Proc. Natl. Aead. Sci. USA, 90, (1993), 8876
[2] Avalle et al., FASEB J., 12, (1998), 1055

S l a c k e n i n g of actin filament elongation and

inhibition of network formation by chlorpromazine
I. DalleDonne and A. Milzani
Dept. of Biology, Univ. ofAlilan, vta Celorta, 26, 1-20133MHan, ltalv.

Chlorpromazine (CPZ), a ealmodulin antagonist widely used as an

antidepressant tranquilliser in the treatmem of psychosis, has been reported
to have a bewildering variety of effects in biological systems. Some of the
CPZ effects have been observed on the cytoskeleton of various cultured
mammalian cell types [t]. More recently, CPZ was found to potently inhibit
motility of human peripheral blood lymphoeytes. Lymphocyte motility is
highly dependent on rapid changes in cell shape. The human T-lymphoma cell
line, MOLT-4, is constitutionally shape-changing and motile, and both of
these properties can be inhibited by CPZ The constant shape-changing
necessary for lymphocyte motility involves cycles of actin polymerisation and
depolymerisation. Inhibition of shape change of lymphocytes with CPZ was
associated with a reduction in F-actin concentration [2]. Although the exact
mode of action of CPZ in MOLT-4 cell system is not known, the ultimate
effect is inhibition of actin polymerisation [3].
We have analysed the effect of CPZ on pure actin. CPZ quenches Trp-79
and Trp-86 fluorescence, the quenching constant K, being 0.17 ~tM"1. In
agreement with an earlier report, CPZ inhibits acfin polymerisation, lowering
the extent of polymerisation. Moreover, novel polymerisation data are
presented indicating that CPZ decreases the ma:dmum polymerisation rate in
a dose-dependent manner as revealed by light seartering intensity changes at
546 nm The assembly inhibition results from the slackening of oligomer
formation during the early stages of polymerisation, as shown by
intermolecular cross-linking with N,N'-l,4-phenylenebismaleimide, of
monomer addition to the ends of growing filaments (i.e. filament elongation)
and of filament annealing. Finally, CPZ strongly inhibits acfin filament
network formation.
An open question is how these biochemical observations of direct effects
of CPZ on actin might relate to the observed effects of CPZ on cell aetin
REFERENCES
[1] Kawahara et at., J. Hepatol., 10(l), (1990), 8.
[2] Matthews et at., Biochem Pharmacol., 50(7), (1995), 1053.
[3] Verschueren et at., J Leuk. Biol., 55, (1994), 552

(We/4.1/034)

Collective and individual dynamics in a photosynthetic

protein
S. Dellerue, M.-C. Bellissent-Funel
Laboratoire Ldon Brillouin (CEA-CNRS). CEA-Saclay,
91191 Gif-sur-Yvette Cedex, France

The structural complexity of proteins leads to the fact that the

dynamical spectrum of those systems is very rich, ranging from femtoseconds
to seconds. The protein we study, the C-phycocyanin, is a soluble
photosynthetic protein involved in light-harvesting and single excitation
transfer to the reaction centre. Its structure is known to a resolution of 1.66 ~,
[1]. Neutrons scattering techniques offer a very large possibility field to study
proteins on a dynamical point of view as well as on a structural one.
This paper presents the results obtained on C-phycocyanin studied by
neutron scattering spectroscopies. In order to obtain the largest possible timerange, we have combined two neutron scattering techniques : the time-of-flight
and the neutron spin-echo spectroscopies. Thus, with the first technique, we
have been able to observe fast individual local motions of the protein (few
picoseconds) and determine the geometry of the motions ; similarly, with the
spin-echo technique, we have observed slow collective motions (50
nanoseconds). The fast individual motions were attributed to diffusion of the
residues which are accessible to the water inside a sphere. This assumption was
confirmed by an analysis of the structure. Coherent slow motions were
attributed to backbone relaxation. Moreover, structural techniques, such as
small angle neutron scattering (SANS) and two-axis spectroscopy, allow to
access to structure information. We show, for example, how the level of
association of C-phycocyanin monomer in solution can be measure by SANS.
Reference
[ l ] M. Duerringet al., J MoL Btol, 217, (1991) 577.

s284

Abstracts FEBS'99

Transport properties of the sodium-D-glucose cotransporter

SGLTI expressed in yeast secretory vesicles
M.A. Fimges, C.T. Lin, R.K.H. Kirme

(We/4.1/035)

(We/4.1/036)

Max-Planck-lnstitut fur molekulare Physiologic, A btedung

Epithelphysiologie, Rheinlanddamm 201, 44139 Dortmund, Germany

In order to characterize the properties of the intracellular (cytoplasmic) and

extracellular (periplasmie) faces of the SGLTI the transporter was expressed in
a yeast strain (NY17, sec 6-4) in which secretory vesicles can be accumulated
by a temperature shift during cultivation. A microsomal fraction was prepared
by differential centrifugation in which the marker enzyme for secretory
vesicles, the vanadate-sensitive H*-ATPase was enriched eightfold. As judged
by Western blot analysis employing an epitope-specific polyclonal antibody,
the mierosomal fraction was also enriched in SGLT1 protein. Transport studies
using a rapid filtration technique revealed striking differences when influx
experiments were compared to efflux experiments. In influx studies the
transport exhibited stereospecificity, D-glucose uptake after 15 see was 2 times
higher than the uptake of L-glucose or 2-deoxy-D-glucose, but no sodium
dependence or inhibition by phlorizin was observed. On the contrary, effiux of
D-glucose compared to L-glucose was strikingly accelerated by intravesicular
sodium and strongly inhibited by intravesicular phlorizin.
These studies demonstrate that SGLT1 - as postulated from genetic and kinetic
studies - is highly asymmetric. Furthermore, the use of the yeast expression
system provides a promising tool to further characterize the cytoplasmic face of
the sodium-D-glucose eotransporter.

Structure of cardosin A, a glycosylated and RGD containing

aspartic proteinase f r o m plant Cynara cardunculus L.
C. Fraz~oa, I. Bentoa, J. Costaa, C. Soaresa, P. Verfssimob, C, Farob, E. Piresb,
J. Cooper~and M. Carrondoa
alTQB-UNL Apartado 127, 2780-Oeiras, Portugal, bDep. Bioqufmica, Univ.
Coimbra, Aparlado 3126, 3000-Coimbra, Portugal, CDiv. Biochem. and MoL
Biology, Univ. Southampton, Southampton, S016 7PX, UK.

Aspartic proteinases (AP) have been widely studied within the living world,
but so far no plant AP was structuraly characterized. Cardosins [1] are AP
from flowers of Cynara cardunculus L. that accumulate at the stigma papillae.
Their milk clotting activity have been exploited since the Roman Era in the
manufacture of traditional cheeses.
The refined cardosin A crystallographic structure (1.72 /~ resolution) [2, 3]
includes two molecules, built up by two glycosylated peptide chains (30 and
15 kDa each). The fold of cardosin A is typical within the AP family. The
glycosyl contents is described by 19 sugar rings localised on the molecular
surface away from the conserved active site, and show a new glycan of the
plant complex type. An RGD trimer was found on the molecular surface
opposite to the active site cleft.
An hydrogen bond between Gin 126 and Man[34 renders the monosaccharide
oxygen-O2 sterically unaccessible to accept a xylosyl residue, therefore
explaining the new type of the identified plant glycan. The crystal structure
suggests a possible mechanism by which cardosin A (localised at the flower
style) might be oriented at the cell surface so that its RGD motif could be
recognised by the pollen receptor.
References:
[1] Ramalho-Santos et al. Planta 203, (1997)204.
[2] Bento et al. Acta Cryst. D 54, (1998)991.
[3] Fraz~_oet al. (1999) submited.

(We/4.1/037)

Sequence analysis and mutagenesis on water channels

and related proteins
A. Froger, V. Lagrrc, J. Gouranton, D. Thomas, C. Delamarche
UPRES-A 6026, Umversit~ de Rennes l, 35042 Rennes cedex.

Membrane channels of the MIP (MMor Intrinsic Protein) family

are distributed in various organisms, including bacteria, insects, plants
and animals. Members of the MIP family cxhibit two main functional
types: water channels or aquaporins (AQP) and glycerol facilitators
(GlpF).
Our work concerns the comparative analysis of MIP proteins,
in order to identity amino acids or motifs responsible for the specific
transit of the two solutes, water or glycerol:
-1 We achieved a database of more than 150 protein sequences of the
MIP family. We used multiple sequence alignments programs
(PILEUP, BLOCKS . . . . ) to classify the sequences. The alignments
were analyzed using various statistical tools. For this purpose we
dcveloped a software (CGD), based on the spreadsheet Microsoft
Excel. We also used correspondence analysis, a method working
without prerequisite for alignment or classification. Five key positions
were clearly identified where the residues are specific of each
functional type and possess very different physico-chemical properties.
-2 To verity these predictions, we constructed mutants by reciprocal
changes of key amino acids in protein models of each functional type.
We show that:
- The substitution of only one key amino acid is sufficient to
inhibit the water or glycerol transport.
- A double mutant of aquaporin loses its function and acquires
glycerol transport properties.

(We/4,1/038)

The crystal structure of a mutant hERct provides evidence

for the molecular mechanism of agonist/antagonist switch
M. Gangloff, M.Ruff, S. Eiler, S. Duclaud, D. Morus.
L G.B.M.C., lllkirch, France.

Estrogens excert their physiological effects in target cells via receptor

binding. We have focused on one of these receptors, the human estrogen
receptor alpha (ER) which is a ligand-inducible transcription factor belonging
to the superfamily of nuclear receptors (NRs) for small hydrophobic ligands
including the steroid hormones, thyroid hormones, retinoids and vitamin D3.
Like other NRs, ER displays a modular structure composed of six domains
(A-F). The N-terminal A/B region bears a ligand-independent transactivation
function AF-1. Region C allows DNA binding and region E, responsible for
dimerization and ligand-binding, carries the ligand-dependent transactivation
function AF-2 on helix H12 [1]. Mutation of cysteine residues into serines in
the ligand-binding domain (LBD) affect the transcriptional activity of both
full length receptor (sharing AF-1 and AF-2) and DEF domains fused to the
DNA-binding domain of GAL4 (without AF-1) in COS cells. Each cysteine to
serine mutation is lowering transactivation by about 20%. This cumulative
effect is leading to a triple mutant with around 40% activity left compared to
the wild-type protein. Moreover the ligand-binding capacity of the receptor is
not impaired by the triple mutation (Kd=inM). Here we report the
crystallographic structures of wild-type and C381 S, C417S and C530S mutant
LBD in complex with the endogenous estrogen, 1713-oestradiol, at 3.2 and 2.2
A. respectively. The main difference between these structures relies on the
position of helix H12 sharing AF-2 function. Indeed the mutant adopts the
same <,antagonist >> conformation as the recently solved LBD/raloxifen
complex [2]. Thus each mutation resides in a key structural region for H12
positioning providing structural evidence for the mechanism of
agonist/antagonist switch.
[1] Moras and Gronemeyer, Curr Opin Cell Biol, 10, (1998) 384.
[2] Brzozowski et al., Nature, 389, (1997) 753.

Abstracts FEBS'99

(We/4.1/039)

NMR Study on Dynamics of the 6FI-IF2 Module

Pair of Fibronectin

s285

(We/4.1/040)

Y. Hashimoto, J. Wemer, ~A. Bouquier, A. Pickford,

B. Marsden, Iain D. Campbell
Department of Biochemistry. University of Oxford. Oxford OXI
3QU, UK.,
~Biomolecular Engineering Research Institute, Osaka 565, Japan

Fibronectin is an extracellular modular protein composed almost

entirely of three types of modules FI, F2, and F3, and is involved in
matrix assembly and cell-cell interaction. Structural studies on a
variety of individual modules and module pairs have provided the
framework for a better understanding of how these different modules
are pieced together to form the functional protein. Further, functional
studies have indicated that target recognition sites of fibronectin may
span several linked modules. Therefore structural studies to
characterize the intermodule interfaces are clearly necessary to
understand function.
Recent NMR studies of module pairs have demonstrated a unique
potential of the technique to elucidate both structural and dynamical
aspects of the module interfaces in solution as well as the different type
of interfaces of module pairs. This may play an important role in the
recognition of the target ligand.
In our presentation, we will present the structural and dynamical
behaviour of the 6FI-1F2 module pair. This pair is part of the collagen
binding region of fibronectin. Its structural and dynamical
characteristics are considered important. 15N TI, T2 and NOE
relaxation studies were carried out to characterize the intermodule
interface and internal dynamics. Methodology for the determination of
the relative orientation between the module by anisotropic diffusion
analysis of the relaxation data will be discussed.

Pancreatic lipase-related protein type 1: a double

mutation restores significant lipase activity

I. Crenoa,S. Jayne,B Ked'elec,J Hermoso*,D. P1gnoland C Chapus

BIP. UPR 9036 CNRS, 13009 Marseille, France. LCCP, CEA-CNRS,
38027 Grenoble, France,*lnstituto Qu~mzca-Flsica "Rocasolano " CSIC.
28006 ,Vladrid, Spain

Besides the active pancreatic lipase (PL) which plays a major role m
dletaD" fat digestion, the presence of pancreatic hpase related protein 1
(PLRP1) has been reported both in the pancreas and pancreatic juice of several
mammals [1]. Surpnsingly, the amount of PLRP1 relative to PL vanes
considerably according to species. In this respect, high amount of this protein
was found in adult dog and cat pancreas in contrast to what was found in rat and
pig.
Despttes the presence of all the amino acids involved in catalysis
(catalytic triad, oxyanion hole, colipase binding, flap) PLRPI displays a very
low lipolytlc activity. Domain exchange experiments bev,veen horse PL and
dog PLRPI showed that the veD, low activity of PLRP1 results mainly from
particular features of the N-terminal domain of this protein.
Therefore, based on sequence comparison and modelling experiments,
five residues located in the vicinity of the active site pocket have been selected
for site directed mutagenesis. The resulting PL and PLRP1 mutants were
expressed in insect cells and subsequently purified. Investigation of the
catalytic properties of the recombinant protems reveals that two substitutions
m positions 179 and 181 in PLRP1 account for the very low activity of the
protein. Substituting Pro 181 for Ala or Ala 179 for Val in PL results in a
significant or complete loss of activity, respectively. As expected restoring a
significant lipolytic activity in PLRPI requires the double mutation Val179Ala
and A181Pro.
In conclusion, th~s work allows to better understand the stmctoral bases
of the lack of lipolytic activity of PLRP1 and brings new insights on the
structure/function relationships of the pancreatic lipase by ~dentifying
important residues. However, the presence of significant amounts of PLRP1 in
some species remains puzzling.
P~elelK;e

[1] Crenot~ I., Foglizzo, E. Kerfelec, B., V4nne, A. Pignol, D.. Hermoso, J.. Bonicel, J
and Chapus, C. (1998) Protein Engng. 11, 135-142.

(This work is supported by Asahi Chemical Industry Co. Ltd. Japan.)

(We/4.1/041)

Study of GImM, phosphoglucosamine mutase

implied in the biosynthesis of peptidoglycan in E.coli.
L. Jolly, D. Mengin-Lecreulx and J. van Heijenoort.
Laboratoire Enveloppes bact4riennes C.N.R.S. Orsay.

In Gram negative bacteria, UDP-N-acetylglucosamine (UDPGIcNAc) is situated at the branchpoint of the biosynthetic pathways of
two essential cell-envelope components, namely, peptidoglycan and
lipolysaccharides. This essential metabolic pathway, which represents
potential targets for new antibacterial compounds, was recently
investigated in detail. The four-step formation of UDP-GIcNAc from
fructose-6-P has bee elucidated in Ecoli. It involves the successive
actions of glucosamine-6-P synthase (GImS), phasphoglueosamine
mutase
(OlmM),
glucosamine- 1-P
acyltransferase
and
Nacetylglucosamine-1-P uridyltransferase activities, the two latter activities
being carried by the same enzyme, GlmU.
The E c o l i phosphoglucosamine mutase has been overexpressed
and purified. Kinetics studies of wild-type and histidine tagged GImM
enzymes from E.coli have been realized. The GlmM enzyme has been
shown to be active under a phosphorylated form and to catalyze its
reaction according to a Ping-Pong bi-bi mechanism. HPLC coupled to
mass spectrometry was used to separate and characterize the
dephosphorylated and phosphorylated forms of the enzyme and sitedirected mutagenesis has allowed us to identify the site of
phosphorylation. The non-specific activity of the enzyme on glucosephosphate isomers has also been investigated.
The level of phosphorylated enzyme may be a regulation factor of
the biosynthetic pathway of peptidoglycan. How the enzyme gets
phosphorylated in the cell remains an open question. Investigations trying
to elucidate this problem have been carried out.
A GImM protein has been identified in two other bacteria :
Helicobacter pylori and Staphylococcus aureus.

(We/4.1/042)

Direct demonstration of thermal dissipation of excess

excitation energy in photosynthetic membranes
L Harnois, W. Yahyaoui, R. Carpentier
GREIB, Univ. du Qudbec d Trois.Rividres, Qud., G9A 5117 Canada,

E x p o s u r e of p l a n t l e a v e s or chloroplasts to i l l u m i n a t i o n that exceeds

their p h o t o s y n t h e t i c capacity l e a d s to p h o t o i n h i b i t i o n a n d
d e g r a d a t i o n of the electron t r a n s p o r t system. To a v o i d p r e m a t u r e
p h o t o i n h i b i t i o n , excessive excitation e n e r g y can be d i s s i p a t e d b y
p r o t e c t i v e m e c h a n i s m s such as d e s c r i b e d b y the e n e r g y - d e p e n d e n t
(non-photochemical) q u e n c h i n g of c h l o r o p h y l l fluorescence. The
protective action is attributed to an increased rate constant for thermal
dissipation of absorbed quanta induced by the formation of a pH gradient
across the thylakoid membrane and by the de-epoxidation of xanthophyll
pigments. We applied photoacoustic spectroscopy, a technique used to
monitor thermal deexcitation processes of absorbed energy, to monitor
thermal dissipation in spinach thylakoid membranes together with
simultaneous measurement of chlorophyll fluorescence in the presence of
inhibitors of opposite action on the formation of ApH across the thytakoid
membrane (tentoxin and nigericin/valinomycin). A linear relationship
between the appearance of fluorescence quenching during formation of the
ApH and the reciprocal variation of thermal dissipation was demonstrated.
Dicyclohexylcarbodiimide which is known to prevent protonation of the
minor light-harvesting complexes of photosystem II significantly reduced
the formation of fluorescence quenching and the concurrent increase in
thermal dissipation. However, the addition of exogenous ascorbate to
activate the xanthophyll de-epoxidase increased non-photochemical
fluorescence quenching without affecting the measured thermal dissipation.
It is concluded that a portion of energy-dependent fluorescence quenching
that is independent from de-epoxidase activity can be readily measured by
photoacoustic spectroscopy as an increase in thermal deactivation processes.
The data also support a clear demonstration that energy-dependent
fluorescence quenching is directly correlated with an increase in thermal
deactivation processes.

s286

(We/4.1/043)

Abstracts FEBS'99

Evidence that the insecticidal crystal from

(Wd4.1/044)

Bacillus thuringiensis has a virus-like structure

H. Kaplan ~, F. R. Clairmont a, V. T. Pham a, and R. E. Milne ~'

i,h3 dology hlvtttute. Acad Si. 370100 Baku. Azerbaqan Medtcal Nohel
l, stitttte /or Bk~hemi~trv.Karolin ska In.~rintte. S- 17177 Stockltohn. Slrede,

"University of Ottawa, Ottawa, Canada KIN 6N5.

~Canadian Forestry Service. Sault Ste Marie, Canada, P6A 5M7.

The insecticidal crystal protein from Bacillus thuringiensis subsp.

kurstaki contains a 133 kDa protein (protoxin) associated with 20-kbp
DNA [ l ]. On ingestion by insect larva the C-terminal half of the protoxin
and the DNA are co-processed by a larval gut protease and nuclease
leaving a 65 kDa toxin derived from the N-terminal half of the
molecule [2]. It was found that the protoxin interacted with the DNA
through a region in the N-terminal toxic moiety. Quantification of the
relative amounts of the 133- kDa protein and DNA in the insecticidal
crystal protein gave one protein molecule per 3.5 base pairs. In order for
so much of such a large protein to interact with the DNA, only a small
region of each protein molecule can make contact with the DNA. A viruslike structure is proposed in which oblong-shaped protoxin molecules are
stacked against each other around a central double-stranded DNA core with
only a small region of the N-terminal toxic moiety interacting with the
DNA. In this model, the C-terminal half of the molecule is projecting
away from the central core with the C-terminal end on the outside of the
structure. The proposed model provides an explanation for two unusual
phenomena observed with the crystal protein. The first is the observation
that the conversion of protoxin to toxin occurs by a series of proteotolytic
cleavages in an apparently obligatory sequential manner starting from the
C-terminal end and proceeding until the 65 kDa toxin is formed [3]. There
is no known protein structural motif which would account for such a
proteolytic process. The second is the loss of insecticidal activity by the
crystal protein on exposure to sunlight.

Superoxide Generating Biological SystemS Produce "OH

Radicals when Going into a State of Low Oxygen Tension
T. Kenmov , S. Kuprin'. A. Hotmgren

EPR spin trap method was used to investigate production of superoxide anion
radicals. O~'-, and hydroxyl radicals, OH', in vit;.-) by flavin containing
enzymes. E.coli Thioredoxm rcductases (TrxR), calf thymus TrxR and the
mutant ScCys498/Cys of rat recon-tbinant TrxR, as well as yeast glutathione
reductase (GR) were studied. TrxRs upon treatment w~th dimtrochlorobenzene
(DNCB) acquired not only the ability to generate superoxide anion radicals
O_~' but also the ability to generate hydroxyl radicab, OH'. The production of
OH" was observed when mixtures of the enzymes with NADPH were reaching
the state of deoxygenation. Superoxide disnmtase did not suppress the OH"
generation while catalase did. The radical production could not be accounted
for the presence of metal ions in buffers. No radicals were detected in
solutions deoxygenated prior the experiments. Similar effects were observed
in case of TrxRs treated by several nitrosylated aromatic compounds including
paraquat and also in case of GR treated by paraquat or DNCB and m case of
xanthine oxidase without additives like DNCB or paraquat and also with
ribofiavin/EDTA oxygen photoreducing system. The results suggest that any
biological or chemical system, which is able to univalently reduce oxygen, is
also able in the absence of Oz to react with hydrogen peroxide giving OH" as
one of the products. The results on OH" production in our experiments may be
responsible lbr the self-deactivalmn of enzymes and necrosis of tissues at
ischaemia.

[1] H.P. Bietlot et at. J. Biol. Chem. 268, (1993) 8440.

[2] F. R. Clairmont et al. J. Biol. Chem. 273, (1998) 9292.
[3] C. T. Chorea et al. Eur. J. Biochem. 189, (1990) 523.

(We/4.1/045)

Tear Protein Adherence to Contact Lenses

Studied by Two HPLC Methods
E. O. Keith ~ and L, E. Janoffb
aMST and bCollege of Optometry, NSU, Ft. Lauderdale, FL USA

The adhesion of tear proteins to contact lenses is a major factor in

lens contamination and deterioration. We have examined the
adhesion of tear proteins to contact lenses with both reverse-phase
and
hydrophobic
interaction
high
performance
liquid
chromatography (HPLC). Six major tear proteins were found to
adhere to contact lenses: albumin, immunoglobulin, lactoferrin,
lysozyme, transferrin, and tear-specific prealbumin (tear lipocalin).
The two methods differed in their ability to resolve these proteins.
In reverse-phase HPLC, the albumin and transferrin peaks
overlapped, while for hydrophobic interaction HPLC, the lysozyme,
lactoferrin and transferrin peaks overlapped, Tear protein adhesion
to contact lenses depends primarily on the type of contact lens
material, and also varies with tear secretion rate and pathology. In
both methods, lysozyme was the major tear protein found to adhere
to both FDA Group 1 (nonionic low water) and Group IV (ionic
high water) contact lenses. Albumin and transferrin were also
found to adhere to these lenses to a significant degree. More tear
proteins adhered to Group IV lenses to than to Group I lenses, as
would be expected due to the ionic nature of the Group IV lens
material. These two types of lenses differed in their accumulation
of tear proteins over time. More tear proteins adhered to Group I
lenses worn for only 2 days than adhered to the same lenses worn
for 2 weeks, while the reverse was true for the Group IV lenses,
where more proteins adhered to lenses worn for 2 weeks. This may
be due to the nonionic nature of the Group I lens material, or to
other tear components, such as lipids and mueins, which may inhibit
protein adhesion to some types of contact lenses. There were also
differences between and within individuals in tear protein adhesion
to both types of lenses. These studies will be of use in studying the
efficacy of lens cleaning methods, as well as in the development of
new contact lens materials.

(We/4.1/046)

Glyeodelin and 13-1actoglobuUn differ in ligand binding

H. Koistinen', R koistinena, M. Seppala' T,V Burovab, y
Choiseff, T. Haehl6 c '
~
" "
OD.ept. of. Obst. & Gyrt, HUCH, Helsinlci~ Finland. Institute of
Biochemicdl Physics Kussian Academy o/Sciences Moscow Russia
CGroupe Protdines, LEIMA - INRA, ~ante~, France'

Human glycodelin [1] belongs to the lipocalin protein superfamily

containing a large group of sn,mll extracellular proteins, Whereas
lipocalins exhibit significant structural and functional diversity, their
overall folding patterns are similar and many of them bind small
hydrophobic ligands. Human glycodelin reveals high amino acid similarity
with 13-lactoglobulins and appears as different glycoforms in endometrium
(glycodelin-A) and in seminal plasma (glycodelin-S). Two glycoforms
differ substantially by chemical composition of oligosaccharides and show
different biological activities [2]. We used Swiss-Model service to deduce
tertiary structure of glycodelin and found it to be highly analogous to the
3D structure of bovine [Mactoglobulin. Despite this structural similarity,
confirmed by circular dichroism spectroscopy, glycodelin-A unlike 13lactoglobulin, does not bind retinoic acid nor retinol. It was not poss~le
to detect any endogenous hydrophobic ligands which could be bound to
glycodelins.
According to high-sensitivity differential scanning
calorimetry both glycodelin glycoforms share almost identical
thermodynamic parameters of reversible denaturation, suggesting that
their folding is not influenced by the differences in glycosylation. Since
almost identical, folding is not the main eanse of the difference between
biological activities of glycodelin-A and glycodelin-S. This indicates that
the functional difference between glycodelin-A and glycodelin-S is
determined by carbohydrate moieties,
[1] Sepp~ta et al., Clin Endocrinol, 46, (1997) 381,
[2] Morris et al., J Biol Chem, 271, (1996) 32159.

Abstracts FEBS'99

(We/4.1/047) The Ferredoxin Receptor on Photosystem I

B.Lagoutte, P.Barth, J. Hanley, P. Setif

s287

(We/4.1/048)

CEA, CNRS URA 2096, SBE, D~partement de Biologie, Bat 532

CEN Saclay, 91 191, Gif sur Yvette Cedex, France

Photosystem I (PSI) is one of the two photosystems which

catalyze light/energy conversion in plants and cyanobacteriae. In PSI,
electrons resulting from this conversion are transferred to a soluble
transporter, often ferredoxin (Fd), a small acidic protein with a [2Fe2S] cluster. The reduced Fd thus obtained is central for the activation
of a large number of metabolic pathways. Mostly embedded in the
photosynthetic membrane, PSI is a 300 kDa complex made up of 11
subunits in the Cyanobacteria Synechocystis PCC 6803. Some
subunits are protruding in the aqueous environment, three of them
contributing to the formation of the receptor site for Fd : PsaC, PsaD
and PsaE. Electron transfer occurs during a transient interaction of
oxidized Fd with these subunits (gsecond time scale), a very fast and
complex reaction which can be accurately characterized by flash
absorption spectroscopy [1]. Based mainly on mutagenesis
experiments, a specific function for these three subuhits is now
emerging. The PsaC polypeptide (9 kDa), which binds the terminal
[4Fe-4S] cofactor of PSI, is likely the direct partner for the electron
transfer itself. PsaD (15 kDa) is essential for the electrostatic guiding
of Fd to its final docking site, mainly acting on the association
constant [2]. It also plays a role in the pH regulation of the affinity via
the single histidine of the entire receptor site [3]. PsaE is more
involved in the regulation of the dissociation rate [2], with the
prominent role of a central arginine.
[ I] S~tif, P and Bottin, H (1995) Biochemistry34, 9059-9071.
[2] Barth P., LagouneB. and S&ifP. (1998) 37, 16233- 1624I.
[3] HanleyJ, Sdif P. Bottin H Lagoutte B. (1996) Biochemistry35. 8563-8571

(We/4.1/049)

Folding and stability of a barley Lipid Transfer Protein

Kresten Lindorff Larsen and Jakob Winther
Department of Yeast Genetics, CarlsbergL~boratorv
Gamle Carlsberg Ve1 10. DK-2500 Copenhagen Valb~. Denmark

We are interested in studying disulphide bond formation during oxidative

folding of proteins both in vitro and in vivo. The barley non-specific lipid
transfer protein encoded by the LtpI gone Is a small (91-residue),
monomeric protein with four disulphide bonds and a known threedimensional structure. We have chosen the LTP1 protein as a model for
studying both oxidative and non-oxidative folding in vitro. We hope that
it will become a useful system for studying functions of Protein
Disulphide Isomerase.
By fluorescence spectroscopy we have studied the denaturant-induced
unfolding/refolding equilibrium of the fully oxidised protein. We find a
one-step, co-operative transition between the native and the unfolded
state. We are currently studying disulphide bond formation during
oxidative refolding of denatured, fully reduced LTPI.

An Aquaporin Acquires Glycerol Facilitator Properties.

V. Lagr6e, A. Froger, S. Deschamps, J-F. Hubert, I. Penerin
UPRES-A CNRS 6026, Universitgde Rennes 1, 35042 Rennes, France.

The MIP (Major Intrinsic Protein) proteins constitute a

transmembrane channel family of currently 150 members that have
been identified in cell membranes of organisms ranging from bacteria to
man. Among these proteins, two functionally distinct subgroups are
welt characterised : the aquaporins that allow specific water transfer
and the glycerol facilitators that are involved in small neutral solute
transport. Using a biochemical approach, we recently showed that
AQPcic (an aquaporin isolated from Cicadella viridis filter chamber )
is tetrameric in cell membranes whereas the glycerol channel of E. coli
(GlpF) is a monomer. These results suggest that oligomerization of
MIP proteins could be involved in transport selectivity. In order to
elucidate molecular mechanisms that are accountable of the protein
selectivity, we have developed a strategy that consists in a systematic
comparison of the physico-chemical properties of amino acids at each
position in multiple sequence alignments. We identified five positions
(PI, P2, P3, P4, P5) corresponding to amino-acid residues conserved
in each subgroup but with highly different physico-chemical properties
in the two subgroups. Our experimental approach has consisted in a
lhnctional study combined to an oligomerization state analysis of
AQPcic, AQPcic mutants and GIpF. We constructed mutants of
AQPcic by substatuting characteristic amino acids of aquaporins by
corresponding glycerol facilitators amino-acids at positions P2
(AQPcic-S205D), P3 (AQPcic-A209K), P4 (AQPcic-Y222P), P5
(AQPcic-W223L) and P4P5 (AQPcic-YW222/223PL). Using a
Xenopus expression system, we show that all the mutations performcd
on AQPcic abolish water transport. Some of these mutations alter as
wcll the oligomerization state of the protein. In addition, the mutant
P4P5 gain competence to transport glycerol.

(We/4.1/050)

Probing the structure of the ORL1 receptor binding

domain with NBD-labeted nociceptin denvahves,
A Lopez, J-L Butour, H Mazarguil, J-C Meumer
In.stltut de Pharmacologwet de Biologte StructuPaledu CNRS,
205 Route de ,Varhonne. 31077 Toulouxe codex4. France.

The heptadecapepnde nociceptin (1) is the endogenous agonist of opioid

receptorqike (ORL1) receptor Here, we have investigated the interactions of
nociceptin and the ORLI receptor by means of the environment-sensitive
fluorescent probe the nitrobenzoxadiazole (NBD) Seventeen [NBD-2,3-Ldlaminopropionyl]-monosubstituted
nociceptine
derivatives,
hereafter
designated DNx ( x = 1,2, 17), were solid phase synthesized, and tested for
ability to inhibit (i) equilibrium binding oftritiated nociceptm, and (ii) forskolinreduced accumulation of cAMP, in recombinant Chinese hamster ovary
(CHO[ORLI']) cells stably expressing the human ORLI receptor The
spectroscopic and photophysical properties (quantum yield and excited-state
lifethne) of DNx (x=l, 4, 8, 10, 15, 17) have been firstly studied in three
solvents, dioxan, methanol, water, characterized by different dielectric constants
(~=2 2, 32.7, 78 4) After which, in the presence of membranes prepared from
('f'I'O[ORLI ] cells, the comparison between the NBD fluorescence signal of
DNx hgands associated with the binding domain of the ORLI receptor and the
corresponding fluorescence signal obtained at the same concentration in
TRIS/BSA solutions have been analyzed. An increase in the relative
fluorescence signal was specifically observed for DNI, 4, 8, and 10 but not for
DNI5 and 17 derivatives This increase was no longer detected in the presence
of excess nociceptin, indicating it resulted from specific interaction with the
ORL1 receptor in the membrane preparation Taking account of the
fluorescence properties of NBD group (2), our results suggest that, within the
complex with receptor, the fluorophore in position 15 and 17 finds itself in a
water-like environment, whereas the fluorophore in positions 1, 4, 8, and 10 Is
located in the hydrophoblc environment, consistent with a recent molecular
model of the ORLI receptor and its complex with nociceptin (3)
Kej'u'ord~. ORLI receptor structttre, nocicepttn, fluorescence o f NBD.
[ Meunier J-C. et a[ ,Nature, 377, (1995) 532
2 Mazeres S o r a l , Biophys. J, 71, (1996) 327.
3 Topham C e / a l , Prot. Eng, 11, (1998) 1163.

s288

(We/4.1/051)

Abstracts FEBS'99

Localization of a hapten link subsite in the combining

site of an anti-estradiol monoclonai antibody.
E. Mappus, U. Denis, T. Blach~re, M. Rol)and de Ravel,
C. Grenot, and C. Y. Cuilleron.

(We/24.1/052) A transmembrane topological model for the GSD Eb protein,

derived from multiple alignment and competition ELISA.
M.C. M6chin & G. Van de Werve
Centre Hospitatier Umvers~tL,de lgronw/tal (Campus Notre-Dame CHUM) &
UniversiM de Mon~at, ~ l . l ~ q l e n r de Nutrition, Montrb.at, Qc, C.,gmada.

1NSERM U 329, Hdpital Debrousse, 69322 Lyon, France

The site of interaction between the hapten link part of a steroid-protein

immunogen and the corresponding antibody binding site, has been studied by
photoaffinity labeling experiments on a mouse monoclonal anti-7-(Ocarboxymethyl)oximinoestradiol antibody (15H11).
In this study, the 7-O-carboxymethyloxime link, employed for preparing the
estradiol-BSA immunogen, was coupled to a 5-azido-2-nitrobenzoyl
chromophore through an ethylenediamine spacer in order to place the
photoreactive nitrene at nearly the same distance as that separating the steroid
from the Ca carbon atom of the derivatized lysine group of the immunogen,
and a radioactive tritium atom was introduced at the 17ct position of the
steroid. The maximal level of specific incorporation of the photoreagent was
0.14 mole of label per mole of antibody. Covalently bound radioactivity was
found exclusively on the heavy chain. Tryptic digestion of the photolabeled
heavy chain, immunopurification with the immobilized antibody, reversephase liquid chromatography, and Edman sequencing, revealed a radioactive
peptide SGNS-(X)-YNENFK derived from peptide 55-65 of heavy chain
whereas chymotryptic digestion gave the peptide SGNS-(X)-Y. In all cases,
the unidentified amino-acid was characterized as the Tyr-59 residue, located in
the CDRz of the heavy chain. Covalent labeling was confirmed by mass
spectrometry of photolabeled peptides which showed molecular ion values
corresponding to the addition of the nitrene photoreagent to the peptides.
These findings support the presence of a subsite interacting with the 7-0carboxymethyloxime hapten link different from that localized earlier with the
chromophore coupled to 6=- or 613-aminoestradiol which was found to label in
both cases the same Tyr 49 residue on light chain [1]. These results provide
useful informations for molecular modelisation and site-directed mutagenesis
experiments [2] aimed at modifying the specificity for the steroid B-ring and
for covalent links inserted between the steroid and labeled molecules in steroid
tracers as required for immunoassays.
[1] Rousselot et al., Biochemistry, 36, 1997, 7860.
[2] Schildbach et al., J. Biol. Chem. 268, 1993, 21739.
(We/4.1/053)

Crystal structures of the small G protein Rap2A

J. M~nrtrey, J. Cherfils
Laboratoire d'Enzymologie et Biochimie Strucmrales,
91198 Gif-sur- Yvette. France,

Small G proteins form a large family of structurally related protein

which have the essential property of cycling between a GDP-form and a
GTP-form, each recognizing separate sets of cellular partners. The molecular
features of the family likeness are well established from crystallographic
studies of various small G protein. The structural differences between the
GDP and GTP conformations have been first described for the H-Ras
oncoprotein and then for Rap, Rho and Arfrespectively.
Rap proteins, which include RaplA, RaplB, Rap2A and Rap2B, have
50% sequence identity with Ras, whose signal transduction function is well
established. Rapl was first identified for its ability to revert the transformed
phenotype of v-Ki-Ras-transformed fibroblasts. In the cell, it probably
controls a signal transduction pathway distinct from Ras.
We present here the crystal structures of the small G protein Rap2A in
complex with GDP, GTPTS and a non-catalytic complex with its natural
ligand, GTP. With this set of Rap2 structures and at the light of the expanding
database of G protein structures, we discuss the contribution of disorder-toorder transitions to the free energy of the GDP/GTP cycle.
[1] Cherfils J. et al., EMBO J., 16, 18, 1997.

Glucose-6-phosphatase (G6Pase) catalyses the last step of gluconeogenesis and glycogenolysis, and is therefore a key enzyme in glucose
homeostasis. However, ira complex structure associated to the
endoplasmique reticulum membrane is not yet totally understood.
Recently, the eDNA involved in glycogen storage disease type Ib (GSD
Ib) that encodes a 46 kDa protein (P46), different from P36 the catalytic
subunit, hut that causes, functional deficiency of G6Pase, has been cloned
and different isoforms have been reported. We investigated the topology
of the GSD/b protein with different new predictive algorithms as well as
with specific antibodies raised against selected peptide sequences of the
GSDIb protein. A BLAST analysis of GSDlb proteins generated 14
sequences (score > 80 and p < 10"6) with a low percentage of homology
(less than 30%). A multiple alignment of each sequence with the human
GSDIb protein was performed. We found (!) identical amino acid
conservations in all of the 14 sequences: tyrosine (Y-25 and Y-60), that
could be involved in phosphorylatinn - dephosphorylation regulation ;
charged residues like arginine (R-28), aspartate (13-72 and D-285) or
lysine (K-212), that may he involved in the function(s) of the GSDIb
protein ; tryptophan (W-246) or glyeine (G-281), that are important for the
structure of the core of proteins. (2) The 10 transmemhrane-predicted
segments (TM-predict), proposed by the recent Hofmann's algorithm,
were found to be very similarly in most of the 14 sequences, suggesting a
topology of the GSDIb protein based on 10 TM segments. (3) Three
domains 7300 from 6 to 106, 36 from 108 to 190 and 7243 from 361 to
435 have been defined using the very recent ProDom algorithm, in at least
8 aligned sequences. Furthermore, with the TM-predict algorithm, the N
and C terminal parts of the GSDIb protein are predicted to be located
outside the cytoplasm, e.i. inside the lumen of the ER. Antipeptide
competition ELISA analysis confirmed the non-accessible N-terminal pa~
of the GSD-Ib protein on intact liver mierosomes. A new transmemb~ane
topological model is proposed for the GSD-Ib protein.

(We/4.1/054)

lntra- and extracellular expression of human prolactin.

~L.Strokovskaya, bJ Michalik, ~I.Kikhno, bE Szolajska,
~A Solomko,bW.Ostoja - Zag6rski.
atMB&G Ukr.VAS. Zabolotnogo 150. 252143 Klev, ~'krame
b1BB, PAS. Ptra'~hsktego 5a. 02-106 ~Iarsa'w.Poland

The expression of human prolactin was done in different insect cell lines Sf9,
Sf21 and l-ff using recombinant baculoviruses, eDNA coding human prolactin
was amplified by PCR using primers suitable for gene trimming flanking
sequences the gene in BamH I and Kpn I restriction sites [1]. In these
restriction sites cDNA-prol was
integrated into baculovirus vector
pFastBacHT, carrying the sequence for His-tag (Bac-to-Bae ,,Gibco"). As a
result of the transfection, a recombinant baculovirus HisProt was obtained.
During infection of insect cell lines with the HisProl the highest level of
prolactin biosynthesis was observed in HF cell lineThe amount of the product
was estimated by SDS-polyacrylamide gel electrophoresis. On examination
under the inverted microscope cultures harbouring HisProl were found to
contain large number of inclusion bodies Previously similar structures were
observed for fusion human prolactin expressed in E coli [2] Such aggregates
could be dissolved only in denaturing conditions (6M guanidineHCl/8M urea).
Secretion of prolactin to the medium was possible when recombinant MelProl
vector was used in which cDNA-prol contained mellitin signal sequence in front
of the prolactin sequence (Bac-N-Blue,"tnvitrogen"). The concentration and
immunoreactivity of the prolactin were determined using radioimmunoassay
The amount of prolaetin present in medium was found to be 10 mg/l culture.
[1]Strokovskaya L.I et al., (1997) ProcNatl.AcadSci Ukr.(in Russian) 36,
166-169
[2]Gilbert M.S et al, (1991) Int J Biochem 23, 107-114

Abstracts FEBS'99

s289

(We/4.1/055) Expression of viral, genome-linked protein, Vpg,

in baculovirus eucariotic system.

(We/4.1/056)

J Michalik', E Szo~ajska", L Strokovskaya b, W Ostoja-Zag6rski a

A. Milzani, I DalleDonne, R. Colombo

~IBB.PAS, Pawtilsk~ego 5a, 02-106 Warsaw,Poland

blA[B&G Ukr,VAS. Zabolotnogo 150.252143 Ktev. Ukraine

The three dimentional model of potyviral genome-linked protein has been

published [1] It suggests a specific RNA-protein interaction as well as
similarity of hydrophobic - hydrophilic distribution to malate dehydrogenase
We wanted to verify these theoretical data by experiments with heterologous
native Vpg protein and compare results of functional as well as structural
tests with theoretical data Vpg protein was synthesized in Bac-to-Bac
expression system The product was synthesized in two- step procedure by
constructing recombinant plasmid pFast Bac with the Vpg gene introduced
between restriction sites BamH I and EcoR I. Alternatively, recombinant
carrying the sequence for Vpg with poly His-tag at C terminus between
BamHl and Pst I sites was prepared. The last construct allows for quick and
specific protein product purification on cations divalent columns Plasmids
were transformed into DH10 Bac competent E.coli cells (Bat-to-Bat,
,,Gihco") Recombinant baemid DNA was used as vector to express
heterologous genes in cultured insect cells The most efficient Vpg synthesis
was observed in High Five cell line from Trichoplusia m
[1]P|ochocka D. et al., (1996) Proc Natl Acad Sci VoL 93, 12150-12154
Supported by KBN Grant No 6 PO 4B 011 13

(We/4.1/057)

Kinetics of KCN binding to cytochrome d in

Intact cells, spheroplasts and membrane
fragments of Salmonella typhimurlum
Dariush Minai-Tehrani, Ezzatoilah Keyhani
Inst Biochem Biophys, Universityof Tehran. Tehran. Iran

Formation of the cytochrome d-KCN complex was studied in the

oxidized state by recording the absorption at 649 nm after addition of

KCN (2, 5, 10 mM) to either intact cells, spheroplasts or membrane
fragments of Salmonella typhimurium.
The effects of pH, various
NaCI concentrations (0 to 1 M) and various sucrose concentrations (0
to 1 M) on the formation of the complex were studied. Maximum binding occured at pH 7 for intact cells and at pH 8 for spheroplasts and
membrane fragments, while binding was negligible at pH 5 and 10 in
all cases. In the presence of NaCI, maximum binding occured in 0.1 M
NaCI for intact cells and in 0.2 M NaCI for spheroplasts and membrane fragments. In the presence of sucrose, maximum binding
occured in 0.1 M sucrose for intact cells, in 0.2 M sucrose for
spheroplasts, while no significant effect was observed for membrane
fragments. In all cases, increased KCN concentrations led to increased binding: complex in 10 mM KCN > complex in 5 mM KCN >
complex in 2 mM KCN. Lineweaver-Burke plot for the formation of cytochrome oLKCN showed that Ks was around 4 mM whether KCN was
added to intact cells, spheroplasts or membrane fragments. In
contrast, Vmax was higher for membrane fragments (10117 pmol/
min/mg protein) than for spheroplasts (234 pmol/min/mg prot.) and
Vmax for spheroplasts was itself higher than for intact cells (14.50.7
pmol/min/mg prot.). These results reflected increased accessibility of
cytochrome d from intact cells to membrane fragments as expected
from the membranal localization of cytochrome d. For spheroplasts,
NaCI and sucrose affected both the values of Ks and of Vmax. Ks
(mM) rose progressively from 40.5 (control) to 9.81.5 in 1 M NaCI
and from 40.5 to 8.71.8 in 1 M sucrose. Vmax (pmol/min/mg prot.)
rose from 234 (control) to 42+2 in 0.2 M NaCI and to 29+5.6 in 0.2 M
sucrose, then dropped to 105 in 1 M NaCI and to 7.51.2 in 1 M
sucrose. For membrane fragments, Ks remained unchanged, regardless of NaCI or sucrose concentrations; Vmax raised slightly to
13211 in 0.2 M NaCI and decreased to 1023.5 in 1 M NaCI but
remained approximately unchanged in sucrose. The effect of various
concentrations of NaCI and sucrose observed on spheroplasts but not
on membrane fragments probably reflected changes in the accessibility of cytochrome d due to changes in spheroplests permeability.

Oxidation of actin Cys-374 by t-butyl hydroperoxide

induces structural changes in the surface loop 39-51.
Univ of~.lllan, Dept. of Biology, vta Celorta 26, 1-20133 l~ltlan, ltalv.

Covalent changes caused by in vivo protein oxidation are primarily

responsible for the accumulation of catalycally compromised and structurally
altered enzymes during ageing [l]. Furthermore, protein oxidation may play
a role in several pathological states, including inflammatory disease,
atherosclerosis, neurological disorders and cataractogenesis [2]
The susceptibility of G-actin to both methionine and cysteine oxidation
when treated with t-butyl hydroperoxide (t-BH) was investigated
The results show that no methionine residue was susceptible to oxidation
by t-BH at concentrations of 1-20 raM, while Cys-374, one of the five
cysteine residues of the actin molecule, was found to be the site of the
oxidative modification. Perturbations in the intrinsic Trp fluorescence and
the decreased susceptibility to limited proteolysis by ct-chymotrypsin and
subtilisin of oxidised actin give indication of some alterations in protein
conformation in helix 74-92, in subdomaln 1, and in the central segment of
surface loop 39-51, in subdomain 2. AG(H20), the free energy of unfolding
at infinite denaturant dilution, derived from urea denaturation curves,
differed by 0 657, 2.437 and 2.757 kcal/mol for native actin and actin
oxidised by 1, 10 and 20 mM t-BH, respectively. This indicates a lower
conformational stability for the oxidised actin. G-actin structural alterations
due to Cys-374 oxidation produced by t-BH result in the decrease in the
maximum rate of polymerisation, the increase in both the delay-time and the
time required for half maximum assembly, the decrease in the elongation rate
and the enhancement of the critical monomer concentration for
polymerisation. The results suggest that oxidation of actin Cys-374 induces
structural alterations in the conformation of at least two different distant
regions of the molecule The involvement of both the C-terminus of the actin
polypeptide chain and the DNase-I-binding loop in the inter-monomer
interactions in the polymer could account for the altered kinetics of
polymerisation shown by the oxidised actin.
REFERENCES
[1] Stadtman, E R., Science, 257, (1992), 1220.
[2] Stadtman, E. R., and Oliver, C N , J. Biol. Chem., 266, (1991), 2005.

(We/4.1/058)

Structural and Functional Analysis of the Somatomedin

B Domain from Human Vitronectin
C. X. Moss, P. A. Underwood b, B. C. MabbutC
"School of Chemistry, Macquarie University NSW 2109, Sydney Australia
"CSIRO Molecular Science, North Ryde, NSW 2113, Sydney Australia

Vitronectin (Vn) is a major component of plasma, where it circulates as a

75kD monomeric glycoprotein. Vn also localises to the extracellular matrix,
where it is deposited as conformatioanlly altered, disulphide-linked
multimers. Vn binds to and stabilises the conformationally labile serpin PAIl, a regulatory element of the plasmin fibrinolytic cascade. A high affinity
PAI-1 binding site has been localised to the cysteine-rich N-terminal domain
of Vn, somatomedin B (SB) [1]. PAI-1 preferentially binds to
conformationally altered, multimeric Vn and is also capable of inducing the
formation of Vn multimers [2]. Although the arrangement of disulphide
bonds in SB is recognised as being important for the interaction with PAI-I,
the pairing of the eight Cys residues in this domain is unknown.
In order to characterise the structure and function of SB, we have produced a
recombinant form of SB as an MBP fusion protein. Interestingly, the SBMBP fusion protein forms dimeric and trimeric complexes, linked by
disulphide bonds. Despite the presence of these intermolecular disulphide
bonds, the SB-MBP fusion protein is biologically active, as determined by
PAI-I binding assays. Reduction and alkylation of all Cys residues within
SB-MBP destroys PAI-I binding activity, indicating that certain disulphide
bonds are critical for maintaining the conformation of the PAI-1 recognition
site. We have used a two-step reduction and alkylation procedure, coupled
with MALDI-TOF mass spectrometry, to identify Cys 9 as being involved in
SB domain cross-linking.
We have mapped the epitope for an anti-Vn monoclonal antibody to within
amino acids 9 and 18 of the SB sequence. This epitope is more accessible in
conformationally altered, multimeric Vn, and cannot be destroyed by
reduction and alkylation. The proximity of this disulphide independent
epitope to Cys 9 suggests a model in which the multimerisation of Vn
exposes a site in SB that undergoes disulphide exchange with Cys residues
from other Vn molecules.
[1] Seiffert D. and Loskutoff, D.J., J. Biol. Chem., 266, (1991) 2824
[2] Seiffert D. and Loskutoff, D.J., .1. Biol. Chem., 271, (1996) 29644

s290

(Wed4.1/059)

Abstracts FEBS'99

Binding of Influenza Hemagglutinin

to immobilized Receptor Saccharides
Kaori Chiba~, Yu Tsutsumi~b Hiroshi Nakanishi~b

(We14.1/060)

Spectroscopic study of the conformational transition of HbA

L C Petcu", G Turcub, N Rosoiu',
Faculty of McdlClUe. "'OvldlIIS" UmversUy. Constanta, Romania.

~Faculty of Physics. BucharestUniversay Ronlanta

a Natl. ln,;t. Bloscz Hum.-Tech.. Tsukuba 305-8566 JAPAN.

b Tokyo Sci Univ. Ntv,la 278-0022. JAPAN

Influenza hemagglutinin (HA) is a large membrane protein (MW

200,000) that consists of three sets of two side chains, HAl and HA2.
HAl is necessary for binding with receptor saccharides on the surface
of the target cell and HA2 has a important role during cell fusion
between the virus particle and the target cell by changing it's structure
dramatically. At the tip of HAl, there exists a pocket-like structure,
with which an influenza virus particle attaches to a target cell via the
receptor saccharides as the first step of infection. Thus, the binding
kinetics and affinity of a certain HA molecule to a certain kind of
receptor saccharide directly determines the probability of infectivity.
For the purpose of elucidating the binding kinetics and specificity,
we have investigated binding reactions between bromelain digested
HA (BHA), purified from one of the most traditionally-studied
influenza virus, PR/8, and the two most typical glycolipids,
sialylneolactotetraocylceramide
and
sialyl(2-6)neolactotetraocyl
ceramide, as model receptor saccharides for birds and human beings,
respectively. We immobilized the glycolipids and monitored the
binding kinetics with the resonance mirror method (IAsys). As results,
we obtained obvious differences in the binding kinetics and
intensities of the resonant signals between the two cases using each
type of receptor saccharides. The results of these molecular-based
experiments well explained the higher infectivity of this virus to birds,
compared to that to human beings.
Usually, types of HA has been classified according to the results of
its polyclonal antibody-antigen reaction. Although this way of
classifying HA is important for vaccination, it dose not include any
information about the binding propensity of the virus particle to the
human-type receptor saccharides. Accumulating the results of these
experiments in many types of HA, it will be able to reclassify the types
of influenza viruses according to its possibility of infection to human
beings.

(We/4.1/061)

Cellular localization of GFP-fused phosphate permeases in S. cerevisiae

J, Petersson ~'b, A.L. Kruckeberg c & B.L. Persson a'b
"Department of Engineering and Natural Sciences Vdxjo Universlt). S-351 95 Vaxjo Sweden
t'Department of Biochemistry, Stockholm Umverstt)., S-106 91 Stockholm. Sv.aden
' E C Slater Institute. Umversd3, of Amsterdam, 1018 TVAmsterdam, The Netherlands

In the yeast, Saccharomyces cerevisiae, inorganic phosphate uptake is

considered to be catalyzed by three different plasma membrane transporters,
allowing the cell to supply itself with phosphate at different external
conditions. Two of these, the derepresslble proteins Pho84 [1] and Pho89 [2],
are expressed and active when low concentrations of external phosphate are
available (in the btM range). These proteins, which transports phosphate
together with protons (Pho84) or sodium ions (Pho89), are the so-called high
affinity phosphate transporters with pH optima at pH 4.5 and pH 9.5,
respectively. The third, so far genetically unidentified, has been suggested to
have a much lower affinity for phosphate and to be active in the mM range.
To gain deeper insight into trafficking and possible degradation pathways of
the proteins, green fluorescent protein (GFP) fused to the phosphate
transporter, Pho84, was used as an intracellular reporter system. This system
studied by fluorescent microscopy allowed for a verification of the plasma
membrane localization of the catalytically active Pho84-GFP chimera.
Furthermore, obtained results strongly suggest a vacuolar degradation pathway
known to be the case for several other S. eerevisiae plasma membrane proteins
[3].

[1] Bun-ya, M., Nishimura, M., Harashima, S. and Oshima, Y. (1991) The
PH084 gene of Saccharomyces cerevisiae encodes an inorganic phosphate
transporter, MoL Cell. Biol. 11, 3229-3238.
[2] Martinez, P. and Persson, B.L. (1998) Identification, cloning and
characterization of a derepressible Na+-coupled phosphate transporter in
Saccharomyces cerevisiae, MoL Gen. Genet.248, 628-638.
[3] Bryant, J.B. and Stevens, T.H. (1998) Vacuole biogenesis in
Saccharomyces cerevisiae: Protein transport pathways to the yeast
vacuole, MicrobioL Mol. Biol. Rev., 62,230-247.

Proteins are macromolecular aggregates whose structure and

tridimensional configuration are strictly determined and directed in order to
perform a certain function inside the organism Small disturbances in protein
structure can cause its dysfunction and the emergence of some biochemical
imbalance in metabolic processes
Hemoglobin is considered to be a functional protein prototype and thus
studies developed upon it reveal the fundamental aspects of the structure function relation for other proteins as well
The purpose of this survey is to highlight certain aspects related to the
structure - function relation that governs the existence of Hh as an allosteric
protein by using spectroscopic methods of absorption and fluorescence and a
number of substances synthesised in the lab known as allosteric effectors of llb
As BPG (2,3-diphosphoglycerate) lacks appropriate characteristics to
allow the study of its binding and releasing from the tetramer when the gaseous
ligand is released or bound, we performed the study of some functional
analogues of BPG, possessing more favourable physical properties the
fluorescent substance HPT (8 hydroxy - 1,L6 pyrenetrisulfonic acid trisodium
salt), respectively IHP (inositol hexaphosphate) We noticed that anothei
allosteric effector of Hb, BZF (bezafibrate) mainly binds to the deoxygenated
state of Hb molecule and dwindles its affinity to the gaseous ligand
Consequently our study puts forth data regarding the replacement ot
HPT from metHb by 1HP, the releasing of HPT by binding CO, the movement of
the allosteric equilibrium when BZF binds, and the dependence of HPT
fluorescence on pH in solutions of Hb derivates

(We/4.1/062)

Characterization of GImU, an enzyme involved in

the peptidoglycan biosynthesis in E. coli.
Pompeo F., Mengin-Lecreulx D., and J. van Heijenoort.
C N R S E P 1088. Universit ~ P a r i s - S u d 91405 Orsay. France

UDP-N-acetylglucosamine plays an important role in the biochemistry of

all living organisms. In gram-negative bacteria, it is situated at the branch
point of the biosynthetic pathways of two essential cell envelope
components . peptidoglycan and lipopolysaccharides. The enzymes
implied in these pathways are potential targets for new antibiotics.
The two latter activities involved in the four-step formation of UDP-Nacetylglucosamine were carried by the bifunctional enzyme GImU. The
enzyme is a GIcN-1-P acetyltransferase and a GIcNAc-I-P
uridyltransferase ( UDP-GIcNAc pyrophosphorylase).
GIcN-I-P + AcCoA

P- GIcNAc-I-P + UTP ' ' ' - ~ UDPGIcNAc

Acetyltransferase

Uridyltransferase

GImU has been purified to homogeneity and shown to exibit a number of

characteristics which suggested that the acetyltransferase and
uridyltransferase activities may reside in separate catalytic domains For
exemple, the acetyltransferase but not the uridyltransferase activity was
inactivated by thiol-specific regeants suggesting that a sulfhydryl group
may play a role in the catalytic mechanism of these activity of GImU
The site-directed mutagenesis on the four cysteines of the protein has
shown that the two cysteines 307 and 324 are involved in the
acetyltransferase activity Site-sirected mutagenesis on three residues of
the uridyltransferase consensus sequence has shown that G14, R18 and
K25 are implied in the catalytic mecanism of these activity.
A structural study of GImU is in progress : crystallization of the entire
protein or of each domain of GImU will be interesting.

Abstracts FEBS'99

(We/4.1/063)

Studies of the role of the motif B in T7 RNA

polymerase functioning
E.E. Ru~akova, S.N. Kochetkov, A. P. Yankin
Engelhardt lnstttute of Molecular Biology ofMolecular Biology,
Russian Academy ofSctences, Vavilovstr. 32,117984 Moscow,Rnssia

Bacteriophage T7 RNA polymerase is known to be one of the

simplest enzymes catalyzing RNA synthesis This enzyme consist of one
subunit and is able to carry out transcription in the absence of additional
protein factors.
Earlier it was found that conservative motifB in T7 RNA
polymerase plays an important role in the catalysis of the transcription, being
probably responsible for binding and correct orientation of nucleoside
triphosphates.
In order to explain the effect of motifB to catalysis, we produced
some mutants with replacements in motif B. It was found that the
replacements of Ser633Ala and Tyr639Phe,Ser641Cys caused an
inactivation of T7 RNAP while its specific interactions with the promoter
were fully retained. Mutants Met635Tyr and
Met635Tyr,Tyr639Phe,Ser641Ala have demonstrated low polymerase
activity (about 5-10 per cents of WT enzyme). These mutations resulted in
the increase of misincorporations during transcription: the mutant enzymes
synthesized full size transcripts on T7 promoter-containing plasmid DNA
using only 3 and even 2 nucleoside triphosphates.
Generally, the replacements of residues 633 and 641 affect the
activity of the enzyme more strongly than of residue 635. The data obtained
suggest the importance of the mutated amino acid residues and further
demonstrate functional role of motif B in T7 RNA polymerase.

(We/4.1/065)

Role of membrane associated guanylate kinase

homologues in cell signalling
O. Spangenberg, M. Konrad
MPlfor Biophysical Chemistry, Dept.of Molecular Genetics,
37077 Gottingen. Germany

The synapse-associated protein SAP97 is a structural protein of the

cortical membrane cytoskeleton [1]. It is a mosaic protein composed of
three PDZ, an SH3, and a guanylate kinase (GK)-like domain. Whereas
the SH3 domain is a well-known structural element for binding of
proline-rich regions in an effector molecule, PDZ domains are novel
motifs that bind with high affinity to specific sites in a number of proteins
including potassium channels, glutamate receptors, and nitric oxide
synthase. The C-terminal GK domain in SAP97 shows a striking
sequence similarity to authentic guanylate kinases from yeast and
mammals. A vital role for the GK domain is suggested by studies on a
Drosophila homologue called discs large gene (dig). It was hypothesized
that the GK domain acts as a putative GMP kinase and, in concert with
NDP kinase, regulates the ratio of GDP and GTP in G-protein dependent
signalling pathways. We set out to evaluate the assumption that SAP97
and its GK domain do encode an enzymatically active GMP kinase [2].
Various fusion protein constructs of the GK domain were overproduced
in E. coll. No measurable guanytatc kinase activity was detected. Our bz
vivo data demonstrate that SAP97 cannot complement the lethal GUK1
disruption phenotype in S. cerevisiae. The GK domain appears to have
evolved a structural role in assembly of macromolecular complexes. This
view is supported by the recent finding of a protein (GKAP) that
specifically interacts with this domain.
[1] Garner, C.C., and Kindler, S., (1996) Trends in Cell Biol., 6, 429-433
[2] Kuhlendahl, S. et al., (1998) Eur. J. Biochem., 252, 305-313.

s291

(We/4.1/064)

Structural Studies on Proteins of the lnterleukin-1 Signal

Transduction Pathway. Sigrid Stuhrmann and Nick Gay
Universi~ of Cambridge, Department of Biochemistry, 80
Tennis Court Road, Cambridge CB2 1GA, England

The cytokine IL-I is thought to function by bringing together a receptor

molecule (IL-IR) and an accessory transmembrane protein (IL-IRAcP) at
the surface of cell, an event that allows binding to be perceJved by
components of the signalling pathway in the cytoplasm. The signal then
gives rise to a complex sequence of events including the activation of the
Rel-like transcription factor nuclear factor kappa B (NFr,J3). The IL-I
receptor associated kmases IRAKI and IRAK2, together with the adaptor
molecule MyD88, are important components in the immediate post-receptor
cytoplasmic signalling pathway. IRAKI and IRAK2 have homologues in
Drosophila (Pelle) and plants (Pto). The Drosophila signalhng pathway
mediated by the Toll receptor is very similar to IL-I signalling and is
responsible for dorsal-ventral pattermng during early embryogenesis of the
fly. In the adult fly it is revolved in innate (non-adaptlve) immunity. The
conservation between d~verse species indicates that the IL-I system
represents an ancient signalling machine critical for responses to
environmental stresses and pathogens. Structural studies of cytoplasmic
components in the 1L-1 pathway will give insights into the mechanism of
stgnal transduction and may also provide a basis for rational design of
inhibitors specific to the IL-I pathway which is responsible for many
inflammatory diseases.
1RAK-I, MyD88, Pelle and Tube have so-called death domains at their Nterminus. They mediate reversible protein-protein interactions. Death
domains were initially recogmsed in components of signalhng pathways
leading to apoptotic cell death. Homology modelling with NMR-structures
of death domains indicates that the death domains in tube and pelle are
hkely to adopt a similar overall structure, which consists of six antiparallcl,
amphipathic a-helices arranged in a novel fold. Pelle, IRAK-I and ILIRAcP are expressed and purified using E. coil and the baculovirus
expression systems for biochemical and structural studies. Recent results
wall be presented.

(We/2.2/000) Recognition of Digitoxigenin-Fluorescein Diastereomers by

Anti-Digitoxigenin Antibodies.
S. Y. Tetin, D. Tsarouhtais, R. J. Himmelsbach, D. L. Cotter
Abbott Diagnostics Division, Abbott Laboratortes,
100 Abbott Park Road, Abbott Park, IL 60064, U.S.A.
Digitoxigenin-fluorescein tracer is used in commercial
fluorescence polarization immunoassays (FP1A) The synthesis
yields a diastereomeric mixture of the tracer. Purification by
RP-HPLC afforded each diastereomer with greater than 95%
purity The structures were confirmed by mass and NMR
spectrometry Both diastereomers, SII and S12, have the same
absorption spectra with molar absorption ~;=50,000 M ~ at 493 nm
They also possess identical fluorescence emission spectra with a
maximum at 525 nm, the same quantum efficiency, and the same
fluorescence lifetime "t=4 I nsec. Furthermore, in affinity
determination experiments SI1 and SI2 demonstrated similar
dissociation constants: 0.21 nM and 0.15 nM, respectively The
most important difference was found in spectral properties of
diastereomers saturated with the antibody Both diastereomers
were modestly (20%) quenched upon binding, which indicates
involment of the fluorescein moiety in the interaction with the
antibody Nevertheless, anisotropy of SII in the bound form was
rm,~=0 191 and anisotropy of SI2 was rm~=0 121 in contrast to the
unbound form where both diastereomers have r,~=0.021. Such a
difference can be explained by the distinct positioning of the
fluorescein moiety in relation to the external surface of the
antibody binding site

s292

(We/4.1/067)

Abstracts FEBS'99

Effect of L-phenylalanine on the stability and domain

interactions in human phenylalanine hydroxylase
Matthlas Thorolfsson , Peter Fojan-, Sffen B. Petersen-, and
Aurora Martfnez

(We/4.1/068)

'Departmel~t of Btochemi,~trr and Molecular Btology, Untversity oJ Bergen,

and 2Delta1tmeJtt of Bioteclm(llogy, Universi O' of Aalborg, Denmark

Human phenylalanine hydroxylase (PAH) is an oligomeric enzyme that

catalyzes tile hydroxylatmn of L-phenylalanine (L-Phe) to L-tyrosine. Each
subunit contains a N-terminal regulatory domain (Ser2-Ser110) with a
phosphorylation site (Ser-I6), a catalytic C-terminal domain (Aspl12Lys427) including a non-heine iron and a tetramerization domain at the Cterminus (Gln428-Lys452) [l]. PAH exhibits a nonhyperbolic (sigmoidal)
dependance of activity on L-Phe concentration reflecting a slow transition
of the enzyme from a low-affinity and low-activity state to a high-affinity
and high-activity state. This conformatmnal change induced by the substrate
results in an increased surface hydropobicity, a change in the dimer
tetramer equilibrium to the tetrameric form, a red shift of the fluoroescence
emission maximum and an increase of the fiuoroscence intensity. Here we
present results on the effect of the subslratc oll the interactions between the
domains in human PAH and on the thermodynamic stability of the enzyme
studied by differential scanning calorimetry and activity measurements.
The denaturation of the recombinant human wild-type PAH resulted in three
transitions in the calorimetric scans, corresponding to the regulatory,
catalytic and tetramerization domains of the enzyme. L-Phe increased the
cooperativlty m the calorimetric scans for the tetrameric wild-type enzyme
indicating dramatic changes in both domain-domain interactions as well as
dimer-dimer interactions. L-Phe was found to increase the stability of the
isolated C-terminal catalytic dolnain, measured as an increase in the melting
temperature (T,,,) in activity and calorimetry measurements. Thus, the
studies indicate that the substrate regulates the interactions between domains
in the enzyme that control eazyme activity.

Structure-function studies of the Na*/I.~

exchanger (NHE- 1)
Touret N., Godart H., Cuiller B., Poujeol P. and Counillon L.
UMR CNRS 6548, UNSA. 06108 Nioe cedex 2. France

Na+/H exchangers are integral plasma membrane proteins which

exchange extracellular Na + for intracellular H . Amongst these
transporters, the NHE-I isoform is widely expressed in eukaryotic
cells, and is responsible for intracellular pH regulation.
To identify the functional domains involved in ion transport
mechanisms, we have used selective tests based on cell resistance to
acidification in the presence of NHE-1 inhibitors (HOE 694, 30 uM).
Different mutants, showing up to a 5000 - fold increase in Ki for HOE
694 were isolated (550/~M instead of 0,1 ,uM for NILE-1 i soform).
The cDNA expressed by the most resistant mutant was then cloned and
sequenced. Our results revealed a point mutation resulting in a
Phe163Ser substitution in the fourth transmembrane domain.
Moreover, the kinetic characterization of this mutant showed an increase
in the apparent Km for external sodium (30 mM for the wild type
isoform, versus 120 mM for the mutant isoform).
Using intraeellular acidification in the presence of low external sodium
concentration (3 mM), we selected revertants for the sodium - low
affinity phenotype.
Two types of revertants can be distinguished :
the first group had recovered a wild type phenotype with a Km for
the sodium of 30 raM, and a Ki of 0,1 /zM for the HOECIIST
compound ;
the second group possesses a Km of 30 mM for external sodium,
and conserved a Ki of 550/M for the HOE 694.
To identify the mutation responsible for the sodium - low affinity
phenotype reversion, we are now cloning and sequencing the cDNA
expressed by these differents isoforms. This study will allow us to map
the aminoacids involved in the sodium binding and transport.

[I]Knappskog, P.M., Flatmark, T., Aarden, J.M., Haavik, J. and Martfnez,

A. (1996) Eur. J. Biocheln. 242, 813-2 I.

(We/4.1/069)

Stability of the bovine brain

Phosphatidylethanolamine-BindingProtein
B. Vallee, J Ramstem, L Serre, N Bureaud and F. Schoentgen
('entre tie Btoplwstque 3Iol~culalre-CL\,'RS, 450-10rl{~ans, l+ance

The Phosphatidylethanolamine-bmding protein family is composed

of proteins highly conserved throughout the nature and found in numerous
tissues from a wide range of species, such as mammals (human, bovine,
monkey, rat and mouse), plants (Anttrrhmum, Arabtdopsts and tomato),
Drosophila, parasites ('lbxocara cat)is) and Saccharomyces cerevtsiae. The
hiologtcal function of each member of this famdy is not yet wellestablished, however the role of some of them is partially known in plants,
the PEBP related protems are involved in morphogenesls and ontogenesis
(florescence determination, alternation between the vegetanve and
reproductwe cycles of the plant meristem), in rat, the protein ms highly
expressed in ohgodendrocytes of developpmg brain and in elongated late
spermatids, revealing that the presence of PEBP is correlated with tlssular
and cellular development
In our laboratory, we extract PEBP from the bovine brain The
bovine PEBP is a cytosolic protem of 21 kDa containing 186 amino ac,ds.
It brads phospholiptds, especially phosphattdylethanolamine, and
nucleotides, such as GTP Recently, the 3D-structure of the bovine PEBP
has been determined by X-ray crystallography, the bovine PEBP exhibits a
new folding, with a [~-sheet core and large loops
In order to get new instght into PEBP characteristics, we have
stud)ed Its stabdlty towards pit, temperature and urea concentrat,on by
fluorescence and circular dlchrolsm spectroscopies and by dffferentml
scanning calonmetry For pit t~tratmn, a cooperatwe two-state transmon ~s
observed with a pK~ of 3 D. PEPB thermal denaturation follows a classical
two-state mechanism with a melting temperature of 54C and an enthalpy
of 119 kcal/mol The chemical denaturation by urea g~ves a stabdlty value
of 4 5 kcal/mol

(We/4.1/070)

The mannose-speeifie |acalin-related iectins: a

novel superfamily of plant lectins
E.J.M. Van Damme, W. Zhang and W.J. Peumans
Laboratory for Phytopathologyand Plant Protecuon, KULeuven,
W. De Croylaan 42, B-300I Heverlee,Belgium

Jacalin-related lectins have originally been discovered in jack fruit

(Artocarpus integrifolia) seeds, which contain large amounts of a
Thomson-Friedertreieh or T-antigen-specific agglutinin, called jacalin.
Due to the apparent confinement of the j acalin-like lectins to Moraceae
species these lectins are usually considered as a minor lectin family.
However, after the identification of a mannose/maltose-speeific lectin in
rhizomes of hedge bindweed (Calystegia sepium), which shares a high
sequence similarity with the precursor ofjacalin it became evident that
jacalin-related lectins occur also outside the Moraceae family.
Moreover, the discovery of the Calystegia sepium agglutinin (Calsepa)
revealed the existence of two subgroups within this lectin family.
Jacalin mad Calsepa have a different molecular structure ( [2 kDa+13
kDa] 4 versus [16 kDa] 2), indeed, and in addition exhibit a totally
different specificity (GalI3(I,3)GalNAc versus mannose). Lectins
similar to Calsepa have been found in Convolvulus arvensis and Ipomea
batatas (Convolvulaceae family) and in tubers of the Jerusalem
artichoke (Helianthus tuberosus, Asteraceae family). Evidence has been
presented that the polypeptides of the so-called myrosinase-binding
proteins from Brassica napus seeds and seedlings contain domains with
a high sequence identity to the jacalin-related lectins. Besides in dicots,
patatwe jacalin-related proteins occur also in several monocot species.
We recently isolated and characterized mannose-binding lectins from
the Gramineae species Hordeum vulgare (barley), Triticum aestivum
(wheat) and Oryza sativa (rice), and from Musa acuminata (banana,
family Musaceae). A comparative study of the biochemical properties,
biological activities and molecular structure indicated that all these
monocot and dicot lectins are closely related to each other. Based on
these findings it is concluded at the subfamily of the mannose-specific
jacalin-related lectins is widespread among higher plants. Using the
available sequence information a model has been elaborated for the
molecular evolution of the family of the jacalin-related leetins.

Abstracts FEBS'99

(We/4.1/071)

Open structure of pig muscle 3-phosphoglycerate kinase

(PGK) with bound MgADP and 3-phosphoglycerate (3-PG)

s293

(We/4.1/072)

M. Vas Kazinczy 1, A. N. Szil~tgyit & M. Ghosh z

tlnstitute ofEnzyatology, BRC, Hung. Acad. Set., Budapest, Hungary &
eLaboratory of Molecular Biophysics. University of Oxford, Oxford, UK
The mechanism of relative domain movements and its role in the catalysed
phospho-transfer is investigated on PGK, a typical two-domain kinase, which
catalyses the phospho-transfer from 1,3-bisphosphoglycerate to MgADP to
form 3-phosphoglycerate and MgATP. Crystals (space group P21) of pig
muscle PGK were grown in the presence of MgADP and 3-PG in 2.5 M
phosphate, pH 8.0, i.e. under the conditions which has resulted in crystals of the
closed, catalytically competent conformation of Trypanosoma brucei PGK
[1].The X-ray data (SRS Daresbury, UK; complete up to 96%) have led to the
structure of PGK*3-PG*MgADP ternary complex at 1.8 A resolution with
R=20.8% and Rfree=24.1%. This structure, however, unexpectedly represents
an essentially open conformation compared to that of T. brucei PGK Inspection
of any possible structural effect of the sequence differences (about 50%) of the
two species has revealed no obvious reason for the largely different relative
domain positions. In the enzyme-substrate interactions difference can only be
observed in the mode of interaction of the phosphates of the nucleotide with
P G K Namely, in the pig muscle PGK structure the 13-phosphate of MgADP
does not have any detectable density, though its presence in the crystal mother
liquor has been confirmed. This can be attributed to the mobility of this
phosphate group, i.e. its interaction with a key element of the interdomain
region (helix 13) is lacking. The absence of this contact with the protein has
allowed us to understand the concerted action of the two substrates during the
domain closure. Systematic comparison of all the available PGK ternary
complex structures with different domain positions, including the ones with 3PG and MnAMP-PNP from pig muscle [2] and from 7hermotoga maritima [3],
both at the level of the secondary structure and of contacts of the conserved
amino acid side chains, indeed, revealed the importance of the contact with the
nucleotide and delineated, at atomic level, a very probable mechanism of the
domain closure. In addition to the previously observed C- and N-hinge points,
the importance of the interdomain helices 13 and 14 as well as the connecting
beta sheet L is now demonstrated and a third hinge point here is proposed.
[1] Bernstein et al., Nature 385 (1997) 275
[2] May et ah, Proteins 24 (1996) 292
[3] Aucherbach et al., Structure 5 (1997) 1475

(We/4.1/073)

Model Calculations of Interaction of Human Serum

Transferrin and Tryptophan-Derivative Enantiomers
Balfizs Visegr~tdy, Ferenc Kilfir
('entrat Research Laboratory..l[edtcal ~~tverstt3 of Pets. S~lget ut 12.
H-7643 P ~ . [[unga(v

Globular proteins interact with others by their surface interaction (or binding
sites Since the knowledge of the structure and positioning of the binding sites i
important for understanding the protein function, a careful characterization c
those is essential The interactions between tryptophan-derivatives (tryptophan
methyl, ethyl-, and butyl-ester) and human serum transferrin have been followe,
by capillary_ electrophoresis [1]. Changes in the migration properties of the sma
molecules and stereoselective separation of enantiomers reflected that at leas
one specific binding (interaction) site exists on the surface of the protei
molecules The enantiomers were separated by iron-free transferrin, but simila
phenomenon were not observed with ~t's iron-saturated form For the bette
understanding and characterization of the binding sites we performed a detaile,
study with compute model calculations Here we present the results obtained b
using the Sybyl version 6 5 (Tripos, Inc, USA) software Different 3I
structures of the protein molecules in the transferrin family having iron o
without the metal were used The most successful ,,docking" results with thre
different structures of a recombinant N-terminal (half molecule) of iron-fre
human serum transferrin [2] showed different orientations of the tryptopha
derivatives on the surface of the protein Every docking can be characterized b
an ,,energy value" of the interaction The energy calculations showed that the t
enantiomers of the tryptophan-derivatives have a more stable interactions wit
the transferrin which fitted well with the results obtained by capillar
electrophoresis We assume that the ,,active site" for the stereoselectiv
separation includes the iron binding amino acid residues, but other side chain
may also play important role.
Acknowledgments: The work was supported by grants OTKA T25527 an,
TKT T-08 394/96. The X-ray diffraction data of human serum transferrin wer
kindly obtained from Edward N Baker

[1] Ferenc Kilar, Electrophoresis, 16, (1995) 1510

[2] Philip D Jeffrey, Biochemistry, 37, (1998) 13978.

Designing enzymes with novel specificities by swapping

target recognition loops between DNA cytosine methylases
G. Vilkaitis. R. Gerasimaite and S. Klima~auskas
Institute ~ Biotechnolog~l Grai(nino 8, LT-2028 Vdnius, Ltthuanla

The /-/hal DNA cytosine-C5 methyltransferase (M.HhaI) recognizes its

G CGC target on DNA via two distinct loops in the protein, that interact with
non-overlapping parts of the target sequence. In this work we constructed a
series of hybrid proteins in which the individual recognition loops and/or
their adjacent regions in M.Hhal have been exchanged with the homologous
regions from the M.Bsp61 (recognition target GCNGC) and M.Lsp1109I
(GC(A/T)GC) methyltransferases. The hybrid proteins were overexpressed
in E. coli and their in vivo methylation phenotypes were studied by:
cleavage of the plasmid DNAs with appropriate restriction endonucleases
(i); transformation of the recombinant plasmids into a E coli strain
containing McrB restriction system (ii); direct mapping of 5-methylcytosine
residues using the genomic sequencing technique (iii). We find, that
exchanges of the first recognition loop leads to inactive proteins, unless
adjacent segments are transferred in concert. Exchanges of the second loop
lead to reductions of target specificity: GCGN in the case of M.Bsp6I and
C~GN(A/T) - for M.Lspl 109I. The appearance of the A/T specificity at the
5 th position of the target site of the HhaI-LspI 109I hybrid suggests that the
second loop of M.Lspl 1091 is responsible for the recognition of the central
A/T base-pair. In aggregate, our results demonstrate a high potential of this
approach for the design of enzymes with novel DNA methylation
specificities.

(We/4.1/074)

Role of the C-terminus alpha helix 9

of Glutathione Transferase A I - I
in protein stability and ligandin function.
L. A. Wallace, H. W. Dirr
Protein Structure-Function Research Programme, Dept of Biochemisto;
University of the Witwatersrand, Johannesburg, South Africa 2050.

Human GST AI-I is a member of the superfamily of cytosolic glutathione

transferases (GSTs, E.C. 2.5.1.18) that function in cellular detoxification and
ligand binding of many hydrophobic endogenous and xenobiotic compounds.
Each monomer of 221 amino acid residues contains an alpha/beta domain I and
an all-alpha helical domain II. The C-terminus (helix 9) forms part of the
hydrophobic electrophile binding site in domain I. Helix 9 plays an important
structural role in the enzyme's catalytic function [1], however, its role in
stabilising and specifying the native state is unknown. The thermodynamic
stability and unfolding kinetics were investigated using wild-type enzyme, a Cterminus deletion (residues 209-221) mutant and F220W mutant. Various
structural (far-UV CD, intrinsic fluorescence and ANS binding) and functional
(catalytic activity) probes were used to monitor urea-induced changes. In
addition, the effects of active site ligands and non-substrate ligands on
stability/function and the conformational dynamics of helix 9 were investigated.
In light of this data, the previously proposed two- state equilibrium
unfolding/refolding process (N2 --> 2U; see [2]) can be modified to
accommodate the dynamics of the helix 9 i.e., N2 (helix9stabilised) --> N2
(helix9 mobile) --> 2U, where N2(helix9stabilised) and N2(helix9mobile) are
the native folded dimer in the presence and absence of active ligand, respectively
and U represents the completely unfolded monomer. Wildtype hGST AI-I
exhibits a three-state unfolding kinetics pathway (N2 --> N2* --> 2U),
where a transient native dimeric intermediate (N2*) with partially dissociated
domains at or near Trp20 was detected [2]. Deletion of helix 9 simplifies this
pathway to a two-state pathway; N2# --> 2U where N2# resembles N2* in terms
of spectroscopic and transition state properties.
[1] Board, P. G. and Mannervik, B. (1991) Biochem. J. 275, 171-174;
[2] Wallace, L. A., Sluis-Cremer, N. and Dirr, H.W. (1998)
Biochemistry 37, 5320-5328

s294

(We/4.1/075)

Abstracts FEBS'99

Aspartate transcarbamylase short time dynamics

studied by inelastic neutron scattering.
J.-M. Zanotti ~, G. Herv6 b and M.-C. Bellissent-Funel a
Laboratotre L~on Brilloum (CEA-CNRS), CEA-Saclay, 91191 Gifsur-Yvette Cedex (France). ~ Biochimie des stgnaux r~gulateurs
cellulatres et mol~culatres (URA 1682 du CNRS), Universit~ Pierre et
Marie Curie, 96, Boulevard Raspail, 75006 Paris (France)

E. coli aspartate transcarbamylase (ATCase) is a 310 kDa allosteric enzyme

which catalyses the first committed step in pyrimidine biosynthesis. The

crystallographic structure of ATCase of E. coli has been determined at 2.5
resolution. The binding of the substrates, carbamyl phosphate and aspartate,
induces significant conformational changes [1]. The protein switches from a T
state (quaternary structure in the absence of ligand) towards a R state
(quaternary structure of ATCase saturated with ligand). The same quaternary
structure change is observed upon binding of the bisubstrate analogue PALA
(N-(phosphonacetyl)-L-aspartate) [2]. The passage of the T state to the R state
results in an increase of 5% of the radius of gyration of the protein, as measured
by small angle X-ray scattering [3].
Owing to the large neutron incoherent scattering cross-section of the hydrogen
atom and the abundance of this element in proteins, inelastic neutron scattering
experiments give a global view of protein dynamics as sensed via the
individual motions of its hydrogen atoms. We have carried out incoherent
inelastic and quasi-elastic neutron scattering experiments, at ambient
temperature and normal pressure, in order to assess a difference in the local
dynamics (few angstroms), at short time (few tens of picoseconds) of ATCase
in T and R conformations. The experiments have been performed on the
Mib6mol time of flight spectrometer (Laboratoire L6on Brillouin) in the 0.4 to
3.0 A-~ Q range. The spectra of two samples and of the corresponding buffers
were recorded: ATCase in T form and in R form (ATCase+PALA). The
experiments enable to assess the short time dynamics, from a fraction to 20
picoseconds, of the T and R forms of ATCase.
[1] Kantrowitz E.R. et al., Science, 241, (1988) 669. [2] Krause K.L. et al., J.
Mol. Biol., 193, (1987) 527. [3] Fetler L. et al., J. Mol. Biol., 251, (1995) 243.

(We/4.1/076)

Characterisation of the human homolog of the

Drosophila Goliath protein.

F. Bulle ~, M. Champ S. Andreae ~, M-G Matte~.% S. Siegrist~

and G. Guel a~#.
"unit~ Inserm 99, Hbpital Hemi Mondor. 94010 Crdteil. Frmlce
~Unitd Inserm 406. H6pital de la Timone, 13385 Mar~eille. France

By systematic sequencing of human testis cDNA clones, we identified new

human transcripts, homologous to lower species mRNAs (yeast, nematode,
Drosophila...), potentially involved in the cell cycle [1]. Among them, we were
interested by one cDNA corresponding to a protein which exhibited homologies
with the Drosophila Goliath protein. In this species, this zinc finger protein could
be a transcription factor involved in the embryo mesoderm formation [2]. The
complete sequence of the human Goliath cDNA reveals an open reading frame of
276 aa coding for a putative 32 kDa protein exhibiting 40,9 % identical aa
residues with the Drosophila protein. Two zinc finger motives (C3H2C3) were
found in the amino acid sequence as for the Drosophila protein. By Northem blot
analysis, this cDNA, used as a probe, detected two mRNAs of 1,8 kb and 2 kb
expressed in variable amounts in 16 human tissues tested. The highest rnRNA
level was found in leukocytes. Semi-quantitative RT-PCR analysis on cell
subpopulations of leukocytes (lymphocytes T and B, polynuclear cells and
monocytes) revealed the major expression of Goliath mRNA in the myeloid cells
(polynuclear cells and monocytes). The simple pattern, obtained on genomic
DNA after hybridization with the Goliath cDNA, suggests that the two
messengers are transcribed from the same gene. By in situ hybridization, this
gene was localized on chromosome 5q35. The human Goliath protein, expressed
in a bacterial system, was used to produce rabbit polyclonal antibodies. By
Western blot analysis, we demonstrate the presence of a 32 kDa Goliath protein
in human leukocytes and we are now using these antibodies in order to determine
the cellular localization of the protein in subpopulations of leukocytes. These
features suggest a functional role for Goliath during hematopo'iesis, this
hypothesis is under investigation in our laboratory.
[1] Pawlak et al., Genomics, 26, (1995), 151.
[2] Bouchard et al., Gene, 125, (1993), 205.

Abstracts FEBS'99

s295

6.3 Gene regulations by nutrients and hormones

(We/6.3/077)

Nueleosides induce the gene expression of fibroneetin and

laminin in liver stellate cells and hepatocyte-cocultures

A.ArnaudI, J.M.L6pez-Pedrosa~, L.Fontana~, M.J.S~iez-Lara~, A.Gil~

'1.Dept R+D, Abbott Laboratories, and ~Dept.Biocherntstry and
Molecular Biology, School of Pharrnacy, Granada. Spare

(We/6.3/078) Regulation of FATP, ACS, and [3-oxidation

gene expression by fatty acids in weanling rats.
F. OuaIi, F. E)jouadi, C. Merlet-B~nichou, B. Riveau, J. Bastin
INSERM L1319, Universit~ Paris 7, Paris, France

We studied regulation of genes involved in fatty acid utilization in heart,

Extracellular matrix proteins such as fibronectin and laminin promote
changes in cell shape, migration, proliferation and differentiation. Cellular
immunity, genetic stability and apoptosis have been shown to be affected by
changes in the intracellular pool of nucleotides.To investigate the effect of
~,ucleosides on extrace!lular matrix proteins, we used two cell culture models:
a hver stellate cell line (LSC), and a co-culture of rat hepatocytes and LSC.
Fibronectin levels measured by Northern and Western blots increased in both
culture systems when nucleosides were added to the medium; this induction was
higher in the LSC model.However, the secretion of fibronectin was not affected.
Nucleosides also induced laminin content in LSC. We also studied whether
fibronectin and laminin inductions might be related to changes in the
proliferative response of LSC. However, assays of DNA content, DNA syntesis,
proliferating cell nuclear antigen (PCNA), protein content and succinatedehydrogenase activity (XTT) showed that nucleosides did not promote LSC
proliferation. These results suggest that nucleosides culture supplementation
stimulates fibronectin and laminin gene expression in LSC alone and in cocultures of LSC and hepatocytes, without affecting the proliferation process and
that exogenous nucleosides could be involved in the differentiation cellular
process.

liver and kidney of weanling rats submitted to high- or low-fat diets or

treated by etomoxir, a blocker of fatty acid import into mitochondria.
Northern-blot analyses were performed using cDNA probes specific for
fatty acid transport protein (FATP), a cell membrane fatty acid (FA)
transporter, acylCoA synthetase (ACS), involved in activation of FA, longchain- and medium-chain acylCoA dehydrogenases (LCAD, MCAD)

which catalyse the first step of mitochondrial FA B-oxidation, and acylCoA

oxidase (ACO), a peroxisomal FA B-oxidation marker. High-fat feeding
from postnatal day 21 to day 28 resulted in a coordinate increase (+58 to
+136%) in mRNA abundance of all genes in heart. In liver and kidney,
diet-induced changes in mitochondrial and peroxisomal B-oxidation enzyme
mRNAs (from +27 to +114%) occured with no change in FATP gene
expression. Changes in fatty acid homeostasis resulting from etomoxir
administration led to a marked increase in cardiac gene expression levels
(+122 to +488%), had lower effects in liver, and did not affect renal
mRNAs pattern. Feeding rats a low-fat diet containing 0.5% clofibrate, a
ligand of peroxisome proliferator activated receptor alpha (PPAR~x),
resulted in similar inductions of B-oxidation enzyme and ACS genes in the
three tissues, while up-regulation of FATP gene was restricted to heart.
Altogether, these data suggest that changes in fatty acid homeostasis in
immature organs can efficiently be transduced to the level of gene
expression, possibly via PPAR~, to dictate tissue-specific adaptations in

the mRNA pattern of various fatty acid utilizing enzymes and proteins.

(We/6.3/079)

Leukemia Inhibitory Factor-Receptor

adipocyte differentiation.

promotes

N. Belmonte, J. Aubert, S. Dessolin~ P. Villageois, B.

Barhanin, R. Nrgrel, G. Ailhaud and C. Dani, Centre de

(We/6.3/080) Differential involvement of PPARa in fibrate and

fatty acid up-regulation of L-FABP gene in the
liver and the small intestine
H. Poirier t, I. Niot I, 0. Braissant 2, P. Costet ~,
C. Meunier-Durmort ~, M.-C. Monnot L,T. Pineau ~,
W. Wahli 2, P. Besnard t

Biochimie. UMR CNRS 6543, Nzce, France.

The effects of Leukemia Inhibitory Factor-Receptor (LIF-R) on

adipocyte differentiation have been investigated. LIF-R seems to play
an important role since the capacity of embryonic stem (ES/ cells
deficient for LIF-R to undergo adipocyte differentiation in vitro is
dramatically reduced compared to LIF-R expressing ES cells. Our
results strongly suggest an autocrine/paracrine mechanism in which
LIF-R activation induces early events which are critical for subsequent
differentiation. This proposal is based upon three observations: i) LIF
is secreted by preadipocytes which express also LIF-R: ii) blocking
LIF-R activation decreases terminal differentiation of preadipocytes:
iii) a short-term exposure to LIF at the preadipose stage is sufficient to
trigger adipose cell differentiation. LIF and a PPARy ligand stimulate in
a synergistic manner the differentiation. LIF stimulates rapidly the
expression of C/EBP 13and ~5which are known to play a critical role in
the transcriptional control of adipogenesis. Two major signalling
pathways from LIF-R to the nucleus have been identified so far in
different cell types: a MAP kinase pathway and a JAK-STAT pathway
where STAT3 is the main transcriptional factor activated. The
contribution of these pathways in the stimulation of C/EBPs by ElF in
preadipose cells has been assessed. For that purpose we generated a
preadipose cell line conditionally expressing a dominant-interfering
mutant form of STAT3 and we made use of a MAP kinase-kinase
inhibitor. The results indicate that LIF-stimulated C/EBPs gene
expression is independent of STAT3 activation but requires activation
of p42/p44 MAP kinases. Altogether, these results implicate LIF and
its receptor in the commitment of preadipocytes to undergo terminal
differentiation by controlling the expression of C/EBPs gene
expression via the MAP kinase cascade.
This work was supported by grants from CNRS. INSERM and ARC
(grant n5065 to CD).

~Physiologie de la Nutrition, UMR CNRS ENSBANA/CESG,

F-21000 Dijon, France
21nstitut de Biologic Animale, CH-1015 Lausanne, Switzerland
~Laboratoire de Pharmacologic et Toxicologie, INRA,
F-31931 Toulouse, France

Liver fatty acid-binding protein (L-FABP) is a cytoplasmic polypeptide

which binds with a strong affinity espeeiaily long-chain fatty acids
(LCFA). It is highly expressed in both the liver and the small intestine
where it is thought to play an essential role in the control of the cellular
fatty acid (FA) flux. Because the L-FABP gene expression is increased
by both peroxisome-proliferator hypolipidemic drugs (PP) and LCFA,
it seems to be under control of PP-/FA-activated transcription factors
termed peroxisome-proliferator activated receptors (PPARs). However,
the precise molecular mechanism by which these regulations take place
remains to be fully demonstrated. Using transfection assays and
PPARc~ null mice, we report here that (i) L-FABP promoter contains
two peroxisome-proliferator responsive element (PPRE) sequences, (ii)
ppARc~ and/or 13/8isoforms are involved in the FA up-regulation of LFABP gene, and (iii) in contrast to the liver, pPARc~ is not absolutely
required to the L-FABP gene regulation by LCFA and PP in the small
intestine. On the basis of these findings, the involvement of PPAR13/8 in
the metabolic adaptation of the small intestine to lipid content of the

diet is discussed.

s296

(We/6.3/081)

Abstracts FEBS'99

Peroxisomal proliferation in yeast: glucose directly

represses the activity of both components of the
Oaflp-Pip2p transcription factor complex whereas
fatty acids activate only Oaflp
U. Baumgartner, B. Hamilton, M. Piscacek, H. Ruis, H.
Rottensteiner

(We/6.3/082)

Alpha-glucosidase in Crustacea : cDNA characterization

P. Le Chevalier ~, D. Sellosb, C. Le Boulay",
P. Lrcher a, A. Van Wormhoudtb
a LUMAQ, IUT, 29334 Qu~mper Cedex, France,
bMNHN/Colldge de France, BP 225, 29900 Concarneau, France

lnstitut f. r Biochemie und Molekulare Zellbiologie and Ludwig Boltzmann

Forschungsstelle der Universit%d Wien, Dr Bohrgasse 9,
A-I030 Wien, Austria

In the yeast Saccharomyces cerevisiae, both the proliferation of peroxisomes

and the expression of t-oxidation enzymes are induced in the presence of fatty
acids.This induction is mediated by an oleate response element (ORE). A
complex containing the Zn2Cys6 zinc cluster proteins Oaflp and Pip2p binds to
this element. Cells deleted at PIP2 or OAF1 do not respond to fatty acids and
therefore, proliferation of the peroxisomal compartment as well as growth on
fatty acids is abolished in such strains. We show here that both proteins, as a
result of their C-terminal activation domains, possess strong transcriptional
activator properties. Fatty acid-specific induction, however, is mediated only via
Oafl p and requires large parts of the protein.
Another level of control is exerted by glucose since it represses OREdependent transcription even in the concomitant presence of fatty acids. We
determined wether the observed repression of OREs was due to Oafl p and Pip2p
being direct targets for glucose repression. Using lexA fusions of both proteins
lexAop-lacZ-reporter gene activities were determined in the presence of glucose,
oleic acid, or both carbon sources. Glucose repressed the fl-galactosidase activity
obtained in oleic acid medium for both proteins. Since the lexA fusions were
expressed from the ADH1 promoter known for its high level expression on
glucose we reason that the activities of both transcription factors were effectively
inhibited by glucose in a direct manner. Potential glucose- repressive domains
were identified by deletion analysis of the lexA-fusion proteins. Further work is
required to identify the component(s) mediating glucose repression whereby one
possibility is that phosphorylation of Oaflp and Pip2p by the Snfl kinase is
required for activation.

(We/6.3/083)

Amino acids control the mammalian CHOP

gone transcription.

Bruhat, A., Jousse C., Ferrara, M. and Fafournoux, P.

UR 238 INRA, INRA de Theix, 63122 Saint Genbs Champanelle
FRANCE

The tropical shrimp, Litopenaeus vannamei, is an omnivorous

species, and it can be assumed that an important proportion of
dietary carbohydrates is necessary for its growth. Together with a
high level of alpha-amylase, alpha-glucosidase (EC 3.2.1.20)
participates in the ultimate liberation of glucose residues from starch
or glycogen. Recent experiments have shown that alpha-glucosidase
is inducible by the level of starch in the food. Alpha-glucosidase is a
glycosylated enzyme with a molecular weight of approximatively
105 kDa, exhibiting maximal activity at pH 6, but, in contrast to
other known alpha-glucosidases, no isomaltase activity was
observed. In order to obtain information on the sequence of the
molecule, a shrimp hepatopancreas cDNA library was screened with
a DNA probe of 549 bp obtained by RT-PCR amplification using
specific primers, one deduced from an internal peptide sequence, the
second deduced ~om the conserved catalytic site. In order to access
the complete sequence, successive PCR experiments using a vector
targeting primer (PT3) and specific primers deduced from the
sequence of the partial sequence were made. A 3 kb cDNA was
obtained, encoding the complete protein sequence of the mature
enzyme. The amino acid sequence of the catalytic site is identical to
that of mammalian sucrase-]somaltase.Characterization of this
cDNA will allow the study of the regulation level as well as the
localization of its synthesis. Moreover, gene structure
characterization will be helpful to understand gene effectors m
Crustacea.

(We/6.3/084)

Expression of a 6-phosphofrueto-2-kinase/fruetose2,6-blsphosphatase from the liver of S p , rus a,rata.

A.Caseras, I. Me~n, D. Mediavilla, I. V. Baanante.
Departamentde Bioquimiea i BiologiaMolecular, Facultatde
Farm/teia,Univexsimtde Barcelona;Barcelona,Spain.

In mammals, plasma concentration of amino acids are affected by nutritional

or pathological conditions. We examined the molecular mechanisms involved
in the regulation of gene expression upon amino acid limitation. We have
previously shown that regulation of C H O P (a CCAAT/enhancer binding
protein (C/EBP)-related gene) mRNA expression by amino acid concentration
has both transcriptional and post-transcriptional components (1). We report
the analysis of the cis DNA elements in the human C H O P promoter region
important for its activation by amino acids. Using a transient expression
assay, we show that the transcriptional activation of C H O P by leucine
starvation requires two DNA elements located both upstream and downstream
from the start site. One is a negative regulatory element (NRE) that ~s
sufficient to repress the activity of a heterologous promoter. The other one is
cis positive element that is essential for amino acid regulation of the C H O P

promoter. This sequence is the first described that can regulate a basal
promoter in response to starvation of several individual amino acids. From
these properties, this DNA sequence can be called Amino Acid Response
Element (AARE). In addition, we show that C H O P A A R E sequence binds/n
vitro members of the C/EBP-ATF transcription factors family. Altogether,

these results suggest that C H O P regulation by leucine may be an example of a

more general regulatory mechanism by which mammalian gene expression
would be controlled by the levels of amino acids.

(1) Bruhat, A., Jousse, C., Wang, X-Z., Ron, D., Ferrara, M. and
Fafournoux, P. (1997) J. Biol. Chem. 272, 17588-17593.

6-Phospho fructo-2-kinase/fructose-2,6-bisphosphatase
(6-PF-2K/fru-2,6P2ase) is a homodimeric bifunetional enzyme
that catalyzes the synthesis and degradation of fructose-2,6bisphosphate, a key regulator of the glycolysisglueoneogenesis pathway.
We have previously cloned and sequenced a 2527 bp
eDNA that encodes the liver isoform of the bifunetional
enzyme in the teleost fish Sparus aurata; the anfmoacid
sequence deduced from the eloned eDNA corresponds to a 55
kD protein. Besides, the regulation of the expression of this
gene was observed in fish liver alter fasting and refeeding [1].
In vitro transeriptiun/translation of the cloned eDNA
generates a single 55 kD protein, which corresponds to the
molecular weight previously reported for each bifunetional
enzyme subonit purified from the liver o f S p a r u s aurata [2].
The cloned eDNA was expressed in prokaryotie and
eukaryotie systems. Pmkaryotic expression of the enzyme
was achieved in E. coli BL21(DE3) with a T7 RNA
polymerase-depundent system; the fish hepatic isoform of
the enzyme was also expressed by transient transfeetion of
COS-7 cells. In both cases, the protein exhibited ldnase
activity. The fish 6-PF-2K/fru-2,6P2ase was analysed by
Western blot with specific polyelonal antibodies and was also
found to have a molecular weight of 55 kD.
[1]. Met&x,I.; Caseras, A.; Mediavilla, D.; Fecn~indez,F.;
Baanaate, I.V. Bioch. et Biophys. Acta 1999 Feb. 16;
1444 (2): 1-13.

[2]. Gareta de Fmtos, P.; Baanaate. I.V. Arct~ Biochem.

Biophys. 1994 Feb.l; 308 (2): 461- 468.

Abstracts FEBS'99

(We/6.3/085)

Possible involvement of a shared motif for insulin- and

glutamine- up- regulation of GAPDH transcription in
HepG2 hepatoma cells
S. Claeyssensac, C. Gangneux"bc, A. Lavoinneac, J.P. Sailerbc
aGBPDN,blNSERM U-519,ClFRMP23,Fac M~decine F-76183 Rouen

Enteral glutamine (Gin) supplementation has been shown to promote an

increase in overall protein syntheses and insulinemia in hypercatabolic
volunteers. Insulin (Ins) and Gin exert similar anabolic effects as they
both increase glycogen and fat synthesis and decrease proteolysis in
hepatocytes studied "ex vivo". Elsewhere, an up- or down- regulated
expression of the so called positive or negative acute-phase protein
(APP) is promoted by Gin and counteracted by Ins. Moreover, the motif
5'-Py-CTTG(A/T)-3' is found in many insulin-sensitive genes, such as
the metabolic gene glyceraldehyde-3-phosphate
dehydrogenase
(GAPDH), and in several APP genes, such as the o~lmicroglobuhn/bikunin precursor (AMBP), ot2-HS-glycoprotein (AHSG)

and albumin. On such grounds, we have investigated whether this motif

is involved in a coordinated effect of Ins and Gin upon the expression of
some of these genes In human HepG2 hepatoma cells cultured in
serum-free medium, Ins (0.5 ng/ml) significantly increased the levels of
mRNAs for AMPB (x1.8), Albumin (x2.5) and GAPDH (x3) but did not
change that of AHSG mRNA. In contrast, Gin (10 mM) significantly
increased the level of GAPDH mRNA (x2 5) only. Furthermore, additive
effects of Ins and Gin upon the GAPDH mRNA level could be observed
Cat reporter assay with the GAPDH promoter that harboured the motif
5'-Py-CTTG(A/T)-3' indicated that the Ins- and Gin-induced upregulation of GAPDH mRNA level took place at a transcriptional level.
This result suggested that the motif 5'-Py-CTTG(A/T)-Y might mediate
both Ins and Gin effects upon GAPDH transcription, which will be
confirmed by mutagenesis experiments.
Key words glutamine, GAPDH, transcription

(We/6.3/087)

Immunoselection of new Peroxisomes Proliferators

Responsive Elements from human genomic DNA
P. Collet, L. Domenjoud, M. Dau~a
EA 2402. Prolif3rateurs de Peroxysomes, UHP Nancy L Facuh~ des
Sciences, 54506 Vandoeuvre-les- Nancy Cedex , France.

PPARs (Peroxisomes Proliferators Activated Receptors) are

transcription factors known to be involved in the regulation of lipid
metabolism and adilx>cyte differenciafion [1].
In order to gain some insight about other processes that could be
controlled by these factors and about the involved genes, we purpose to
clone the DNA target sequences of PPARs. Fragments of human genomic
DNA were cut with Sau3A and incubated with human HepG2 cell nuclear
exuract that contains PPARs. The PPRE target sequences can be retrieved,
after binding to PPAR through immunoprecipitation with a polyclonal antiPPAR antibody which recognizes all PPAR subtypes [2]. The selected
sequences were amplified for cloning before characterization. The selection
amplification process has been repeated several times and the selected
fragments have been cloned. More cycles should eliminate the low affinity
bound fragments as PPAR is in limiting amount in the nuclear extract used
for the immunoprecipitation.
Two libraries of fragments have been constructed after one and five
immunoselection-PCR cycles, respectively. The sequences of some clones
have been analyzed. Many clones do not exhibit the classical DR1 site
(corresponding to the consensus sequence TGACCTXTGACCT. But other
configurations like DR0 or widely spaced halfsites have been encountered.
These results should lead to the discovery of new PPAR target genes as well
as to new kinds of PPRE regulatory elements.
1. H.N. S~3RENSEN, E. TREUTER & J.A. GUSTAFSSON - Vitamins and
Hormones, 54, 121-166, 1998.

2. O. BRAISSANT,F. FOUFELLE, C. SCOTTO, M. DAUt~A & W. WAHLI Endocrinology, 137, 354-366, 1996.

s297

(We/6.3/086)

Role of the CCAAT binding protein (NF-Y) in the

transcriptional regulation of the peroxisomal multifunctional
protein-1 (MFP-I).
S. Desaint, M. Cherkaoui-Malki, M C, Clemencet, N, Latruffe

Umversit~ de Bourgogne Dijon, France

Peroxlsomal MFP-1 plays a central role in the IS-oxidation of long

and very long chain fatty acids [1, 2]. Gene encoding rat MFP-1 lacks
typical TATA and CCAAT sequences. Further analysis of 5'-flanking region
in the proximal promoter shows a putatif NF-Y binding site in addition to
adjacent GC-rich boxes. NF-Y or CBF (CCAAT binding Factor) is
ubiquitously expressed as trimeric transcriptional factor, that binds to
inverted CCAAT box : ATTGG in a number of eukaryotic promoters [3].
The purpose of the current studies is to further define the role of NF-Y m the
regulation of peroxisomal MFP-1 gene expression.
Using rat liver nuclear extract and gel mobility shift assay, we show a
specific binding of NF-Y to the ATTGG sequence. For functional analysis,
we carried out luciferase assay using various deletion constructs in
peroximal promoter of the reporter gene. HepG2 cells were transfected with
these reporter genes and cultured in presence or absence of sterols. A
statistically significant decrease in luciferase activity was observed m the
presence of cholesterol. These results strongly suggest that the 120 bp region
between - 121 and + 1 contains the cis-acting element(s) necessary for sterol
mediated transcriptional regulation of peroxisomal MFP-1 gene. This
contrasts with the up regulation mediated by peroxisome proliferators (such
ciprofibrate, an hypolipemic agent) through PPAR/PPRE (Peroxisome
Proliferator Response Element) interactions [4]
[1] Calra et al., FEBS lett., 378, (1996) 57
[2] Calra et al., Biochem.J., 330, (1998) 1361
[3] Mantovani, Nucl. Acid Res., 26, (1998) 1135.
[4] Bardot et al., Biochim. Biophys. Res. Commun., 192, (1993) 37
LBMC. Umversit~ de Bourgogne. Faculty; des Sciences Gabriel. 6 Bd Gabriel. 21000 Dijon,
France

(We/6.3/088)

Combined effects of xenoestrogens and growth factors

on cultured cells.
I Gaumond.J Rousseau.M Graham,M-G Martinoh,L Cossene
Universitedu Quebeca "l'rois-RJvleres.Canada

Many chemicals accumulated in the environment over the years are now
suspected to have estrogenic actwity Several studies using artificial
reporter systems as well as m vtvo assays support the possible actions of
these xenoestrogens on the development and/or function of the
reproductive systems in animals Often times however, the
concentration of such chemicals in the environment does not appear
compatible with any biological action. Since these effects are
particularly observed after m utero or early age exposures, and since
second messengers seem more and more involved m some estrogenic
responses, we decided to test for possible interactions between the
estrogenic signaling pathway used by these xenoestrogens and those of
growth factor stimulation
We therefore investigated the ability of two insecticides suspected to
have estrogenic activity and persistant in the environment (endosulfan
and chlordane) to affect two estrogen-dependant mechanisms: Using
MCF-7 and GH~ cell lines, we respectively examined the growth or
prolactin (PRL) mRNA levels m these cells after exposure to different
doses of endosulfan and chlordane alone or after IGF-I/EGF
stimulation Using metabolic actwity labeling (MTT) or quantitative
RT-PCR, we observed an increase of growth and PRL mRNA
expression after treatment with different doses of endosulfan or
chlordane. We also observed that such increase was amplified with a
treatment with growth factors.
This study demonstrate that the effects of estrogenic contaminants can
be modulated by the growth status of cultured cells, suggesting that
some interactions exist between estrogen and growth factor signaling
pathways. Furthermore, it suggests that cell systems reflecting the
normal interactions between cell signaling pathways in estrogenresponsive cells is an important complement for the evaluation of
estrogenic activity.

s298

Abstracts FEBS'99

(We/6.3/089)

Zur operon in Listeria monocytogenes

Dalet K.a, Gouin EP, Cossart P~', Cenatiempo Y~and

H~chard y.a

Zur operen in Listetia monacyto~enes

Dalet K.a, Gouin EP, Cossart P., Cenatiempo Yaand
Hechard Y.~

(We/6.3/090)

~IBAI1G, CTZR,~k'lences, 4OAr. du recteur Pmeau, 86022 Po~tlers, France

bUntte des mleracttons Bact~rtes-cellules, Instttut Pasteur, Parts, France.

Listeria

mono~ytogenes

is a Gram positive pathogenic bacteria. Iron

~]BAIIG, UFR Sczences. 40 Av du recteur Pineau, 86022 Poltwrs, France

bUntt~ des mteracttons Bactertes-eellules, Instttut Pasteur, Parts, France.

Listeria

monocytogenes

is a Gram positive pathogenic bacteria, iron

contribute to both the growth of this intracellular bacterium and its

contribute to both the growth of this intracellular bacterium and its

virulence. In many organisms, regulation by iron is mediated by Fur : Ferric

virulence. In many organisms, regulation by iron is mediated by Fur. Ferric

Uptake Regulator. This transcriptional regulator with iron as cofactor,

Uptake Regulator. This transcriptional regulator with iron as cofactor,

represses expression of genes involved in the uptake of this metal ion.

represses expression of genes involved in the uptake of this metal ion.

However, little is known about metal uptake regulation in Listeria

However, little is known about metal uptake regulation in Listerm

monocytogenes.

We describe, here, the first operon involved in such

monocytogenes

We describe, here, the first operon involved in such

mechanism The three ORFs encode an ABC transporter, its associated

mechanism. The three ORFs encode an ABC transporter, its associated

hydrophobic membrane protein, certainly involved in zinc uptake, and a Fur

hydrophobic membrane protein, certainly involved in zinc uptake, and a Fur

family protein named Zur In Listeria m o n o c y t o g e n e s , Zur has strong

family protein named Zur. In Listeria raonocytogenes, Zur has strong

homologies with ZurR of Bacillus subtilis which is implicated in zinc

homologies with ZurR of Bacillus subtilis which is implicated in zinc

uptake. Moreover, zinc repressed expression of the z u r operon in Listeria

monocytogenes.

Thus, we could hypothesized that Zur, with zinc as

cofactor, repressed the expression of its own operon.

(We/6.3/091)

Differential control of UCP-I, -2 and -3 gene expression

by sympathetic innervation in rat brown adipose tissue
F. Denjean, J. Lachuer, A. G~lo~n, F. Cohen-Adad, C.
Moulin, H. Barre and C Duchamp
U3/115578 ( "NR,';-~r(~BL- 1,-69622 I "diettrbamw. ["r~mce

BroWn adipose tissue (BAT) activation is a characteristic adaptive

response to cold in small rodents. The mechanisms of BAT
thermogcnesis are well known to be hnked to the BAT specific
expression of a mitochondrial uncoupling protein (UCPI) under the
control of BAT sympathetic mnervation. More recently, other potential
UCPs have been described with a more ubiquttous expression but the
control of their expression is still uncompletely known The respective
control of these UCPs rnay be investigated m BAT since this tissue ts the
only one expressing the three protems.
The control of UCP-I, -2 and -3 mRNA levels by sympathetic mnervation
was investigated m rats reared at thermoneutrality (TN, 25C) or exposed
to cold (CE, 7 days, 4C) by specific and sensitive RT-PCR assays. In TN
rats, unilateral surgical sympathetic denervation of interscapular brown
adipose tissue (BAT) markedly reduced UCP1 mRNA level (-38%) as
compared with the contralateral innervated BAT pad, but was without
significant effect on UCP2 and 3 mRNA levels. Cold-exposure markedly
increased UCP-1 (+180%), UCP-2 (+115%) and UCP-3 (+195%) mRNA
levels in mterscapular BAT. Unilateral sympathetic denervatton
prevented the cold-induced rise in BAT UCP1 and UCP2 mRNAs, but
not that m BAT UCP3 mRNA.
These data indicate a differential endocrine control of UCP1, UCP2 and
UCP3 gene expression in rat BAT both at thermoneutrality and during
prolonged cold-exposure

uptake. Moreover, zinc repressed expression of the zur operon in Listeria

monocytogenes.

Thus, we could hypothesized that Zur, with zinc as

cofactor, repressed the expression of its own operon.

(We/6.3/092) Green Fluorescent Protein gene may be used as a reporter for

estrogen regulation in cell cultures.
P. Dulieu, S. Berthault
Laboratotre de Biochimte et Biologie Mol~culaire, Universitd de Franche
Comte, 16 route de Gray, 25030 Besanon, France

Gene regulation by estrogen is classically studied in vitro by

performing transient transfections with vectors encoding CAT
(chloramphenicol acetyl transferase) or luciferase reporter gene under the
control of an appropriate promoter and enhancer sequence. In order to avoid
cultured cell lysis after stimulation and to study temporal stimulation effect,
we investigated the suitability of gfp (gene encoding the green fluorescent
protein) as a reporter for studying gene regulation by estrogen. MCF 7 cells
were maintained for 48h in DMEM containing 5% Dextran-coated charcoal
stripped fetal calf serum. They were then transfected with a plasmid vector
encoding GFP under the control of HSV thymidine kinase promoter and
two copies of the ERE (Estrogen Response Element) consensus sequence of
the vitellogenin A2 gene [I I. Twenty for hours after transfection of
unstimulated cells, background fluorescence was estimated both by FACS
analysis and video analysis. Cells were then stimulated by adding 17[3estradiol (10-8 M) to the medium. FACS analyses performed 48 h after
estrogen stimulation indicated a 3 fold induction factor for the GFP
fluorescence. Normalization was performed with cells transfected with a
vector encoding GFP under the control of the constitutive CMV promoter.
Experiments were performed in the same conditions with CAT as reporter
system and comparable fold induction was obtanined.
These results are very promissing and show that gfp could also be
used as a reporter system for studying gene regulation. Whereas CAT assay
represents the mean stimulation effect for a cell population, FACS analyses
allow to discriminate between cells which are more sensitive to the
stimulation when compared to the mean value.
[1] Klein-Hitpass et al., Cell, 46, (1986) 1053

Abstracts FEBS'99

(We/6.3/093)

PPARy ligand binding domain (LBD) characterization

G. Ferry, M. Goussard, J. P Nicolas, M. Rodriguez, J Imbert,
J.P. Galizzi, E. Caner, JA. Boutin

s299

(We/6.3/094)

Laboratoire Glaxo Wellcome, 25, avenue du Qudbec. 91951 Les Ulis France

lnstitut de Recherches Servler. 125 chemin de ronde.

78290 Crots~ sur seine, France

PPARy is a member of the nuclear receptor superfamily of transcription

factors. It functions as a master regulator of adipogenesis. PPARy is
highly expressed in adipocytes and mediates the differentiation of several
preadipocyte cell lines. PPARy is a target for obesity related therapeutical
programs. Natural ligands of PPARy comprise prostaglandins such as
PGJ2.
The ligand binding domain (LBD) of PPARy has been expressed in E coli
as a fusion protein with glutathion S-transferase. The protein was then
extracted and purified. An original technique for binding measurement
based on a separation by gel filtration in a 96 well format, has been
assessed to study the affinity constants for reference natural and synthetic
ligands. The data obtained with the present assay compare well with those
reported in the literature. Our screening program being a cell based
reporter gene assay, the present technique is used as a complementary
assay for the discovery of non thiazolidine-dione agonists and antagonists
aiming at obesity.

Smad3 and Smad4 but not Smad2 bind

directly to a TGFI3-responsive element in the human
PAI-I promoter / J.-M. Gauthier, S. Dennler, S. Huet

Smad proteins play a key role in the intracellular signalling of TGF[5. Upon
receptor activation, Smad2 and Smad3 are phosphorylated and form
heteromeric complexes with Smad4. These complexes translocate to the
nucleus where they control expression of target genes. The human
Plasminogen Activator Inhibitor Type-1 (PAl-l) gene is transcriptionnally
regulated by TGF[3. We have identified a Smad3/Smad4 binding sequence,
called the CAGA box, present in three copies in the human PAI-I promoter
and required for TGFl3-induced transcription [1]. This CAGA sequence is
also found in other TGFI3-regulated promoters. Furthermore, this
Smad3/Smad4 binding sequence confers TGFI3 stimulation to a heterologous
promoter. Gel shift experiments indicate that Smad3 and Smad4 binding is
direct. Although it presents an overall 92% identity with Smad3, Smad2 is
functionally different since it does not bind to the CAGA sequence.
Mutagenesis studies have shown that a thirty amino acid domain absent in
Smad3 and corresponding to exon 3 in Smad2 prevents Smad2 from binding
to the Smad3/Smad4 binding sequence [2]. This suggests that Smad2 and
Smad3 may have different subsets of target genes explaining TGFI3
pleiotropic effects. Other transcription factors have been shown to be
regulated by TGFI3. We are currently investigating potential cross-talks
between the transduction pathways involving Smads and some of these
factors.
I- Dennler et al, EMBO J., 17, (1998), 3091
2- Dennler et al, Oncogene, in press

(We/6.3/095) RXR-RAR heterodimer exists in a gradual shifting between

repressive and active states determined by RAIl ligands
P. Germain, P. Balaguer. A-M. Boussioux, J-C. Nicolas
INSERM U439, 70, rue de Navucelles, 34090 Montpellwr France

Retinoic acid receptors (RARs and RXRs) form heterodimers and act as
ligand-inducible transcription factors that regulate genes expression.
Previous studies have shown that both heterodimerization partners can
participate to the transcriptional activity of thc RXR-RAR
heterodimer. In this study, we characterize synthetic retinoids that
exhibit potent RAR antagonism but different profiles of RAR-mediated
transactivation in combination with an RXR-selective agonist.Thus we
show that the effect of synthetic RAR retinoids can be restricted to
heterodimer-mediated effects in three ways : (i) some synthetic
retinoids can act as agonist (ii) some RAR antagonists can activate the
heterodimer in association with an RXR agonist (iiil some RAR
antagonists block the heterodimer whatever the ligand-binding status of
RXR. RAR antagonists able to synergize with an RXR agnnists lead to
an inhibition of the MCF-7 human breast cancer cells prnliferatioo, in
combination with an RXR agonist as efficient as an RAR agonist alone,
demonstrating a good correlation between transcriptional results and
proliferation data. Study interaction in the RXR-RAR heterodimer
context show that co-regulators are implicated in the different profiles
of transcriptional effect of antagonists. Moreover proteolysis digestion
of RAR in presence of synthetic retinoids indicates that different
conformations are induced in RAR bF antagonists. The finding that
different RAR-antagonists are able more or less to synergize with RXRselective agonist in the RXR-RAR hctcrodimer context supports the
idea that the RXR-RAR heterodimer exists in a gradual shifting between
repressive and active states which is determined by the RAR ligand.
Such compounds can be used for increasing the understanding of the
mechanism of transcriptional activation and the respective contributions
of the different retinoic receptors in governing the biological effects of
the natural pan-agonist acid retinoic.

(We/6.3/096)

Activation of NF-r~B Is Regulated in the Developing

Mammary Gland and Inhibits STAT5-Signaling
S. Geymayer and W. Doppler
lnst for Med Chemistry & Biochemistry, Unlverstity of lnnsbruclg A ustria

The NF4cB family of transcription factors regulates diverse cellular

functions, such as immune response, cell growth and development, and
has been reported to be constitutively active in a variety of mammary
carcinoma cell lines. However, its role in normal mammary gland
development has not yet been addressed. In our study, we detected
developmentally-regulated NF-KB activity in the mammary gland of
mice. During pregnancy, the DNA binding activity of NF-IcB p50/p65
increased until day 14 post coitum, and decreased with the onset of
lactation due to inhibited translocation of p50/p65 complexes into the
nucleus. Experiments performed with the mammary epithelial cell line
HCI 1 revealed the milk protein gene L3---casein as a potential NF-rB
target, which is inhibited by p50/p65 heterodimers: Expression of both
the endogenous 13-casein gene and a transfected 13-casein gene promoter
reporter construct inversely correlated to NF-KB p50/p65 activity.
Furthermore, an NF-KB binding site in the hormone response region of
the 13-casein gene promoter was identified. Cotransfection experiments
employing 293 cells demonstrated that NF-rB p50/p65 inhibited
13-casein gene promoter activity by interfering with the prolactin receptor
/ JAK2 / STAT5 pathway. Our results provide evidence for a role of
NF-v,B in normal mammary gland development, and indicate its function
as a negative regulator of 13-casein gene expression during pregnancy.
Supported by the Austrian Science Fund (F209)

s300

(We/6.3/097)

Abstracts FEBS'99

PPAR13 signaling pathway is important for neuronal

differentiation of ES cells.
I.Grillier, P.Escher, T. Lemberger, W Wahli, B. Desvergne.
Insntut de BiologwAmmale. UniversitOde Lausanne, CH1015Lausanne,
Sulsse.

The Nuclear Receptors PPARs ot and y are involved in energy homeostasis

by modulating the expression of key enzymes for lipid metabolism. So far no
clear function has been found for the third member of this family PPAR13. In
contrast to ~ and y, PPAR13 displays an ubiquitous tissue distribution. During
rat embryogenesis, PPAR13 mRNA appears early (day 8.5) and before
PPARs oc and y (day 13.5). It peaks during neuronal differentiation especially
in the forebrain and stays high throughout embryogenesis, during which its
expression level is higher than in the adult tissue. To examine the role of
PPAR[5 in the early stages of the central nervous system (CNS)
differentiation we have differentiated totipotent mouse embryonic stem cells
(ES) into neuronal ceils. This system is very close to the mouse embryonic
CNS development. After 10 days of treatment, 90% of the cells were
expressing the neurofilament NF160, specific of the fully differentiated cells.
Semi-quantitative RT-PCR reactions showed that the mRNAs of calbindin
D28, NMDR and GAD, which are neuronal specific markers, are detectable
as soon as day 7 and 8. The mRNA expression level of PPARct is low and
does not present significant variations during ES cells neuronal
differentiation; the mRNA ofPPAR yis not detectable. In contrast, mRNA of
PPAR[3 is already strongly expressed in the ES cells. It peaks during the early
phases of differentiation and slowly decreases during neuronal differentiation.
To analyze the effect of an inhibition or a forced expression of PPAR~3 on
neuronal differentiation of ES cells, we infected ES cells with retroviral
vectors containing PPAR13 cDNA in the antisense or sense orientation. At an
early stage (day 6), only 3% of the ES cells infected by the PPAR]3 antisense
vector display a neurite length over 300,t,tm, while 70% of the neurites of the
cells infected by PPAR[3 sense vector and 25% of the neurites of the non
infected cells or empty vector infected cells had already reached this length.
These differences diminished in later stages of differentiation. We are now
using specific ligands of PPAR [3 to complete our study.

(We/6.3/099)

Amino acid limitation induces chop through a specific

pathway independent on the unfolded protein response
C.Jousse", A.Bruhat ~, H.Harding b, D.Ron b, P.Fafournoux ~.
"UNCM, INRA de Theix, 63122 St Gen~s Champanelle, France
~' Skirball Institute, NYU Medical Center, New York, USA.

All cells regulate gene expression in response to changes in the

external environment such as nutrient availability. In mammals, plasma
concentrations of amino acids are markedly affected by dietary or pathological
conditions, with levels falling several-fold in cases of malnutrition. The
current understanding of molecular mechanisms involved in amino acid
dependent control of mammalian gene expression is limited. Our ignorance
extends both to the identity of the genes implicated in the response to amino
acid deprivation and to the pathways that mediate it.
One of the genes strongly induced by an amino acid limitation is the
chop gene (C/EBP-Homologous Protein). Chop encodes a transcription factor
that regulates certain aspects of the response of cells to stress. Recent work
has implicated a signaling pathway responsive to accumulation of ma]folded
proteins in the endoplasmic reticulum, the so called unfolded protein response
(UPR), in activating chop expression in response to many stimuli. This
response is associated with the transcriptional activation of the genes encoding
ER chaperones such as the immunoglobulin binding protein BiP (GRP78).
Indeed chop and BiP are coordinately regulated during the response to
different stress inducers. However, the possible role of the UPR in mediating
the effects of amino acid limitation has not been addressed to date.
To determine if the UPR is also implicated in chop induction in
response to amino acid deprivation, we compared the induction of chop and
the well-characterized UPR marker BiP in response to amino acid limitation,
chop expression is strongly induced when cells were placed in media lacking
the essential amino acid leucine, whereas BiP mRNA levels remained
unchanged. Uncoupling of the response to amino acid limitation from the
UPR is also supported by observations made on a cell-line with a temperature
sensitive mutation in leucyl tRNA synthetase: shift to the non-permissive
temperature induces chop whereas no change in BiP mRNA is observed.
Moreover, we show that amino acid starvation and ER stress regulate the
chop promoter activity using distinct cis elements. We conclude that the
induction of chop by amino acid deprivation proceeds by a pathway that is
independent of the UPR.

(We/6.3/098)

GLUT2 mediates a glucose-responsive signal in hepatic

cells
G. Guillemain, M. J. Munoz-Alonso, M. Loizeau, J. Girard, A.
F. Burnol and A. Leturque
CNRS UPR 1524 9 rue Jules Hetzel Meudon France

How eukaryotlc cells sense and respond to glucose still remains to be

solved. However, recent works suggest that the glucose transporter GLUT2
mtght be lmphcated not only in glucose transport, but also in the transmtsslon
of glucose signals by metabolic pathways or protein-protein interactions
To test this hypothesis, we have overexpressed in hepatoma cell line
mhAT3F, two intracytoplasm~c domains of GLUT2, the third loop, between
transmembrane domains 6 and 7, and the C-terminus domain, tagged with a
green fluorescent protein (GFP) The overexpresslon of both domains have no
effect on [14 q-glucose mcorporanon into glycogen However, the
overexpresslon of the mtracytoplasmm loop of GLUT2 led to the loss of the
effect of glucose on the transcnptlon of two hepanc genes, GLUT2 and Lpyruvate klnase Moreover, the GFP-GLUT2 loop fusion protem was
specifically locahzed into the nucleus On the contrary, the GFP-GLUT2-Cterminus was locahzed m the cytoplasm, and Its overexpresston had no effect
on glycogenesls or on the glucose transcrtptton effect on gene This suggests
that a cytoplasmic factor brads to the GLUT2-1oop, and pamc~pates to the
import into the nucleus
Furthermore, ~t has been shown, using the two-hybrid system, that the
loop of GLUT2 was able to interact ;wth lmportm ~2, a protein revolved m
nuclear import of cytoplasmic factors. This mteracnon was highly specific,
since the mtracellular loop of GLUTI and GLUT4, and the C-termmus of
GLUT1, GLUT2 and GLUT4 were not able to brad to the importm or. The
GLUT2-1mportm ~. interaction was also demonstrated in vwo using crosshnking experiments in hepatocytes
In conclusmn, the overexpressmn of the GLUT2-1oop created dominant
negative mutant cells for the transcnpnonnal effect of glucose, suggesting that
the GLUT2-1oop ~s involved m glucose signalling The loop of endogenous
GLUT2 anchored the lmportin et We propose that a stgnal releases the
importer that translocates factors mediating the glucose signaling pathway
This pathway starting from GLUT2, would conduct to the snmulanon of
some hepatic genes transcription

(We/6.3/100)

Repression of estradiol-1713 transcriptional effect in

cultured endometrial cells. M. Jouvenot, I. Lascombe, J.F.
Musard, B. Cypriani, P. Adami. Laboratoire de Biochimie-Biologw
Moldculaire. UFR S.T.. 240 Route de Dole, 25030 Besan#on cedex. France

Estradiol-1713 (E2) can induce c-fos gene expression in breast cancer cell
lines and in the uterus in vivo, but not in cultured guinea-pig endometrial
ceils [ 1]. On the basis of our previous results in this culture model, we have
postulated that a labile repressor could prevent the E2 transcriptional effect on
the guinea-pig c-fos gene [21. The aim of the present study was to investigate
this hypothesis. We thought to determine whether E2 effect is absent on
transfected human c-fos recombinants which are known to be E2-responsive
in cancerous Hela cells. Three h-fos-CAT plasmids were used: pFC1-BL,
pFC2-BL and pFC2E. These plasmids contain different human c-fos gene
regulatory sequences and the human c-fos gene promoter inserted upstream
of the CAT gene: pFCI -BL contains c-fos sequences ranging from -2250 to
+41, pFC2-BL the sequences ranging from -1400 to +41 and pFC2E the
sequences from -1300 to -1050 and from -230 to +41. All these
recombinants bear the c-fos estrogen response element (ERE, -1200).
Stromal (SC) and glandular epithelial cells (GEC) were transfected with a hfos-CAT plasnlid and a control [3-galactosidase plasmid 13] and then,
incubated in chemically defined medium with or without the appropriate
stimulus (I0 -8 M E2, 100 ng/ml EGF, I0 ~g/ml insulin, 10 -7 M ICI
182,780, alone or in association). In both cellular types, pFCI-BL was not
induced by E2, even in the presence of growth factors. The lack of E2 effect
was not due to unfunctional endogenous estrogen receptors since an EREwttk-CAT plasmid, containing the ERE from vitellogenin A2 gene, was induced
by E2 in both cell types. The pattern of pFC2-BL and pFC2E expression
was strikingly different and depended on the cellular type: pFC2-BL and
pFC2E induction was restricted to GEC and did not occur in the SC. We
argue for a repression of E2 action which is dependent on the ERE
environment and also cell type-specific involving DNA/protein interactions
with cellular type-specific factors.
I I I M. Jouvenot et al., Mol. Cell. Endocrinol., 72, (1990) 149
I2I I. Pellerin et al., Endocrinology, 131, (1992) 1094
13I I. Lascombe et al., Biotechniques, 20, (1995) 88

Abstracts FEBS' 99

(We/6.3/4101)

Ligand complexes of the nuclear retinoic acid receptor

hRART at 1.4 A resolution provide the molecular basis
for ligand selectivity.
B. P. Klaholz, A. Mitschler and D. Moras

s301

(We/6.3/102)

Molecular characterization of the 5'-flanking region

of the human KE4 gene.
I. Lana~, A. Vergara a, C. de Miguelb and I. Encioa

'Dpto Ciencmsde la Salud, UniversidadP~blica de Navarra, 31008 Pamplona, Spare

bDpte de Btoquirnica y BiologfaMolecular, Universidadde Navana, 310S0 Pamplona,Spam

lnstitut de G f n e t l q u e et de Biologic Moltculaire et Cellulaire, Biologic Structurale,

1, rue Laurent Fries BP i63, F-67404 lllkirch Cedex, France

We present the crystal structures of several ligand-complexes of the human

retinoic acid receptor hRAR% As a member of the nuclear receptor
superfamily, RAR plays an important role in the ligand dependent
regulation of transcription. Retinoids are used in the treatments of chronic
myelogenous leukemia and skin diseases including psoriasis, acne and skin
cancer. To adress the question of ligand selectivity which is important in the
view of the side-effects of the natural Iigands, we have cocrystallized the
complexes of the hRART ligand-binding domain with several synthetic
ligands. The choosen retinoids are either agonists for all RARo,13,ysubtypes or possess PART-selectivity. The structural comparison of these
complexes provides evidence that hydrogen bonds trigger the ligand
selectivity. Indeed, the hydroxyl moiety of RAR'~-selective ligands form a
hydrogen bond to the sulfur of the RAR~/-specific Met272, which
corresponds to an isoleucine in RAR0t and 13. In contrast, natural or
synthetic ligands devoid of a hydrogen bond donor show no RART-

The HKE4 gene lies on the centromeric side of the HLA class 1I
region, on the short arm of chromosome 6 at 6p21 3, and encodes a
transmembrane protein with two histidine-rich charge clusters. In the
mouse, it has been shown that the KE4 and retinoid X receptor beta
genes are tightly linked, only separated by a CpG-rich island that
works as a bi-direetional promoter. Here we describe the molecular
cloning and characterization of the HKE4 Y-flanking region. As in the
mouse, the region has a high GC content, lacks obvious TATA box,
and contains numerous putative binding sites for the transcription
factor Spl The CAP site was mapped in primer extension
experiments, and several transcription initiation nucleotides were
identified. To determine whether this region has functional promoter
activity luciferase reporter plasmids, constructed with restriction
fragments and deletion fragments, were transiently transfected into
HeLa and CV-1 cells. Transcription activity was observed for
constructs carrying either of the two possible orientations. Deletion of
a single Spl binding site caused considerable loss of transcription
activity in both directions, suggesting that this site is important for
promoter activity.

selectivity. Our high resolution data provide structural details of the ligand
binding pocket, which are important for the reliability of structure based
drug design of retinoids, hopefully less prone to side-effects.
B. P. Klaholz, J.-P. Renaud, A. Mitschler, C. Zusi, P. Chambon, H.
Gronemeyer, D. Moras; Conformational adaptation of agonists to the human
nuclear receptor RART. Nat. Struct. Biol. 1998, 5, 199-202.

(We/6.3/103) Functional study of a hexose transporter gene involved in

grape ripening. M. Leterrier, R. Atanassova, C Galliard, L.
Laquitaine, L. Fillion, P. Coutos-Thevenot & S. Delrot
ESA CNRS 6161, Laboratoire de Phystologie et Btochimle Vdgdtales,
Universitd de Poitters, BdttiraentBotamque 40 we. du Recteur Pmeau, 86022
Poitiers cedex, France

The ripening of grape berry involves a strong sugar accumulation

mediated by hexoses transporters. Screening of a genomic library of Vitis
vimfera resulted in a complete clone, Vvhtl, containing the genomic
sequence corresponding to the cDNA ofa hexose transporter and 2.5 kb of
its promoter region.
In order to study the regulation ofgene expression conferred by the
Vvhtl promoter, we cloned the promoter sequence and the produced 5'
deleted promoter fragments in front of the GUS and GFP reporter genes as
transcriptional fusions, were introduced into Tobacco cells culture was
stably transformed with these chimeric constructs. Biolistic transient
transformation of grape berries at different stages of ripening are achieved
for rapid check of maturation stage-dependent expression conducted by the
Vvhtl promoters. Analysis of Vvhtl promoter driven expression during
the development and berry maturation in stably transformed grapes
(already produced) does complete this study
Histochemical GUS staining and GUS fluorimetric test revealed a
different relative activity between truncated promoters studied in
transgenic Tobacco cells. GFP reporter gene expression are visualised by
confoeal microscopy. Different effectors, as sugars and phytohormones,
are used to functionally characterise Vvhtl promoter cis-acting elements,
responsible for transcription inducibility in Tobacco cell culture.
In addition, the study of Vvthl gene physiological role is underway
by sense and antisense strategies in Tobacco and Grapevine cells and also
in plants of both species.

(We/6.3/104)

Adrenergic stimulation affects gene expression of PPARy2

in brown adipocytes
E. Lindgren, J. Nedergaard and B. Cannon
The Wenner-Gren Institute, The Arrhenius lxzboratorles F3, Stockholm
Umversity, S-106 91 Stockholm, Sweden

Peroxisome Proliferator Activated Receptor y2 (PPAR',(2) is a nuclear, liganddependent transcription factor responding to thiazolidinediones (antidiabetic
drugs) and the natural ligand 15-deoxy-A ~2'~4prostaglandin Jv PPAR',/2 is
predominantly expressed in adipose tissue, both white and brown. PPARy'2
together with C/EBPcc is important for the initiation of differentiation of
white adipocytes [1]. When mice are exposed to cold, brown adipose tissue
becomes activated and starts to generate heat. It is the unique and tissuespecific protein UCP1 that is responsible for this mechanism. This activation
is mediated by the endogenous neurotransmitter norepinephrine which is
released upon cold exposure. Both cold exposure and norepinephrine
treatment induce an increase m UCP1 mRNA levels and elevate UCP1
protein content. Treatment with the PPARy2-1igand pioglitazone induces an
increase of brown adipose tissue mass, mRNA levels of UCP1 and UCPI
protein content m mice [2]. Sears et al. [3] showed that PPAR)'2 binds to the
UCP1 enhancer, from which it can activate transcription. PPAR'(2 has been
suggested as the master controller of several fat specific genes and of
adipocyte differentation. However, we show in this study that mice exposed
to cold show no changes m mRNA levels of PPARy2. In primary cultures of
brown adipocytes PPARy2 is spontaneously expressed and the differentiation
process only marginally alters the mRNA level. Norepinephrine treatment of
primary cultures of brown adipocytes induced a dose-dependent decrease in
the PPARy2 mRNA level. The ECs0 value was calculated to 0.1 ~M and
maximal decreasing effect was reached with I p,M norepinephrme. The
influence of norepinephrine in decreasing PPARy2 mRNA levels is observed
after 1 hour and reaches its maximal at 2-4 hours (40% of control). The
decrease in PPART2 mRNA levels is transient and the level is back to control
levels after 21 hours. The effect of norepinephrine on coactivators of PPARy'2
has also been investigated. Based on these investigations the significance of
PPARy2 for norepinephrine dlfferentation is discussed.
References: [1]: Tontonoz et al., Cell, 79, (1994) 1147, [2]: Foellml-Adams
et al., Biochem, 52, (1996) 693.,[3}: Sears et al,. Mol Chem Biol, 16, (1996)
~410

s302

(We/6.3/105)

Abstracts FEBS'99

Insulin Regulation of Hepatic

Glucose-6-Phosphatase Gene Transcription
P. A. Lochhead, K. S. Walker and C. Sutherland

(We/6.3/106)

MRC Protein Phosphoo'lation Unit, Universtty of' Dundee. Scotland.

Glucose-6-phosphatase (G6Pase) catalyses the final steps of

gluconeogenesis and glycogenolysis thus is a key enzyme in controlling
blood glucose levels. Overexpression of the catalytic subunit of G6Pase in
hepatocytes produces a metabolic profile resembling that of liver cells from
patients with non-insulin dependent diabetes mellitus (NIDDM) [1],
therefore dysregulation of the G6Pase catalytic subunit could contribute to
the aetiology of the disease. G6Pase is controlled in part at the level of
gene transcription. In the fasting and diabetic model there is an increase in
G6Pase nuRNA, while in the fed state there is a decrease. In hepatoma cell
lines the addition of cAMP and/or glucocorticoids increases G6Pase gene
transcription. The addition of insulin completely inhibits both basal and
cAMP and glucocorticoid induced transcription, therefore insulin has a
dominant negative effect. These actions of insulin are phosphatidylinositol
3-kinase (Pl3-kinase) dependent and do not require p70 $6 kinase or the
MAP kinase pathway [2], this is identical to the regulation of PEPCK by
insulin [3]. We have investigated the potential role of molecules
downstream of Pl3-kinase in the regulation of G6Pase gene transcription
by insulin.
[1] Sconac, J. et al., J. Biol. Chem., 272, (1997) 26972.
[2] Dickens, M. et al., J. Biol. Chem., 273, (1998)20144.
[3] Sutherland, C. et al., J. Biol. Chem., 270, (1995) 15501.

(We/6.3/107)

Transcriptional regulation of stromelysin-3 gene

expression by retinoic acid.
M.G. Ludwig, P. Basset. P. Anglard
lnstitut de G~;n~tique et de Blologie Mol~;culatre et Celhtlalre,
CNRS/INSERM/ULP, BP163, 67404 Illktrch cedex. France

Matrix metalloproteinases constitute a multigene family of homologous

proteolytic enzymes that are capable of degrading virtually all components
of the extraeellular matrix and basement membranes, mid that are implicated
in a variety of physiological and pathological tissue modelling processes. In
this family, stromelysin-3 (ST3) expression has been associated with the
progression of most human invasive carcinomas and its contribution to
tumor progression is based on both clinical and experimental evidence. In
the present study, we have characterized the murine ST3 gene structure
together with its promoter. The transcription initiation site was mapped
by primer extension and comparison of the first 150 bp of the proximal
promoter sequence to its human counterpart revealed 80 % identity. Three
retinoic acid responsive elements (RAREs) including one of the DRI type
and two of the DR2 type were found at position -367, -2141 and -2340.
respectively. Although these RAREs differ from the consensus sequence
by one bp, gel shift experiments showed that RAR-RXR heterodimers
bind efficiently to both DR2-RAREs: a weaker binding was observed for
the DRI-RARE. In transient transfection assays, these elements, either
isolated or in the context of the ST3 promoter, were found to mediate
transactivation by RARs and RXRs in the presence of their ligand.
Consistent with these data. 9-cis retinoic acid increases ST3 mRNA levels
in F9 cells. As ST3 expression is also detected during mouse development,
the absence of its expression in specific sites of RARc~-RXRy knock-out
mice indicates the physiological relevance of its regulation by RA.

TGF~ activates the NF~zB pathway

T. L6pez-Rovira. E. Chalaux, J.L. Rosa, R.Bartrons.
F. Ventura
Umtat de Bioquimwa, Campus Bellvitge, Umversttat de Barcelona, Feixa
Llarga s/n, 08907 Hospltalel de Llobreg:at. Spain.

Transtbrrning growth factor 13 (TGF[3) is the prototypic member of a

large family of signalling molecules that have critical roles on cell
proliferation, differentiation and migration. TGF~ signalling from the
plasma membrane to the nucleus is mediated by the Smad family of
intermediate effector proteins. Smad proteins are phosphorylated by
activated receptors and translocate into the nucleus where they interact
with the transcriptional machinery acting as coactivators as well as
transcription factors.
We report that TGF]3, but not BMP2, is able to induce activation of a
NF~B-driven reporter construct, synergizing with other activating
stimuli. Time-course assays show that this activation proceeds as an
early-response effect. TGF~ induction requires an intact NF~cB pathway
since inhibition of I~:B degradation or expression of dominant negative
l~Bt~ blocks the TGF[3 effects. Smad 3 and Smad 4 overexpression
thrther increased reporter construct activity, supporting the notion that
transcriptional activation of the reporter requires Smad activity.
Altogether. these data support the idea that transcriptional regulation
by TGF[3 involves cross-talk between distinct transcription factors
tamilies.

(We/6.3/108)

Different induction of gulonolactone oxidase in aromatic

hydrocarbon responsive or unresponsive mouse strains
J6zse~MandL TamfisKardon, Mild6s Csala, C~bor Bknhegyl
and Lkszl6 Braun

Department of Medical Chemistry, SemmelweisUniversityof Medicine, P O.

Box 260, 1444 Budapest, Hungary
The role of aromatic hydrocarbon receptor (AhR) mediated signal
transduction pathway was investigated in the regulation of ascorbate
synthesm by using Ah-responsive and Ah-unresponsive mouse strains treated
m w v o by 3-methylcholanthrene It is demonstrated that gulonolactone
oxidase, the final enzyme of ascorbate synthesis, is induced by 3methylcholanthrene. To evaluate the possible participation of redox
mechanisms in the response, the effect of the glutathione depleting
buthionme sulfoximine was also studied. Both treatments increased hepatic
and plasma ascorbate concentrations In the Ah-responsive strain, while in the
unresponsive strata only buthionine sulfoximine treatment was effective.
Gulonolactone oxidase mRNA level in the liver and the microsomal
ascorbate production from p-nitrophenyl glucuronide, D-glucuronic acid or
gulonolactone were originally elevated m A/a-responsive mice which were
further increased upon m vivo addition of 3-methylcholanthrene. In Ahunresponsive mice the effects of 3-methylcholanthrene treatment were
absent. Administration of buthionine sulfoximine was ineffective with respect
to hepatic gu[onolactone oxidase mRNA level and ascorbate production from
p-nitrophenyl glucuronide, D-glucuromc acid or gulonolactone in liver
microsomes in both strains. These results suggest that the transcription of
gulonolactone oxldase is regulated by AhR mediated signal transduction
pathway, thus the gene encoding gulonolactone oxidase may be an additional
member &the Ah-battery in mice.

Abstracts FEBS'99

s303

(We/6.3/109) P F K F B 4 : the gene coding f o r h u m a n testis 6-pho~phofructo2-kinase/fructose-2,6-bisphosphatase.

A. Manzano, A. Navarro-Sabatd, L. Riera, A.J. Lange*and R. Bartrons

B~ochemist~ Untt, Dept Ctellctes ptalologlques II, Cimlpu~ de Belhttge ~'mve*~ttltt ~A,
Barcelona C, Fet.ra Llarga s tl E-08907. Bo*celo~la. SP Ilk

(We/6.3/l10) A n g i o t e n s i n o g e n
deficient
mice
exhibit
alterations in adipose tissue development
F. Massiera l, K. Murakami 2, A. Fukamizu 2, R.
Negrel 1, G. Ailhaud 1 and M. Teboul 1'
1 UMR CNRS 6543, Nice,France 2 Unlversa3' of Tsukuba, Japan

Synthesis of the potent vasoactive peptide angiotensin II (ANG II)

6-Phosphofructo-2-kinase/frt.~ctosc-2,6-bisphosphatasc (PFK2/FBPase-2) is a bifunctional enzyme that catalyzes the synthesis and
degradation of t?uctose-2,6-bisphosphatc, a key regulator of
glycolysis. Several genes have been described in mammals coding
for different PFK-2/FBPase-2 isol'orms that differ in tissue
distribution and enzymatic properties.
The complete human cDNA of testis isozyme has an open reading
frame of 1,407 nucleotides, coding for a plotcin of 469 mnino acids.
The functionality of this protein of 54 kDa has been tested m COS-1
cells, driving the expression of an active isozvmc. The gene
codifying for the testis isozyme (PFKFB4) has been localized by
fluorescence in situ hybridization (FISI I) in thc short arm of human
clu'omosome 3 at p21 -p23.
Northern blot analysis shows one band of 2.4 kb that is testis
specific and modulated during dcvelopmcot. Another band present
also in testis of 4.8 kb corresponds to the isolbrm of PFK-2/l-l:~l'ase2 ubiquitously expressed. 'fhc results obtained by ,7 .situ
hybridization show the specific localization of the testis isoform in
germinal cells.
A hunlan genomJc clone, containing the scqt.lCl]CCS ol' the 5"
flanking region and the first intrml, has been isolated and sequenced.
Putative binding motifs for different regulatory factors are localized.
A detailed molecular study of the binding sequences can help to
elucidate the role of this isoform in testis naetabolism.

has recently been described in adipose cells. ANG II is known to

stimulate the production and release of prostacyclin which, in turn acts
as a potent adipogenic signal in promoting the terminal differentiation of
preadipocytes to adipocytes. Adipose tissue has been shown to be one
major source of angiotensinogen (AGT), the substrate from which
ANG II is formed.
In order to get insight in the role of AGT in adipose tissue
development in vivo, we compared white and brown adipose tissues of
wild-type (WT) and AGT deficient mace (agt -/-).
Interestingly, agt -/- mice have a decreased weight and smaller
epididymal fat pads than WT mace. Cellularity studies show that in agt
-/- mice i)both white and brown adipose cells exhibit hypertrophy and
ii) a higher fat cell lipid content is accompanied by a reduced number of
fat cells in epldidymal fat pad. The observed hypoplasia paralled by a
compensator adipocyte hypertrophy might result from a blockade in the
recruitment of preadipocytes. Analysis of adipose cell markers in
epidldymal fat pad indicate that the expression of leptin RNA is higher
in agt -/- mice whereas the expression of PPAR ~ is unaffected.
Altogether these results demonstrate the crucial role of AGT in adipose
tissue development in vivo.

*B,ochcmistry Department, Me&cal School UMN. 4-225 M,Illard ]lall,

435 Delaware Street SE, Minneapolis, MN-55455. USA

(We/6.3/lll)

Glutamine and regulation of the expression of the z2M

gene in rat hepatocytes
D. Meisse a, I. Dusanter-Fourtb, A. Hussona, A. Lavoinnea
aG B.P.D.N . IFRMP n23, Facult~ de M~decine, 76183 Rouen, France,
b l N S E R M U-363, 1CGM. H6pital Cochin, 75014 Parts, France

Glutamine has been shown to up-regulate the expression of the ~2macroglobulin (oc2M) gene, at least in part, through the glutamineinduced cell swelling [1]. Since cell swelling per se stimulated the ot2M
gene transcription [2], the aim of this work was to test the possibility
that the transcription factors STAT might be involved in the effect of
cell swelling in cultured rat hepatocytes.
Cell swelling induced by hypoosmolarity (-50 mM NaCI) activated
STAT3/3- and STAT3/1 dimers but not STAT1/I homodimers.
Maximal activation was observed after 60 min of culture (x6, STAT3/3 ;
x3, STAT3/3 , n = 3). Similar results were obtained by culturing the
cells in the presence of 100 U/ml interleukin-6, the major known inducer
of o~2M gene. This strongly suggested that hypoosmolarity induced the
expression of the o~2M gene through the activation of STAT proteins.
The effect observed in hypoosmotic condition was due to cell swelling
since (i) raffinose addition totally blocked the activating effect of
hypoosmolarity on STAT proteins and (ii) the culture of cells in the
presence of 10 mM 2-aminoisobutyric acid also activated STAT3/3- and
STAT3/1 dimers. By contrast, we were unable to detect any activation
of STAT4-, STAT5- and STAT6 proteins. Thus, cell swelling
specifically activated STAT1 and STAT3 proteins as homodimer 3/3 and
heterodimer 1/3 in primary rat hepatocytes.
[1] A. Lavoinne et al. Biochimie 80, (1998) 807.
[2] D. Meisse et al. FEBS Lett. 422, (1998) 346.

(We/6.3/112)

Expression of the haptoglobin gene in rat liver under

acute- phase conditiones involves participation of
CEBPalpha,-beta and -delta
T. Milosavljevic, I. Grigorov, I. Cvetkovic, M. Petrovic
Institute for biological research Sinisa Stankovic~ Belgrade, Yugoslavta

Turpentine-induced acute-phase (AP) response in rats is followed by an

increase in the synthesis of haptoglobin (Hp) in liver. Analysis of the rat Hp
gene promoter sequence implicated an involvement of some members of the
liver-enriched CCAAT/enhancer-binding protein (C/EBP) family in the
regulation of this gene expression. This study was aimed at elucidating whether
an interaction of C/EBPs with the Hp gene promoter elements corresponding to
the C/EBP binding site correlates with induced expression of this gene. By using
a combination of Western blot and DNA-binding assay, we found that
C/EBPalpha was a predominant C/EBP isoform in the nuclear extract (NE) of
untreated rats whereas C/EBPbeta and C/EBPdelta became major isoforms after
turpentine treatment. High pool level in the control NE and DNA-binding activity
of four C/EBPalphas with molecular masses 42, 30, 20 and 14 kD decreased
significantly under AP conditiones. Concomitant with this decrease, the binding
activity and pool level of 35-, 32- and 20-kD C/EBPbetas and 32- and 27-kD
C/EBPdeltas increased in response to the turpentine treatment. Our results
suggest that induction of C/EBPbetas and C./EBPdeltas as well as repression of
C/EBPalphas binding activities and/or pool levels may be regarded as an integral
part of the AP-related induction of the rat Hp gene expression.

S304

(We/6.3/113)

Abstracts FEBS'99

CONCERTED EXPRESSION OF BK VIRUS LARGE TAND SMALL T-ANTIGENS STRONGLY ENHANCES

ESTROGEN RECEPTOR-MEDIATED TRANSCRIPTION.
U. Moel!s.M Van Ghelue, B. Johansen, and O. M. Seternes Instituteof
Medical Biology,Universityot Tromso,N-9037TromsO,Norway.

Previous studies have shown that the human polyomavirus BK genome

contains an oestrogen response element (ERE). This isolated element binds its
cognate receptor in vitro and can mediate 17~-oestradiol-induced gene
expression when linked to an heterologous promoter. The roles of the EREand the AP-I binding sites in oestrogen receptor-directed transcription from the
complete BK virus promoter/enhancer (Dunlop strain) have been examined and
the effects of the general co-activator CBP and large T- and small t-antigens on
estrogen receptor-mediated transcription have been investigated. A constitutive
activated oestrogen receptor stimulated BKV promoter activity in HeLa cells.
Mutations in either the ERE or the AP-I binding sites did not impaired
oestrogen receptor-induced activation of the BKV Dunlop promoter, while
mutations in both binding motifs almost completely abolished oestrogen
receptor-induced transcription. Simultaneous expression of large T- and small
t-antigens strongly activated oestrogen receptor-mediated transcription. When
expressed separately, only large T-antigen moderately stimulated oestrogen
receptor-mediated transcription. The stimulatory effect of large T-antigen on
the activity of the oestrogen receptor is probably indirect because no physical
interaction between the two proteins was detected in a two-hybrid assay. Large
T-antigen abrogated the synergistic effect on transcription between this nuclear
receptor and the general co-activator CBP. The findings that the BKV early
proteins amplify oestrogen receptor-mediated transcription may have important
biological implications in individuals with raised oestrogen concentrations.

(We/6.3/115) Regulation of the human ubiquitous 6-phosphofructo-2kinase/fructose-2,6-biphosphatase

L. Riera, A. Navarro-Sabate, A. Manzano and R. Bartrons
Umtat Bioqulmwa. Departament Obnc~es FtszolOg~quesIL Campus Bellv~tge
Universitat Barcelona.08907 L "Hosp~taletde Llobregat, Barcelona, SPAI:\~

6-phosphofructo-2-kinase/fructose-2,6-biphosphat ase (PFK2/FBPase-2) catalyzes the synthesis and the breakdown of fructose
2,6-bisphosphate The bifunctional enzyme is regulated by different
effectors, covalent modification as well as by the control of its
expression. Several isozymes and different mRNAs coding for the
PFK-2/FBPase-2 have been described in humans Complete cDNA
human sequences are available for heart, liver, muscle, testis and
brain lsoforms.
We have recently reported the complete human cDNA
sequence for the PFK-2/FBPase-2 brain isoform The results obtained
by fluorescence in situ hybridization (FISH) showed that this gene
maps in the short arm of human chromosome l 0, at p 14-p 15, which is
found to be different from the other genes localized until now. On the
other hand, Northern blot analysis showed the expression of this
isoenzyme in the different human tissues studied
Furthermore, the expression of this ubiquitous PFK-2/FBPase2 has been analyzed by Northern blotting in different proliferative cell
lines (HT29, MCF7, HeLa, NP29 and NP9). The results obtained
show that all these cell lines express ubiquitous PFK-2/FBPase-2
mRNA The incubation of HT29 cells in the presence of lpM insulin
or I00 ng/ml of PDB (Phorbol 12,13-Dibutyrate) increase the levels
of the mRNA of this isoform and also Fru-2,6P2 concentration. These
changes are followed by increases in lactate production.
Ubiquitous PFK-2/FBPase-2 isozyme contains a significantly
high kinase/phosphatase activity ratio which points out an important
role of this isoform in the control of glycolysis of the proliferative
cells The characterization of the promoter region of this isozyme as
well as the study of the factors involved in its regulation may
contribute to elucidate the role of this enzyme in proliferative tissues.

(We/6.3/114)

Endocrine control of glucagon receptor gene expression

in brown adipose tissue of cold-exposed rats
A. Morales, J. Lachuer, C. Duchamp, A. G~lo~n,
B. Georges, H. Barre
UMR 5578 ('NRS-UCBLyonl. P-69622 l'tlleur&nme cede.r, l, rance

Glucagon is well known to regulate hepatic carbohydrate metabolism and

white adipose tissue lipolytic activity. This pancreatic hormone may also
have thermogenic effects in brown adipose tissue (BAT), the specialized
thermogenic tissue of small rodents. On target cells, the actmn of
glucagon depends on its interaction with specific membrane receptors. It
is not known whether the sensitivity of BAT to glucagon is modulated in
parallel with the thermogenic activation of the tissue induced by cold
exposure. If this ts the case, it may be triggered by the two main stimuh
known to actwate BAT thermogenesis during cold exposure.
noradrenaline released by the sympathetic innervatmn of BAT and / or
3,5,3'-triiodo-L-thyronme (TO production by an activated BAT specific
5'-desiodase. The aim of our study ~as to investigate the impact of
prolonged cold exposure on glucagon receptor gene expression m rat
BAT and the potenUal control of thts effect by either thyroid hormones or
noradrenergic innervation.
Using a semi-quantitative reverse transcriptase-polymerase chain reaction
(RT-PCR) assay, it was shown that I) cold exposure (7 days 4C)
markedly decreased the relative abundance of glucagon receptor mRNA
in interscapular BAT. 2) Pharmacologically-induced hypothyroidism at
thermoneutrality resulted in a marked reduction of BAT glucagon
receptor mRNA, a phenomenon rapidly reversed by exogenous T~. 3)
Unilateral surgical sympathetic dencrvatlon of BAT prevented the coldinduced decrease in glucagon receptor mRNA.
The present results therefore indicate that, during cold exposure, the
negative effect exerted by catecholammes on the relative abundance of
BAT glucagon receptor mRNA predoroinates other the positive effect
exerted by thyroid hormones.

(We/6.3/116)

Functional analysis on the peroxisome proliferator

response element (PPRE) of the rat thiolase B gene
V.Nicolas-Frances ~, V.K.DasarP, E. AbruzzP, T. Osum?& N. Lalruffe ~
aUaiversityof Burgundy,Dilon FrancebHime]lInstituteof Technology,HimejtJapan

The peroxisomal 3-oxoacyl-thiolase is involved in the final reaction of

long and very long chain fatty acids 13oxidation leading to the production of
acetyl-CoA, a major precursor for fatty acids and cholesterol synthesis Rat
peroxisomal thiolase is encoding by two genes termed A and B and only the
thiolase B gene is induced in rat liver by peroxisome proliferators (PP) [1 ].
It is now well established that induction by PP depends on the nuclear
receptor PPAR ct and on a sequence termed PPRE localized in the promotor
of the inductible genes. The sequence analysis of the thiolase B gene
revealed a putative PPRE at -681 to -668 [2]. The aim of this work was to
determine if this PPRE could he functional in thiolase B gene induction.
By gel retardation we show that thiolase B gene PPRE could interact
with the hepatic nuclear factor HNF4 and probably with PPAR ct. Using
cell transfection in hepatic cell line HepG2 and in non hepatic cell line COS
we found that PPAR ct and HNF4 transactivate a reporter gene luciferase
under the control of thiolase B PPRE. The respective role of HNF4 and
PPAR ct on thiolase B gene expression will be discussed.
1 : Hijikata et al., J.BioLChern., 265, (1990), 4600-.
2 : Kliewer et al., Nature, 358, (1992), 771-.
" Universi~ ofBurguncly, LBMC, Faculty of Sciences Gabriel 6, Bd Gabriel
21000 Di/on, France.
b Himeji lnsitute of Technology, Faeul~, of Life Sciences, Laborato~ of
Molecular and Cel Biology, 1479-1 Kanafl. Kamigori Hyogo Himeji, Japan.

Abstracts FEBS'99

(We/6.3/117)

Synergistic activation of the LAMC2 promoter by TGFbetal and EGF

J. Olsena and P.Simon-Asmannb

s305

(We/6.3/118)

Biochemistry. Lab. C. The Panum Institute. Copenhagen N, Denmark

blNSERM U381, 3 avenue moliire, 67200 strasbourg, France.

The small intestinal epithelium is organised into flask-shaped crypt regions

surrounded by protruding villi. Epithelial stem cells located near the bottom of
the crypts are responsible for the constant renewal of the epithelium which
occurs throughout life and from which emerge daughter cells undergoing
differentiation. The development and maintenance of the intestinal mucosa
depend on interactions between the epithelium and the subepithelial
fibroblasts.The two cellular layers are separated by extracellular matrix
molecules organized into a basement membrane (BM: laminins, perlecan,
collagen IV and nidogen) which are produced by both cell types.
Laminin-5 (laminin a3,p3,~/2 chains) is found in the intestinal BM where it is
thought to be important for the migration of the epithelial cells. We have
recently used the LAMC2 gene, encoding the laminin 72 chain, as a model to
study cytokine regulation of BM molecules in the intestine. The LAMC2
promoter is strongly inducible by hepatocyte growth factor (HGF) and
moderately inducible by TGF-II1 and epidermal growth factor (EGF). The HGF
response occurs via the most 5' of two closely spaced AP-1 elements in the
promoter.
In the present work we have investigated the regulation of the LAMC2
promoter by combinations of cytokines in human intestinal epithelial cells. We
find that the stimulation by TGF-II1, occurs via a SMAD like cis-element, and
that the TGF-II1 signalling synergenize with signals elicited either by HGF or
EGF. In contrast no synergy was found after simultaneous treatment by HGF
and EGF.
As HGF is only highly expressed under certain circumstances, such as during
early development and wounding, the intestinal co-expression of TGF-[II and
members of the EGF family, might secure epithelial LAMC2 expression at a
reasonable level in the absence of HGF.

(We/6.3/119)

Expression of rat-liver serine dehydratase

during chronic acidosis
I. L6pez-Floresa, J.B. Barrosoa, R Valderrama~. F.J. Esteban-Ruiz",
M A. Peinadoa; J.A. Lupi~fiezb, J Perag6na
a Dpt of Experimental Biology, Untv Ja#n, Spain,
b Dpt of Biochemistry and Molecular Btology, Umv Granada, Spare

Serine dehydratase (EC 4.2.1.I3, SerDH) is an enzyme involved in the

regulation of gluconeogenesis from serine in the liver of rat. The activity,
protein and mRNA levels of this enzyme change in response to diet
composition, the hormonal status of the rat and circadian rhythm. In this
work, we have investigated the effect of chronic metabolic acidosis on the
kinetics, tissue location and protein- and mRNA-expression levels of the rat
liver SerDH. Chronic metabolic acidosis is a situation in which the
degradation of muscle proteins and amino acids are significantly stimulated
and in which SerDH could probably be modulated. Experimental acidosis
was induced in rats of 370g by their ingestion of 0.28M ammonium chloride
solution for I0 days. The degree of acidemia caused by this treatment was
determined by measuring the aortic blood pH value and serum bicarbonate
concentration. Acidosis significantly decrease (P<0.05) the SerDH activity at
all substrate concentrations used. Both in control and acidosis, the saturation
curve of SerDH was hyperbolic without evidence of sigmoidicity. The mean
value of maximum rate of SerDH in acidosis was 42% lower than in control,
however, the value of the Michaelis constant was not significantly altered.
The content and tissue location of SerDH was determined by Western-blot
and immunohistochemical analyses. The results obtained with both
techniques showed that, in acidosis, the amount of SerDH was significantly
lower than in control. Moreover, the level of SerDH mRNA was estimated
using reverse transcription and amplification by the polymerase chain
reaction (RT-PCR) and Southern-blot procedures. According to the
behaviour observed in the activity and specific content of SerDH during
acidosis, the level of SerDH mRNA was significantly lower than in control.
These results showed that during experimental acidosis a repression of ratliver SerDH gene transcription could occur, leading to a lower amount and
activity of this enzyme.
This work was supported by grant # CV1-157 from the PAI (Plan
Andaluz de Investigaci6n) Junta de Andalucia, Spain.

Lineage specific modulation of calcium pump expression

during myeloid differentiation.
S. Launay, M. Gianni, T. Kov/tcs, R. Bredoux, A. Bruel, P.
G616bart, F. Zassadowski, C. Chomierme, J. Enoufand B Papp
~z 348 1NSERM. IFR 6, 8, Rue Guy Patin, 75475 Parts Cedex 10. France

Calcium is accumulated from the cytosol into the endoplasmic reticulum

by Sarco-Endoplasmic Reticulum Calcium transport ATPase (SERCA)
enzymes. As calcium stored in the endoplasmic reticulum is essential
for cell growth, differentiation, calcium signaling and apoptosis, and
because different SERCA enzymes possess distinct functional
characteristics, in the present work we explored SERCA expression
during m vitro differentiation of the human myeloid/promyelocytic cell
lines HL-60 and NB4, and of freshly isolated acute promyelocytic
leukemia cells. Two SERCA species have been found to be coexpressed
in these cells: SERCA 2b and another isoform, SERCApLIM,
recognized by the PLIM430 monoclonal antibody. Induction of
differentiation along the neutrophil granulocytic lineage by all-trans
retinoic acid or cyclic AMP analogs led to an increased expression of
SERCApLIM, whereas the expression of the SERCA 2b isoform was
decreased. The modulation of SERCA expression was manifest also on
the mRNA level. Experiments with retinoic acid receptor isoformspecific retinoids indicated that SERCA expression is modulated by
retinoic acid receptor alpha-dependent signaling. SERCA expression of
retinoic acid-resistant cell variants was refractory to treatment.
Differentiation along the monocyte/macrophage lineage by phorbol ester
resulted in an increased expression of both SERCA isoforms. In
addition, when cells were treated by phorbol ester in the presence of the
glucocorticoid dexamethasone, a known inhibitor of monocyte
differentiation, a selective blockage of the induction of SERCApLIM was
observed. Altered SERCA expression modified the functional
characteristics of calcium transport into the endoplasmic reticulum.
These observations show for the first time that the modulation of
calcium pump expression is an integral component of the differentiation
program of myeloid precursors, and indicate that a lineage-specific
remodelling of the endoplasmic reticulum occurs during cell maturation.
In addition, these data show that SERCA isoforms may serve as useful
markers for the study ofmyeloid differentiation.

(We/6.3/120)

Cloning of the mouse Sodiam Iodide

Symporter (mNIS) from mammary gland
B. Perron, A.-M. Rodriguez, G. Leblanc, T. Pourcher
L.R.C..C.E,4. 16V, LP.M.C., Univ. Nice Sophia-Antipolis,
C.N.R.S., BP 68, 06238 Villefianche sur mer, France

Iodide is concentrated in the thyroid by means of a Sodium

Iodide Symport (or co-transport) mechanism activated by TSH.
Recently, cDNA encoding rat and human Sodium Iodide Symporters
(NIS) were cloned using thyroid cDNA libraries and showed that NIS are
multispanning membrane proteins of 618 amino acid and 643 amino
acids respectively. Iodide is also actively transported in extrathyroidal
tissues (mammary gland, placenta, intestine...) under the control of tissuespecific hormones.
Iodide is secreted acmss the mammary epithelium and
concentrated in milk by a mechanism regulated by prolactin. As an
essential constituent of the iodide-containing thyroid hormones, iodide is
required to the newborn for normal maturation of his nervous system.
We chose the mouse as a model to compare the molecular
mechanisms of iodide accumulation in mammary gland and thyroid and
analyze their hormone control. To this end, it was first essential to
identify the gene coding for the mouse NIS. mRNA were extracted from
mammary gland of lactating mice. Using primers to the rat cDNA
sequence we amplified a large part of the open reading frame of the
mouse cDNA by RT-PCR. The extremities of the cDNA were obtained
by RACE-PCR. The amino acid sequence of mouse NIS shows an
homology of 95% to the rat NIS sequence. Our data are in agreement
with the recent report [1] showing that the same gene encodes the NIS
transporters of the human mammary gland or thyroid. These results
most likely imply that different transcription factors are involved in the
tissue-specific regulation and hormonal control of NIS in both tissues.
[1] Spitzweg et al, J. Clin. End. Met., 83, (1998) 1746.

s306

(We/6.3/121)

Abstracts FEBS'99

PACAP and cAMP stimulate promoter activity of the rat

GnRH receptor gene via a bipartite response element.
H. Pincas, D. Duch~ne, J.N. Laven'i~re, R. Counts.

(We/6.3/122)

ECMR, ESA 7080, Universtt~ P. & M Curte, 75252 Palt~, France

Negative cAMP response elements in the promoter

of the L-pyruvate kinase gene
M. Raymondjean, L. Gourdon, D-Q. Lou,
M. Vasseur-Cognet, A. Kahn
INSER~4 Ul29. CHU Cochin. 24 rue du Fbg St Jacques. F-75014Parts

The GnRH receptor (GnRHR) is primarily involved in the control of the

synthesis and release of the pituitary gonadotropin hormones LH and FSH
and, thus, plays a crucial role in the regulation of the reproductive function.
Under various physiological or physiopathological conditions, the number of
GnRHR and related level of GnRHR mRNA are differentially modulated. To
assess the importance of transcriptional regulation, we have ~solated and
functionally characterized a 3.3 kb DNA fragment corresponding to the promuter region of the rat GnRHR gene. We have established that gonadotrope
cell-specific activity is conferred by cis-elements localized in both proximal
(-500 to +1 relative to the ATG codon) and distal (-1100 to -750) regions of
the promoter (1). To further investigate the transcriptional regulation of the
GnRHR gene, we have now examined the possibility that the cAMPdependent protein kinase A (PKA) pathway and laituitary _adenylate cyclase'_activating 12olypeptide (PACAP) participate in the regulation of the promoter
activity.
Transient transfection of alpha T3-1 cells, a gonadotrope-derived cell line
which possesses a type I PACAP receptor, with the GnRHR promoter fused
to the chloramphenicol acetyl transferase (CAT) reporter gene established
that cholera toxin (Ctx) activation of the PKA pathway resulted in a strong
snmulation of CAT activity (2.8-fold). We also demonstrate that PACAP
increased the activity of the GnRHR promoter. Maximal stimulation was
obtained at concentrations as low as 1 nM (2.8-fold). Progressive 5"-deletion
analysis revealed that PACAP- or Ctx-stimulated GnRHR-CAT activity were
consistently retained after deletion at position -515, -433, or -316 but were
significantly decreased after subsequent truncation of the promoter from -316
to 222 (1.3-fold). In addition, 3'-deletion analysis indicated the presence of
putative ClS-element(s) between -96 and -55. Taken together these data
suggest that the response elements for both PACAP and cAMP reside at two
different sites: between -316 and -222 and between -96 and -55. The colocalization of PACAP and cAMP response elements on the GnRHR
promoter together with the suppressive effects of a specific inhibitor of
protein kinase A (PKI) to the response of either stimulator suggest that
PACAP action is mediated primarily by the PKA pathway.
1- H. Pincas, Z. FOlTaL S. Chauvin, J.-N. LaveiTi~:re, and R. Counts. Mol Cell
Endocrinol. 1998," 144.'95-108

(We/6.3/123)

133-Adrenoceptor expression in white adipose tissue

during pregnancy
P. gamos, M. D. Crespo, E. Herrera
Facul~, of Experimental and Teehmcal Setences,
L'no.'ersi~' San Pablo-CEU, AIadrld, 5)~atn

Insulin and catecbolamines are of major imponance for the regulation of

lipolysis in adipose tissue in response to metabolic demands While
catecholamines stimulate lipolysis through activation of 133-adrenergic
receptors, insulin counteracts this adrenergic stimulation by as yet not
completely elucidated mechanisms During pregnancy, modulation of lipolysis in white adipose plays an important role in maintaining the availability
of fuels to the fetus. However, given that during late-pregnancy there are
increased plasma insulin levels and insulin resistance, it remains unclear how
white adipose tissue lipolysis responds to insulin during pregnancy. To
address this issue, we analyzed isolated lumbar adipocytes from virgin and
late-pregnant (20 days) Sprague-Dawley rats for catecholamine-mediated
lipolysis and [33-adrenoceptor expression by quantifying glycerol release and
Western-blot analysis, respectively. Concentration-response curves with
different 13-agonists showed that the sensitivity of adipocyte lipolysis to
catecholamines was not significantly altered during pregnancy. However, the
maximal lipolytic response in adipocytes from 20-day pregnant rats to 13adrenergic agonists was markedly reduced as compared to age-matched
virgin animals It was tempting to speculate, therefore, that 133-adrenoceptor
expression may be downregulated at the end of pregnancy. In support of
this hypothesis, we found a 2-fold reduction of 133-adrenoceptor expression
in membranes of adipocytes from pregnant rats as compared to virgin
controls. These data provide evidence that the inhibition of catecholaminedependent lipolysis during pregnancy occurs, at least in part, at the level of
133-adrenergic receptor expression. Furthermore, present data indicate that,
despite the development of insulin resistance during pregnancy, white
adipose tissue responds to maternal hyperinsulinemia with reduced
catecholamine-dependent lipolysis.

The L-type pyruvate kinase (L-PK) gene is transcriptionally activated by

glucose and insulin and inhibited by fasting and glucagon; this inhibition is
reproduced in hepatocytes by cAMP analogues. While transcriptional
activation by glucose/insulin is a slow phenomenon, inhibited by
translation inhibitor's, the inhibition by glucagon/cAMP is a very rapid
phenomenon (less than 10 rain) insensitive to cycloheximide. We have
previously demonstrated that the glucose response element (GIRE) located
in the L-PK promoter, built around two noncanonical E boxes (L4),
functions in close cooperation with a contiguous HNF4 binding site (L3)
to assure in the L-PK context a full inhibition by cAMP. We have recently
shown that the binding activity of the orphan nuclear receptor HNF4, was
decreased by PKA-dependent phosphorylation. Therefore, the respective
role of the GIRE and HNF4 binding site in the negative response to cAMP
could be questioned. Therefore, we have investigated the cAMP response
of artificial promoters (-54PK/lucifemse), consisting of oligomerized LPK dements, either the GIRE or the L3 binding site, by transient
transfection in hepatocytes in primary culture. Both constructs were
inhibited by cyclic AMP or cotmnsfection with a PKA catalytic subunit
expression vector while only the G1RE conferred, as expected, a strong
positive response to glucose/insulin. The inhibition was maximal when the
reporter gene was directed by both IA and L3 dements (L4L3)3-54
PK/luci. The identification of HNF4 binding sites as potential negative
cAMP response elements was confirmed replacing with an other HNF4
binding site. Transactivation of the (L3)3-54PK/luc by HNF4
overexpression was abrogated by treament with cAMP and overexpression
of the PKA catalytic subunit. Overexpression of a HNF4 negative
transdominant mutant resulted in inhibition of both basal activity of the
promoter and cAMP negative action. However, HNF4 mutants whose
putative PKA phosphorylation sites have bee~ mutated still conferred
cAMP-sensitive transactivation. Overexpression of the CBP transaaivator
increased the HNF4-dependent transactivation, but this effect remained
sensitive to cAMP inhibition. In conclusion, both the GIRE and the HNF4
binding sites of the L-PK promoter are negative cAMP response elements
which act synergistically in the natural promoter. PKA-dependent
inhibition through the HNF4 binding sites requires HNF4 binding but its
precise target (HNF4 itsdfand (or) a coactivator) remains unknown.

(We/6.3/124)

The structure of the Rat Phenylalanine

II)droxylase Gene Promoter
D Rees,' I L McDov, all, M LFishcr,' KB.O'Hare'
( "heater ( '<.,liege, England

I.Ivet?~ool(:mversm, l'2nglwld

The enzyme phenylalanine hydroxylase (L-phenylalanme,

tetrahydropteridine, oxygen oxidoreductase (4-hydroxylatmg); EC
114.16 [) (PAH) catalyses the conversion of phenylalanlne to
t3rosme m man and other mammals The classtcal inherited
metabolic d~sease phen31ketonuria is a consequence of decreased
PAIl expression The highly homologous nature of rat and human
enzvme~ ha'~.e allowed man':, inferences about normal and
pathologlcal phcnylalamne metabolism m humans to be gained
from studies of the rat enzyme We have demonstrated prewously
that the major long-term regulation o f PAH actlvit) is at the level
of transcription however, the molecular basis of PAH regulation
has not been fully elucidated To this end, a 2.4kb fragment
containing the TAATA-less rat PAH gene promoter has been
cloned and several putative regulator' elements identified [1], In
addition, recent studies on both the human and mouse PAH genes
[2,3] have )dentlfied an upstream enhancer
regmn at
approxm~atcly -3.5kb, ~h~ch contains consensus sequences ('or the
binding of the tissue specific transcription factor HNTI ThJs
cnhancer appears esse)mal for efficient transcription of the mouse
PAIl gone but ma) not explain the full pattern of expressmn in the
human PAt,~ gene [2]. We report here that sequence analysis on the
analagons region el'the rat PAH promoter has been used to ident@
the plc>ence ofa sml~larl?,, hlghI~ conserved enhancer and suggest
that th~s region ma 5 be slgnlf)cant )n the tlssuc-speclfic
transcrlrJtmn of the rat PAll gent
1 X:lcDm~all let ~/) I995, (5('no, 153, 289
2 Lc~ X-D. (~'t ~d) 1998. ]'roc.,\)a/. :t~od. So:. ~ X4. 95, 1500
) fatl,I [) M (e: ~t/) 1996, Ahd.( "e//]hM. 16, 3125

Abstracts FEBS'99

(We/6.3/125) Cytochromes P450 from family 4 in the sea bass

C. Sabouraulta, M. Amichotb, J.-P. Girard a, J.-B. Bergeb

s307

(We/6.3/126)

a LPTE, UNSA, BP 71, 06108 Nice, France

b hVRA-IFR 38, BP 2078, 06606 Anttbes, France

Cytochromes P450 belonging to the family 4 (CYP4) are

involved in mammals in endogenous metabolism, such as o~or (o~-l)-hydroxylation of fatty acids, prostaglandins, and
leukotrienes. Most of the corresponding genes are expressed
in liver and are inducible both by endogenous compounds
(fatty acids, hormones) and by xenobiotics (namely
peroxisome proliferators). Their gene expression is regulated
by transcription factors that are members of the nuclear
hormone-receptor family, the peroxisome proliferatoractivated receptors (PPAR).
On the contrary, hydroxylation of lauric acid preferentially
occurs at positions (o~-1) to (0)-4) in the sea bass,
Dtcentrarchus labrax. We have cloned two genes belonging
to the CYP4 family. One, named C Y P 4 T 2 , shared 54 2%
identity with the rabbit CYP4B1, which is expressed in liver
and lung. The second, named C Y P 4 F T , shared 53.9%
identity with the human C Y P 4 F 3 , which catalyse the cohydroxylation of leukotrienes B4 in polymorphonuclear
leukocytes. C Y P 4 F 7 is only detected in kidney and is not
inducible by any of the xenobiotics tested. CYP4T2 is highly
expressed in kidney and small intestine. This isoform is not
inducible by clofibrate but strongly by phthalate esters
(DEHP). The physiological role of these CYP4 is still
unknown in fish.

(We/6.3/127)

Progesterone receptor (PR)-immunohistochemical

localization in porcine ovarian cells.
M.S~omczyfiska, M.Krok, Z.Tabarowski
Lab Animal Endocrinol & Tissue Culture, Dept. Animal PhysioL,
Institute of Zoology, Jagtellonian University, Cracow, Poland.

The modulation of ovarian fiJnction by progesterone has been discussed

by many investigators. The actions of progesterone on the ovary are
thought to be mediated indirectly through the hypothalamus and
pituitary gland or directly by interaction with the progesterone receptor
(PR) within the ovary. PR has been detected in rat, human and monkey
ovaries [1,2]. The aim of our work was to localize PRs in the porcine
ovary and to find out if their expression is hormonally regulated.
Porcine ovarian follicles and corpora lutea were isolated from porcine
ovaries, fixed in PFA and routinely embedded in paraplast. To
investigate the influence of LH on PR expression, fragments of ovaries
were incubated for 12 hours in medium containing 100 ng LH/ml and
later processed in the same way as described earlier. For
immunohistochemistry, 6 lam slides of ovarian tissues were stained with
monoclonal anti-PR antibody (Novocastra, England) followed by
secondary biotinylated antibody (Vector, USA) and Strept ABC/HRP
detection system (Dako, Denmark). The most intensive staining in nuclei
of granulosa cells was observed in large luteinizing follicles and early
corpora lutea. High level of PR was observed in small and medium
follicles which also stained positively for DNA leddering, indicating
apoptosis. The distribution of PR within those follicles was different
from that characteristic for healthy ones.
We have reported that ovarian cells undergo quantitative changes in PR
levels during the oestrous cycle and those changes are hormonally
controlled.
[1]. Iwai Met al., Endocrinology 129, 1991, 1621.
[2]. White M. M. et al., Endocrine Rev, 16,1995, 739.
Sponsored by K B N 6 P 0 4 C 039 14 grant.

Pancreatic gene and protein expressions in male

and female mice - Sexual dimorphism.
D. Sanchez, B. Vall~e, N. Baeza, C. Figarella, O. Guy-Crotte
GRGE, Facult~de M~decine, 13005-Marseille,France.

The pancreatic gland, by its dual function may be involved in many metabolic
processes and an increasing number of works deal with transgenic and/or
knock-out mice; yet little is known on the mouse pancreatic genes and the
corresponding proteins. Some reports have shown a differential pancreatic
behaviour between male and female mice in experimental pancreatitis or
diabetes. Therefore, after the characterization of some mouse pancreatic
exocrine proteins, we compared the levels of pancreatic genes expression and
of the corresponding proteins in male and female mice. The pancreas were
independently studied by dot-blot hybridization. A difference was observed in
the expression of four pancreatic genes. Amylase gene expression was
significantly higher in females than in males whereas trypsinogen and lipase
gene expression were significantly lower. For chymotrypsinogen, the
difference was not significant. These results contrast with those found in rat
pancreas where no differences were observed between females and males in
the levels of pancreatic enzymes mRNAs. If we compare the levels of the four
corresponding enzymes expressed in female and male mice pancreatic
homogenates using slot-blot and antibodies directed to the closely related
human pancreatic enzymes, no signficant difference was observed between the
levels of protein expression. These data show a sexual difference in mice at the
level of pancreatic genes expression but not in proteins expression. They
suggest a difference in the regulation of the pancreatic proteins at the
transcriptional level. This sexual dimorphism calls into question the role of sex
hormones in the mice pancreas.

(We/6.3/128)

Changes in PDE4 subtypes expression in hum,in cell lines

under different culture conditions
CM. Szilagyi, M. Artola, S Lardon, S. Trouilhet, J Allen
IRJ/PARKE-DA [~7,S"3-9 Rue de la Loge BPIO0 - 94265 Fresnes ('edex
(France)

PDE4 activity is regulated both short term by phosphorylation or activation by

phosphatidic acid and long-term by de novo enzyme synthesis following gene
activation. Different types of regulation can occur in the same cell-type.
Studies were conducted to attempt to control subtype expression in different
cells to help understand the role and regulation of a single PDE4 subtype For
this purpose we used 3 agents known to act through different pathways a
combination of cAMP-elevating agents (salbutamol/rolipram), a phorbol ester
(PMA) which stimulates PKC or dexamethasone which modulates gene
transcription through nuclear receptors. Changes in PDE4 gene expression
were evaluated by semi-quantitative RT-PCR Expression of the PDE4C
isozyme was not induced by any of the treatments in any cell line In Molt-4
cells the expression level of PDE4A was slightly down-regulated by PMA (-1.5
fold) and up-regulated (+2 fold) by dexamethasone; no change occurred for the
other subtypes. In Jurkat cells, PMA led to the down-regulation of PDE4B (-2
fold) and PDE4D (-1 9 fold) without affecting PDE4A levels In U937 cells
treated with salbutamol/rolipram, PDE4A was induced 3.2-fold and PDE4B 2fold. For PDE4D, both 4DI/4D2 were up-regulated whereas the 4D3 form was
unaffected by this treatment. Dexamethasone treatment was without effect on
subtype expression in this cell type. In HL-60 cells the increase of intracellular
level of cAMP induced by salbutamol/rolipram up-regulated expression of
PDE4A (+3 3 fold) and PDE4D essentially through the variants DI and D2
whereas PDE4B levels were unaffected. PMA led to an up-regulation of
PDE4A (+2.3 fold) and complete loss of the PDE4B and PDE4D transcripts. In
HT-29, PDE4D3 was the predominant PDE4 subtype but 4D1/4D2 and 4A
were present to a lesser extent with only trace of PDE4B transcript; neither
salbutamol/rolipram nor dexamethasone affected expression, but PMA upregulated both PDE4A and PDE4D expression PMA was a very potent up
regulator of PDE4D expression in HK-293 cells (+3.7 fold) but did not affect
any other subtype studied. In this cell line, salbutamol/rolipram increased the
expression of PDE4B by two-fold. In Caco-2 cells only PMA exhibited a slight
up-regulation (+1.5 fold) of total PDE4D expression. In conclusion, basal
expression of PDE4 subtypes varies considerably between cell types and the
response to pharmacological manipulation is cell type dependent

s308

(We/6.3/129)

Abstracts FEBS'99

T F E 3 and T F E B as Transcriptional Activators

o f T y r o s i n a s e and TRP1 Genes.
C. Verastegui, C. Bertolotto, K. Billc, P. Abbe, J.P.
Ortonne and R. Ballotti*.

(We/6.3/130)

'Dpto Chm~as de la Salud, Umv~'sldad Pliblica de Navarra. 31008 parnplotla. Spare

Bloquirmca y Biologia Molecular, Untvctsldad tic Navarra, 31080 Pamploaa. Spare

~pto

INSERM U3&5ave de Valombrose, 06107 Nice, France.

Microphthalmia, a basic helix-loop-helix-lencine zipper (bHLH-Zip)

transcription factor involved in the development of the melanocyte lineage
has been shown to play a key role in the transcriptional regulation of the
melanogenic enzymes, tyrosinase and TRP1. Noteworthy, among the
transcription factors of the bHLH-ZIP family, TFE3 and TFEB show a
remarkably elevated homology with microphthalmia, particularly in the
bHLH and in the N-terminal transactivation domains. These observations
prompted us to investigate the role of TFE3 and TFEB in the regulation of
tyrosinase and TRP1 gene transcription. Here we show that TFE3 and
TFEB are expressed in melanocyte. As previously reported for
microphthalmia, TFE3 binds to Mbox (AGTCATGTGCT) and an Ebox
motifs (CATGTG) surrounding the TATA box and stimulates the
tyrosinase gene transcription. Further, TFE3 and TFEB, through their
binding to the Mbox motif, stimulate the TRP1 promoter activity. These
data indicate that TFE3 and TFEB are able to regulate tyrosinase and TRP1

Characterization of the promoter of the human retinoid x

receptor [31 isoform
A. Vergaraa, A. Corella'. C. de Miguelband I. Encio"

Retinoic acid and its natural and synthtetic derivatives play an

important role in cellular differentiation, embryonic development
and homeostasis. These effects are mediated by two classes of
nuclear receptors, the RARs and RXRs. Three genes, respectively
termed a, [3 and 7, have been described for each of the two families.
Each subtype gene encodes several isoforms that are generated by
alternative splicing and/or differential promoter usage. Here we
report the characterization of the hRXRI31 5'-flanking region. The
cap site was mapped by primer extension. The putative promoter
region is GC rich, and contains putative binding sites for the
transcription factor Spl as well as sequences closely resembling the
consensus RARE, TRE and VDRE. However, no obvious TATAor CAAT-box were detected. Various lengths of the putative
hRXR[31 promoter sequences were ligated to the luciferase gene
and tested for their ability to drive transcription, showing that a
single Spl binding site is sufficient for basal level of luciferase
activity both in HeLa and CV-I cells. The transfected cells were
then treated with either T3, all-tRA, 9eRA or TNF-a, and negative
regulation of the hRXR[31 gene by TNF-a was observed

expression, making of these transcription factors potential actors in the

regulation of melanogenesis and in the differentiation of pigmented cells.

(Wd6.3/131)

Serial microanalysis of the renal transcriptome

B. Virlon, L. Cheval, E. Billon, A. Doucet and J.M. Elalouf
URA-CNRS 1859, SBCe, CEA-Saclay, 91191 Gif/Yvette Cedex. France

(We/6.3/132)

Role of defined domains of glucocorticoid receptor

in the synergism with Stat5A
a
M. Windegger,
J. Liden,b D.B. Starrc and W. Dopplera
alnsitute of Meal Chemist~ & Biochemistry lnnsbntck Austria" bKarolinska
Institute, Huddinge. Sweden: CUniversi~ o f California. San Francisco, USA

We used the method of Serial Analysis of Gene Expression (SAGE) [1] to

simultaneously analyse a large number of kidney mRNAs, and thus progress
toward the characterisation of the renal transeriptome. SAGE rests on the
isolation of transcripts-specific 10-bp tags, which are subsequently
concatened, cloned, and sequenced. The number of tags for each transcript is
proportional to its abundance, and therefore provides quantitative data about
gene expression. We first used 5gg of poly(A) RNA to generate SAGE
libraries from the whole mouse kidney and 12 000 tags, corresponding to 4800
different mRNA species, were sequenced. The expression profile that we
obtained was characterized by high levels of mitochondrial transcripts as well
as kidney-specific nuclear mRNAs. Since mammalian tissues consist of several
different cell types, it is most desirable to scale down the SAGE approach for
studying well delineated tissue fragments. A mieroadaptation of SAGE was
therefore set up and referred to as SADE [2]. SADE potentiality was first
tested by preparing libraries from a wide range of tissue amounts (from 250 to
0,5mg of tissue) and analyzing the tags in each case. A highly significant
correlation 0=0,88) between tag abundance in macro- and microlibraries
prepared from the same tissue was obtained, demonstrating that representative
libraries can be obtained from small tissue samples. We then analyzed the
transcriptome of different nephron portions isolated by microdissection (50
000 cells). Gene expression profiles obtained in these different segments
revealed abundant cell-specific mRNAs, several of which had no GenBank
entries. Additional applications include transcriptome analysis during
embryogenesis, and its modulation by gene targeted disruption (50 000 tags
analyzed in E6.5 mouse embryos). SADE procedures for generating libraries
from tiny amounts of cells thus open new fields for large scale expression
studies in eukaryotic tissues.
[1] V e l c u l e s c u V . E . e t a l . S c i e n c e 2 7 0 ( 1 9 9 5 ) 484.

[2] ChevalL. et al. Differentialgeneexpression:a practical approach(in press).

For the efficient induction of the expression of the [3-casein gene the
functional interaction between the glucocorticoid receptor (GR) and the
signal transducer and activator of transcription 5 (Stat5) is required.
ha this work we determined the functional effect of defined domains of GR
on the synergism with Stat5A. A panel of mutated constructs of GR was
transiently co-transfected with Stat5A employing CVI cells and the effect
on the expression of a J3-casein gene promoter luciferase reporter gene was
measured. The responsiveness of the MMTV promoter to the effect of
mutated GR in the absence of Stat5A was tested in comparison to the 13casein gene promoter. We found that the pattern of effects caused by the
mutations was similar between the two promoters: Deletion of the DNA
binding domain (DBD) and amino acid changes in the extended region of
the first zinc finger, in the D-loop and in the distal part of the second zinc
finger of the DBD resulted in a complete or partial loss of transactivation
suggesting that the integrity of various parts of DBD are prerequisites for the
synergistical expression of the [3-casein gene, Deletion of the transactivation
domain zl resulted in a decrease of transaetivation. We conclude that an
intact GR is required for both the synergistical transactivation of the [3casein promoter together with Stat5A and the activation of the MMTV
promoter. One different property of GR in combination with Stat5A is its
response to RU486. There RU486 acts as a partial agonist in the activation
of [3-casein gene transcription, whereas without Stat5A it acts fully
antagonistic on the MMTV promoter. Stat5A apears to alter the balance
between corepressor and coactivator bound to the GR-RU486 complex.

Abstracts FEBS'99

s309

8.3 Protein translocation

(We/8.3/133)

In-vitro and structural aspects of the peroxisomal

(We/8.3/134)

membrane protein Pexl3 SH3 domain

P.Barnett, G.Bottger, A.Klein, B.DisteI and H.Tabak
Department of Biochemistry, Academic Medical Centre, University of
Amsterdam. Meibergdreef 15, 1105 AZ, The Netherlands

The SH3 (SRC-homology 3) domain is a non-catalytic protein domain

mediating protein-protein interactions that are important for functions such
as; intracellular signaling, substrate recruitment for enzyme catalysis,
metabolic regulation and subcellular protein localization. The peroxisomal
membrane protein Pexl3 contains one such domain at its cytosolic Cterminus.
Two-hybrid and in-vitro assays have clearly demonstrated the ability of the
Pexl3 SH3 domain to bind to the purified cytosolic/peroxisomal PTSI
receptor, Pex5, and the peroxisomal membrane associated protein Pexl4. All
of these components play an essential role in the import of proteins into the
peroxisomal matrix.
Although crystallographic analysis of the Pexl3 SH3 domain is now
underway, it has been possible to build a theoretical 3D homology model of
this domain with its Pexl4, PPTLPHR, binding ligand in place. This model
has offered insight into such aspects as the natural Pex 13 peroxisomai import
mutant E320K and provides basis for site directed mutagenesis. Less defined,
however, is the Pex5p interaction, Pex5p being devoid of a recognizable
PXXP motif, common to SH3 domain interactions, that could support a
classic SH3 interaction. However, using error prone PCR, we have screened
a Pex5p mutant library and picked up several clones which have started to
shed some light on this interaction.

(We/8.3/135)

Routing, processing and export of rat pituitary prolactin:

identification of a disulphide-bridged preprolactin.
F Bollengier, A Mahler, L.Vanhaelst
I'~Tkgroep Bto~'hemie e , l'akgroep l-armacologte, l ~lje ( b, vt,r~ttelt Brux~el,
Laarbeeklaan. 103, B-I090 Brossel. Belgtd

Recently [1] we investigated the modulation of both the molecular size

heterogeneity and the relative distribution of rat prolactin (rPRL) variants,
synthesized and secreted by rat pituitary cells under different physiological
stimuli. We showed that these parameters are considerably influenced by age
and physiological stimuli. Intriguing facts were the high degree of variability
of PRL isoforms in storage and release, the presence of transient polymeric
rPRL (33-90kDa Mr) and the modulation of the glycoform in peri-and
postnatal life. This in sharp contrast to basal adult life, where throughout the
mature lifespan monomeric rPRL was always the dominant variant and
26kDa rPRL the main glycoform. As yet the nature of polymeric PRL and
the transient variants is unclear, and the significance of molecular size PRL
modulation in the cellular processing of the hormone is largely unknown.
Therefore, to gain an insight in the routing, processing and export of rat
PRL, rat pituitary cells were cultured in serum-free medium in the presence
of cycloheximide, carbonyt cyanide m-chlorophenylhydrazone, brefeldin A
and monensin. The potential influence of these perturbants, whose well
documented effects are the altering of protein synthesis and transport, was
studied on rPRL isoforms appearing in cellular extracts and culture medium.
The outcome of the experiments as recorded in immunoblotting, thiol
gradient electrophoresis and densitometry was as follows. At the cellular
level we were able: 1) to characterize a novel 36kDa protein as a disulphidebridged oligomeric precursor PRL which is presumably rapidly transformed
in the cis/medial Golgi. Formation of oligomers during maturation is a
feature of many secreted and membrane-bound proteins and it has been
suggested that disulphide-bridging in proteins favour internal mobility i.e.
transport to the Golgi stacks, whilst contributing to the stability of the protein
in the fluctuating extracellular environment; 2) to identify monomeric rPRL
as an early Golgi protein, and 3) to assign the main processing of the
glycosylated 26kDa rPRL to the cis/medial Golgi. Indeed, increasing the time
of residence in the ER did not alter the glycosylation pattern of 26kDa rPRL,
which strongly suggests that the formation of this isoform is a pure
posttranslational event and reinforces the evidence we accumulated in the last
decade of 26kDa rPRL being an O-linked glycoform [2]. The secretion mode
indicates that rPRL isoforms are released via the regulated pathway in the
presence of the perturbants used.
1] F.Bollengier et al., J Neuroendocrin, 8, (1996) 721.
2] F.Bollengier et al., Endocrine, 3, (1995) 61.

NMR study of the C-terminal domain of the ToIA protein

orE. coil involved in the infection process of colieins
C. Deprez*, L. Blanchard*, J.-P. Simorre*, F. Guerlesquin ,
M. Jaquinod*, R. Lloubrs', C. Lazdunski and D. Marion*
f*) 1BS- CNRS CEA Grenoble (*~ IBSJk( CNRS Marseille / FRANCE.

The Tol system of Escherichia coli is constituted of 5 proteins :

TolA, TolQ, TolR, Tol B and Pal forming contact sites between the inner
and outer membranes of the bacteria. This system is required to maintain the
integrity of the outer membrane. A pleiotropic phenotype has been
observed for tol and p a l mutants which are leaky for periplasmic proteins
and hypersensitive to drugs and detergents.
The Tol system is also parasitized by colicins. These bacterial toxins are
produced by and active against E. coli and closely related bacteria. Three
steps are necessary for their infection process. 1/Binding to a specific
receptor of the outer membrane. 2/Translocation of the outer membrane
using the Tol system - especially interaction between the C-terminal domain
of TolA (TolAIII) and the N-terminal domain of the colicin (AT 1). 3/Lethal
activity of the toxin (nuclease/pore-forming colicin) [ 1].
In order to understand the translocation mechanism of colicins, the
first step is to determine the 3D structure of the C-terminal domain of YolA
(TolAIII). For the NMR study, a ISN labelled domain of 103 residues was
overexpressed in E. coli. After reduction of the molecule, IH and ]SN
chemical shift assignments were obtained and the determination of the
solution structure of TolAIlI is now in progress. Mapping of the interaction
site between TolAIII and A T I is carried out using 2D heteronnclear NMR
experiments.
[ 1] Lazdunski et aL, J. bacteriol. 180 (1998) 4993

(We/8.3/136)

Hspg0 promotes the export of 60 S ribosomal subunits from rat

liver resealed nuclear envelopes
J Gallas, E Jacobsen. T Langer, S Rosmus. H Schlattcr and H Fasold
Instttute for BIochemtsto~, Bsocentre of the J lI%Goethe-Unlvermty, Alane-('ut~eSir 9, 60439 Franifurt am Alath, Germany, e-mad gallas[a~em,urn-frankfurt de

Ribosomal subunits are assembled in the nucleolus and mature 40 S

subunits and 60 S subunits are stoichiometrically exported into the cytoplasm
Knowlegde about signals and mechanisms for ribosomal subunit export into
the cytoplasm is very limited We used the resealed rat liver nuclear envelope
system [1] and microinjection techniqus with X e n o p u s oocytes to study the
export of 60 S snbunits During the preparation of the vesicles and extraction
of the chromatin, 60 S subunits can be introduced into the envelopes, and after
resealing remain stably included, The export of 60 S subunits is an energyconsuming process that can be strongly enhanced by a cytoplasmatic protein
complex of about 800 kDa [2] The main activity for promoting 60 S subunit
export in this complex was attributed to a protein identified as the heat shock
protein 90 (Hsp90). The export-promoting activity of Hsp90 can be
furthermore enhanced by addition of a protein of approx. 30 kDa, also isolated
from the 800 kDa complex that was identified as the high mobility group I
(HMG-I) protein Coinjection of monoclonal antibodies against Hsp90 in
X e n o p u s oocytes leads to a significant decrease of nuclear export of the 60 S
subunits Similar, polyclonal antibodies raised against Hsp90 alone or the 800
kDa cytoplasmatic protein complex, drasticly reduces export of 60 S subunits
from resealed nuclear envelopes. Using fluorescently-labeled Hsp90, a ,,rim"staining of the nuclear membrane of reseated vesicles or digitoninpermeabilized HeLa-cells can be observed Furthermore we were able to
localize gold-labeled Hsp90 at the NPCs of the reseated nuclear envelopes,
indicating that Hsp90 exerts its export promoting effect by interacting on the
cytoplasmatic side of the NPCs These results suggest that Hsp90 facilitates
nuclear export of 60 S subunits, probably due to chaperone protein
interactions during the export process, which requires maximum dilatation of
the nuclear pore complex
[1] Riedel, N & Fasold, H , Biochem J , 241 (1987), 203
[2] Hassel, Iet al, Eur J. Biochem, 241 (1996), 32

Abstracts FEBS'99

s309

8.3 Protein translocation

(We/8.3/133)

In-vitro and structural aspects of the peroxisomal

(We/8.3/134)

membrane protein Pexl3 SH3 domain

P.Barnett, G.Bottger, A.Klein, B.DisteI and H.Tabak
Department of Biochemistry, Academic Medical Centre, University of
Amsterdam. Meibergdreef 15, 1105 AZ, The Netherlands

The SH3 (SRC-homology 3) domain is a non-catalytic protein domain

mediating protein-protein interactions that are important for functions such
as; intracellular signaling, substrate recruitment for enzyme catalysis,
metabolic regulation and subcellular protein localization. The peroxisomal
membrane protein Pexl3 contains one such domain at its cytosolic Cterminus.
Two-hybrid and in-vitro assays have clearly demonstrated the ability of the
Pexl3 SH3 domain to bind to the purified cytosolic/peroxisomal PTSI
receptor, Pex5, and the peroxisomal membrane associated protein Pexl4. All
of these components play an essential role in the import of proteins into the
peroxisomal matrix.
Although crystallographic analysis of the Pexl3 SH3 domain is now
underway, it has been possible to build a theoretical 3D homology model of
this domain with its Pexl4, PPTLPHR, binding ligand in place. This model
has offered insight into such aspects as the natural Pex 13 peroxisomai import
mutant E320K and provides basis for site directed mutagenesis. Less defined,
however, is the Pex5p interaction, Pex5p being devoid of a recognizable
PXXP motif, common to SH3 domain interactions, that could support a
classic SH3 interaction. However, using error prone PCR, we have screened
a Pex5p mutant library and picked up several clones which have started to
shed some light on this interaction.

(We/8.3/135)

Routing, processing and export of rat pituitary prolactin:

identification of a disulphide-bridged preprolactin.
F Bollengier, A Mahler, L.Vanhaelst
I'~Tkgroep Bto~'hemie e , l'akgroep l-armacologte, l ~lje ( b, vt,r~ttelt Brux~el,
Laarbeeklaan. 103, B-I090 Brossel. Belgtd

Recently [1] we investigated the modulation of both the molecular size

heterogeneity and the relative distribution of rat prolactin (rPRL) variants,
synthesized and secreted by rat pituitary cells under different physiological
stimuli. We showed that these parameters are considerably influenced by age
and physiological stimuli. Intriguing facts were the high degree of variability
of PRL isoforms in storage and release, the presence of transient polymeric
rPRL (33-90kDa Mr) and the modulation of the glycoform in peri-and
postnatal life. This in sharp contrast to basal adult life, where throughout the
mature lifespan monomeric rPRL was always the dominant variant and
26kDa rPRL the main glycoform. As yet the nature of polymeric PRL and
the transient variants is unclear, and the significance of molecular size PRL
modulation in the cellular processing of the hormone is largely unknown.
Therefore, to gain an insight in the routing, processing and export of rat
PRL, rat pituitary cells were cultured in serum-free medium in the presence
of cycloheximide, carbonyt cyanide m-chlorophenylhydrazone, brefeldin A
and monensin. The potential influence of these perturbants, whose well
documented effects are the altering of protein synthesis and transport, was
studied on rPRL isoforms appearing in cellular extracts and culture medium.
The outcome of the experiments as recorded in immunoblotting, thiol
gradient electrophoresis and densitometry was as follows. At the cellular
level we were able: 1) to characterize a novel 36kDa protein as a disulphidebridged oligomeric precursor PRL which is presumably rapidly transformed
in the cis/medial Golgi. Formation of oligomers during maturation is a
feature of many secreted and membrane-bound proteins and it has been
suggested that disulphide-bridging in proteins favour internal mobility i.e.
transport to the Golgi stacks, whilst contributing to the stability of the protein
in the fluctuating extracellular environment; 2) to identify monomeric rPRL
as an early Golgi protein, and 3) to assign the main processing of the
glycosylated 26kDa rPRL to the cis/medial Golgi. Indeed, increasing the time
of residence in the ER did not alter the glycosylation pattern of 26kDa rPRL,
which strongly suggests that the formation of this isoform is a pure
posttranslational event and reinforces the evidence we accumulated in the last
decade of 26kDa rPRL being an O-linked glycoform [2]. The secretion mode
indicates that rPRL isoforms are released via the regulated pathway in the
presence of the perturbants used.
1] F.Bollengier et al., J Neuroendocrin, 8, (1996) 721.
2] F.Bollengier et al., Endocrine, 3, (1995) 61.

NMR study of the C-terminal domain of the ToIA protein

orE. coil involved in the infection process of colieins
C. Deprez*, L. Blanchard*, J.-P. Simorre*, F. Guerlesquin ,
M. Jaquinod*, R. Lloubrs', C. Lazdunski and D. Marion*
f*) 1BS- CNRS CEA Grenoble (*~ IBSJk( CNRS Marseille / FRANCE.

The Tol system of Escherichia coli is constituted of 5 proteins :

TolA, TolQ, TolR, Tol B and Pal forming contact sites between the inner
and outer membranes of the bacteria. This system is required to maintain the
integrity of the outer membrane. A pleiotropic phenotype has been
observed for tol and p a l mutants which are leaky for periplasmic proteins
and hypersensitive to drugs and detergents.
The Tol system is also parasitized by colicins. These bacterial toxins are
produced by and active against E. coli and closely related bacteria. Three
steps are necessary for their infection process. 1/Binding to a specific
receptor of the outer membrane. 2/Translocation of the outer membrane
using the Tol system - especially interaction between the C-terminal domain
of TolA (TolAIII) and the N-terminal domain of the colicin (AT 1). 3/Lethal
activity of the toxin (nuclease/pore-forming colicin) [ 1].
In order to understand the translocation mechanism of colicins, the
first step is to determine the 3D structure of the C-terminal domain of YolA
(TolAIII). For the NMR study, a ISN labelled domain of 103 residues was
overexpressed in E. coli. After reduction of the molecule, IH and ]SN
chemical shift assignments were obtained and the determination of the
solution structure of TolAIlI is now in progress. Mapping of the interaction
site between TolAIII and A T I is carried out using 2D heteronnclear NMR
experiments.
[ 1] Lazdunski et aL, J. bacteriol. 180 (1998) 4993

(We/8.3/136)

Hspg0 promotes the export of 60 S ribosomal subunits from rat

liver resealed nuclear envelopes
J Gallas, E Jacobsen. T Langer, S Rosmus. H Schlattcr and H Fasold
Instttute for BIochemtsto~, Bsocentre of the J lI%Goethe-Unlvermty, Alane-('ut~eSir 9, 60439 Franifurt am Alath, Germany, e-mad gallas[a~em,urn-frankfurt de

Ribosomal subunits are assembled in the nucleolus and mature 40 S

subunits and 60 S subunits are stoichiometrically exported into the cytoplasm
Knowlegde about signals and mechanisms for ribosomal subunit export into
the cytoplasm is very limited We used the resealed rat liver nuclear envelope
system [1] and microinjection techniqus with X e n o p u s oocytes to study the
export of 60 S snbunits During the preparation of the vesicles and extraction
of the chromatin, 60 S subunits can be introduced into the envelopes, and after
resealing remain stably included, The export of 60 S subunits is an energyconsuming process that can be strongly enhanced by a cytoplasmatic protein
complex of about 800 kDa [2] The main activity for promoting 60 S subunit
export in this complex was attributed to a protein identified as the heat shock
protein 90 (Hsp90). The export-promoting activity of Hsp90 can be
furthermore enhanced by addition of a protein of approx. 30 kDa, also isolated
from the 800 kDa complex that was identified as the high mobility group I
(HMG-I) protein Coinjection of monoclonal antibodies against Hsp90 in
X e n o p u s oocytes leads to a significant decrease of nuclear export of the 60 S
subunits Similar, polyclonal antibodies raised against Hsp90 alone or the 800
kDa cytoplasmatic protein complex, drasticly reduces export of 60 S subunits
from resealed nuclear envelopes. Using fluorescently-labeled Hsp90, a ,,rim"staining of the nuclear membrane of reseated vesicles or digitoninpermeabilized HeLa-cells can be observed Furthermore we were able to
localize gold-labeled Hsp90 at the NPCs of the reseated nuclear envelopes,
indicating that Hsp90 exerts its export promoting effect by interacting on the
cytoplasmatic side of the NPCs These results suggest that Hsp90 facilitates
nuclear export of 60 S subunits, probably due to chaperone protein
interactions during the export process, which requires maximum dilatation of
the nuclear pore complex
[1] Riedel, N & Fasold, H , Biochem J , 241 (1987), 203
[2] Hassel, Iet al, Eur J. Biochem, 241 (1996), 32

s310

Abstracts FEBS'99

(We/8.3/137)

Effect of steroidal or non-steroidal antiandrogens on

trafficking of androgen receptor in living cell.
V. Georget, B. Tdrouanne, JC Nicolas, C Sultan

(WedS.3/138)

u439 INSERM, 70, rue de Navacelles; 34090 Montpellier France

AntIandrogens are indicated in hyperandrogenism and in androgen-dependent

pathologies such as prostate cancers. The action of the antiandrogens is based
on their potential to compete with androgens for androgen receptor (AR)
occupancy and they subsequent induced a blockade of one of the different
steps in the action of AR. One of these steps could be the transport of AR
from the cytoplasm to the nucleus. The aim of this study was to evaluate the
ability of various antiandrogens to block the AR into the cytoplasm and to
define their mechanism of action.
We developped in a previous work a model based on a fusion protein between
the AR and the green fluorescent protein (GFP) to study the intracellular
dynamics of AR. The fusion protein GFP-AR conserves the principal
biochemical and functimlal properties of AR. The nuclear transfer process of
AR is hormone-binding dependent and energy dependent.
We tested different steroidal antiandrogens (cyproterone acetate and RU2956)
and non-steroidal antiandrogens (hydroxy-flutamide, inocoterone, bicalutamide
and nilntamide).
To study the AR transloca~.ion, cells were transfected with GFP-AR and
observed directly under microscope. The AR remained in large part in the
cytoplasm only in presence of bicalutamide or nilutamide. These latter
antiandrogens prevented also the nuclear transfer normally cansed by
androgen. The transcriptional activity of GFP-AR was measured by
cotransfection of GFP-AR and an androgen-regulated gene reporter. The
bicalutamide and nilutamide exhibited any agonist activity and a high
antagonist activity. These drugs could be defined as pure antiandrogens. One
hypothesiq to explain the blockade of A R into the c3toplasm could be that the
AR bound to the bicalutamide or the nilutanaide does not dissociate the heat
shock proteins normally dissociated by agonist.
Because the AR localization seems to be the hmiting step in the bioactivity of
the antiandrogen, this highlights the high interest of the dynamics study of the
AR. GFP-AR model represents an ideal tool for screening of anliandrogens
and could be applied more particularly in the screening of new antiandrogens.

(We/8.3/139)

The S-layer protein of C o r y n e b a c t e r i u m g l u t a m i c u m :

translocation and interaction with the cell wall.
C. Houss~ D.T.Nguym,M_~
E. ~
~d N. Bayart
~

desl~manlrnne~ Unive,~ deP~is )~,,91405Oray oMe~Frc~nce.

Evidence of the role of PKC during aclacinomycin

induction of erythroid differentiation in 1(562 cells
R. Gillet, C. Dupont, P. Jeannesson and C Trentesaux
Laborctotredel~odf~e a l~ologte~~ ,
L~ ~ e
- IFR 53
51, rue~ a y
51096ReonsCed~ Franaz E.cnal. repnddg i ~ m x f l "

At subtoxic concentrations, anthracycline drugs are effective in

controlling erythroid differentiation of K562, a human erythroleukemic
cell line, leading to the appearence of hemoglobinized cells.
Aclacinomycin stimulates this process by an enhancement of specific
erythroid gene transcription implicated in hemoglobin synthesis.
Nevertheless, elements about early events, in particular signal
transduction pathways responsible of the molecular variations of the
cells, remain to be elucidated.
To better understand this process, we used bisindolylmaleimide
(GF109203X), an inhibitor with a high selectivity for PKC, a family of
phospholipid dependent serine-threonine kinases, which has been shown
to play a key role in the processes of proliferation / differentiation of a
wide variety of malignant cells. We showed that GF109203X inhibits
aclacinomycin induction of erythroid differentiation on K562, evidenced
by a strong reduction of hemoglobinized cells. This inhibition was
accompanied by a marked decrease of mRNA rates of erythroid genes
and also by a decrease of the protein level of GATA-I, a transcription
factor specific of erythroid differentiation.
To establish firmly PKC involvement, we also verified that
aclacinomycin stimulates rapid translocation of its activity, from the
cytosolic to the membrane compartment. By Western-Blot analysis, we
showed that after short induction times, PKCct was the most implicated,
as early as five minutes. Further studies will permit a better
understanding of the relationships between transduction pathways and
activation of transcription factors, that lead to a specific response of the
cell in function of the stimulus.

(We/8.3/140)

Colicin A interacts with a third component of the

Tol/PAL complex, TolR, upon its import into E. coli cells
L. Joumet, E. Bouveret, A. Rigal, C. Lazdunski and
H. Brnrdetti. L1SM-CNRS, 31 chemm J.Aiguier,
13402 Marseille Cedex 20. France.

Bacterial cell envelopes are complex structures which play a fundamental

role in the interaction of the cell with its environment. Corynebacterium
glutamicum is a Gram positive bacterium whose cell envelope is similar to
that of Mycobacteria. It is composed of a cytoplasmic membrane surrounded
by a thick peptidoglycan layer to which mycolic acids are covalently bound
and probably organized in a membrane like structure. Additionally to this
basic structure, Corynebacterium glutamicum cell surface displays twodimensional ordered arrays composed of a single protein called PS2. Because
of its location and of its high level expression, PS2 is a good model for
investigating mechanisms involved in protein translocation and in protein
interactions with the cell wall.
PS2 is synthesized with an N-terminal extension which is cleaved during
transport across the envelope. The transport of PS2 from the cytoplasm to the
outermost part of the cell wall has a quite slow kinetic (t V2of 3 mn) and is
abolished in the presence of CCCP (a protonophore) or sodium azide.
Deletion of a large C-terminal domain of the protein (300 aa out of 510)
increases its rate of transport (t '/2 < 1ran) and leads to the secretion of the
truncated polypeptide (PS2Ac) in the external medium. Association to the cell
wall is partially restored (70%) when a polypeptide chain, corresponding to
the last 90 aa of PS2, is fused to PS2Ac, and is fully restored when the fused
chain is extended to the last 200 aa of PS2. This result suggests that at least
two distinct domains of the C-terminal part of PS2 cooperate to anchor the
protein on the cell surface. Systematic deletions inside of the first domain
show that a hydrophobic sequence of 31 aa length, located at the C-terminus
of the protein, is essential since its deletion completely abolished cell wall
association. We propose that this hydrophobic tail anchors PS2 on the outer
membrane-like structure upon its arrival, in an unfolded state, at the surface
of the cell. This interaction would prevent secretion of PS2 and may help the
protein to diffuse laterally and interact with other monomers while folding in
its stable conformation. This will result in the formation of two-dimensional
ordered arrays and in the stabilization of the interaction between PS2 and the
underlying hydrophobic structure through weak bonds involving the whole Cterminal anchoring domain.

Key words : bacterial toxin, importation, protein-protein interaction

Colicins are bacterial toxins produced by and active against Escherichia coli.
They are divided in three distinct structural domains, each of which having a
particular fonction : the central domain is responsible for binding of the
colicin to its receptor at the cell surface of the target cell, the N-terminal
domain is involved in the import process across the bacterial envelope (also
called translocation step), and the C-terminal domain carries the lethal
activity. Group A colicins have parasitized an envelope complex, the Tol/Pal
system, to perform their translocation step. The Tol/Pal system consists of six
proteins : TolA, TolQ, and TolR are inner membrane proteins ; TolB and
Orf2 are periplasmic; and Pal is an outer membrane lipoprotein. Previous
work demonstrated that the N-terminal domain of colicin A interacts with
TolA and TolB, and that the N-terminal domain of colicin E3 interacts with
TolB. These interactions are necessary for the import of colicins in target
cells. The precise regions of colicin A N-terminal domain interacting with
TolA and TolB have been determined.
The interaction of TolR with colicin A translocation domain was investigated.
In vitro cross-linking with purified TolR periplasmic domain (TolRII) and
colicin A translocation domain showed that TolRII interacts in vitro with
colicin A translocation domain. Furthermore, in vivo cross-linking and coimmunoprecipitation experiments performed directly on cells producing
colicin A N-terminal domain in their periptasm showed that TolR interacts in
vivo with colicin A translocation domain. The precise region implicated in the
interaction with TolR is being determined by site directed mutagenesis. It
already appears that the interaction with TolR is required for colicin A import
into bacteria.
These results provide the evidence that colicin A interacts with a third protein
of its translocation system, TolR, during its translocation step. Implications of
this result on our understanding of the molecular mecanisms of group A
colicins translocation will be discussed.

Abstracts FEBS'99

(We/8.3/141)

Pex5p, the peroxisomal import receptor for PTSI containing

proteins: structure-function analysis
A.T.J. Klein, P. Barnett, G, Bottger, B. Distel and H.F. Tabak
Department of Biochemistry, Academic Medical Center, University of
Amsterdam, Meibergdreef15, l l 05 AZ Amsterdam, The Netherlands.

Peroxisomes are celorganelles in which a number of different metabolic

processes take place. In S.cerevisiae the complete 13-oxidation of fatty acids is
localized inside the peroxisomal compartment. Proteins which are involved in
the 8-oxidation of fatty acids are directed to the peroxisome via a peroxisomal
targeting signal (PTS). For the import of peroxisomal matrix proteins two
different targeting signals have been identified; PTS1 and PTS2.
Pex5p is a mobile receptor that binds proteins with a PTS1 in the cytosol and
directs them to the peroxisome. Docking at the peroxisomal membrane occurs
through the binding of Pex5p to a protein complex located at the peroxisomal
membrane.
By using the two hybrid system the interaction between Pex5p and PTS 1 can
be reconstituted. The exact binding site in Pex5p for PTSI is not known but it
has been suggested that the seven TPR domains in the C-terminal part of
Pex5p are important for this interaction. TPR domains are found in a lot of
proteins and they are involved in protein-protein interaction. To identify the
residues in Pex5p that are involved in the binding of PTS 1 proteins we
randomly mutagenized the PEX5 gene by error prone PCR.
We have employed two different approaches to screen for Pex5 mutants that
are affected in the interaction with a PTSI protein. By using the two hybrid
system we have isolated Pex5 mutants which have lost the interaction with
PTS 1. We have also used the two hybrid system to isolate suppressor
mutations in Pex5 that restore the interaction with the mutated PTS 1.
In the second screening method we have made use of a protein with a mutated
PTS 1 that cannot he imported into peroxisomes by wild-type Pex5p. Pex5p
mutants were isolated which were able to import this protein with the mutated
PTS 1.
For all these Pex5 mutants the site of the mutation was determined.
Only recently the first 3D structure of a TPR containing protein has been
resolved [1]. Mapping of the mutations on the TPR domain structure should
provide insight in the molecular basis for Pex5p mediated PTS 1 recognition.

(We/8.3/143) Protein secretion in Pseudomonas aeruginosa: a complex

machinery
G. Michel, A. Filloux and A. Lazdunski
LISM,CNRS,UPR9027, IFR1,31 cheminJosephAiguier, 13402
MarseilleCedex20. France.
The opportunistic pathogen P s e u d o m o n a s aeruginosa is a Gramnegative bacterium able to secrete numerous proteins into the extracellular
medium. Most of these proteins were found to be virulence factors. We
have previously cloned and identified the gene products that constitute the
secretion machinery. These genes are conserved among Gram-negative
bacteria secreting proteins by the a two-step secretion pathway, defined as
Type II, or General Secretory Pathway (GSP). Many of the 12 components
of the secretory system, named XcpP-Z and XcpA, probably assemble in
the cell envelope into a proteinaceous complex [ 1].
Some of the Xcp components are not directly involved in the
secretion process but are required for assembly of the secretion machinery.
Interestingly, a subset of these Xcp proteins show homologies or are
identical to proteins involved in the assembly of type IV firnbriae (Pil
proteins in P. aeruginosa) [2]. The current knowledge on the molecular
architecture of the Xcp machinery, via the identification of protein-protein
interactions, will be discussed.
The Xcp complex contains a so-called secretin, which is the
ultimate pore for the passage of exoproteins across the outer membrane
[3]. The central cavity of such a pore is sufficienly large to allow the
translocation of folded proteins. The gating of this pore appears to be a
prerequisite for preserving cell integrity. In addition, some of our results
lend support to the idea of envelope compartmentation suggesting that the
machinery may be localized at specific areas of the cell envelope.
[t] Filloux et aL, FEMS Microbiol. Rev., 22, (1998), 177. [2] Strum et al.,
Annu. Rev. Microbiol., 47, (1993), 565. [3~ Bitter et al., Mol. Microbiol.,
27, (1998). 209.

s311

(We/8.3/142)

Nuclear transport of histone 23 in rat liver is mediated by a signal

and energy-requirerlng pathway independet of importio ct / 13
T Langerand H. Faseld
Instttute for B~ochemlst~. t3rocentre of the J. W .(;oethe-Umversiiy, Ala~ve-CurTeStr 9, 60439 Frankfurt am 3[atn, Germany, e-mad tlanger~q~stud urn-frankfurt.de

The nuclear envelope exhibits a sieve like permeabilty against inert

molecules such as dextrans. For transport of molecules > 60 kDa presence of a
nuclear localization signal (NLS) is mandatory These signals exist of one or
more short amino acid stretches, with a predominance of basic amino acid
residues. The NLS-bearing protein binds to the NLS-receptor which is known
as importin o~ Ttus complex is then recognized by a member of the importm 13family Formation of the transport complex and docking to the nuclear pore
complex (NPC) is energy independent whereas transport through the NPC is
an energy-consuming process and requires the small GTP-binding protein Ran
Since histones are small proteins with a molecular mass between 1l and 23
kDa they might be expected to enter the nucleus solely by diffusion Histones
bear a high content of basic amino acids, so that many sequences can fit into
the consensus NLS-sequencas
Histone 2b nuclear transport was investigated using digitonin permeabilized
cells and the rat liver reseated nuclear envelope system [1] The reseated
vesicles still respond to the importin signal for the uptake of NLS-bearing
proteins [2] During the preparation of the vesicles chromatin is extracted in
cold hypotonic heparin-containing buffer. This allows investigation of the
import mechanism independent of intranuclear binding to chromatin In
permeabilized cells, H2b uptake in nuclei reaches maximal levels only by
adding cytosol and an appropriate energy source Addition of recombinant
importin ct, importin !3, Ran, ATP and GTP does not fully restore uptake of
H2b neither in resealed vesicles nor in nuclei of permeabilized cells as it is the
case for nucleoplasmin Import of H2b cannot be blocked by addition of
excess of a synthetic NLS-bearing peptide or Nucleoplasmin but it can be
diminished by chilling or energy depletion These results suggest that H2b
uptake is mediated by a special energy-requiring transport mechanism
independent oftbe receptor for classical NLSs, importin ct and importin J3
[1] Riedel, N & Fasold, H, Biochem J, 241 (1987), 203
[2] Hassel, I et al, Eur J Biochem, 241 (1996), 32
(We/8.3/144) CNBr cleavage of the overexpressed mitochondrial pF113. A
new strategy to construct an import competent preprotein
P.F. Pavlov. P. Moberg, X-P. Zhang and E. Glaser
Department of Btochemlst~, Arrhemu~ l~tboratortes for Natural Sctences
Stockholm Umverstt 3, 10691 Stockholm, S~eden

We have isolated a soluble import-competent 15 kDa N-terminal fragment

of the overexpressed Nicotianaplumbaginifolia Fll3 precursor of the ATP
synthase (N 15pFl13). The isolation was achieved after cyanogen bromide
chemical cleavage of the insoluble precursor collected in inclusion bodies,
followed b3 purification of the fragment using ion-exchange
chromatography. Purity of the final product was estimated to be > 99%.
The N 15PF 113 fragment contained a presequence of 54 amino acids (except
for the N-terminal methionine) and 82 N-terminal amino acids of the
mature protein. The NI5pF113 fragment was shown to be imported into
isolated potato tuber mitochondria and to be processed by the isolated
mitochondrial processing peptidase (MPP) integrated into the cytochrome
bc 1 complex of the respiratory chain. Addition of the N 15PF 113 fragment,
at ~M concentrations, resulted in the inhibition of import of the in vitro
transcribed and translated Fl[3 precursor (PFl[3) and alternative oxidase
(pAOX) precursor into isolated mitochondria as well as processing of
these precursors catalyzed by the isolated MPP/bc 1 complex. The
N[ 5pFl[3 fragment conjugated via biotin link to avidin stocked import
sites even after reisolation of mitochondria and inhibited import of the
mitochondrial precursors indicating that it can be used as a substrate for
generation of a stable translocation intermediate. Our results present a
novel procedure tbr production of an N-terminal fragment of the F 113
precursor which contains all information necessary for mitochondrial
targeting and processing and which can be used for structural and
functional studies of the mitochondrial protein import system. This
procedure has a general value as it can be used for production of chemical
quantities of any mitochondrial import substrate and presequence peptide.

s312

(We/8.3/145)

Abstracts FEBS'99

Translation in plant mitochondria: the need for import

of aminoacyl-tRNA synthetases AND tRNA
N. Peeters, K. Akashi. A. Chapron, D. Lancelin, 1. Small
Station de Gdndtiqtw, hVRA, Ver~aille~. Flalle

Despite a larger genome than other mitochondria, plant mitochondria still only
code for a few of their own proteins. The aminoacyl-tRNA synthetases
(aaRSs), key components in protein synthesis, make up some of the numerous
proteins encoded by the nucleus and imported into mitochondria. Interestingly,
the aaRSs in plant mitochondria can have different phylogenetic origins: in
Arabidopsis thaliana mitochondria, three examples are representative of this
variety: alanyl-tRNA synthetase is cytosolic-like, asparaginyl-tRNA synthetase
is chloroplastic-like and cysteinyl-tRNA synthetase is mitochondrial-like. For
more details, see the taaRSAt database developed by I. Small and co-workers
(abstract and poster).
Not only the aaRSs are imported but also some tRNAs. It has been shown that
all plant mitochondria tested import tRNAs, and the subset that is imported
varies between plants [1]. We hypothesise that aaRSs could be implicated in the
import of their cognate tRNA into the mitochondria. There is some evidence for
this fi'om both yeast [2] and plants [3]. Indeed, when imported tRNAs and the
corresponding aaRSs are both of cytosolic origin (which is generally the case in
plants) it is even possible that the two molecules are co-Emported via the protein
import channel. A general involvement of aaRSs in deterreming the specificity
of mitochnndnal tRNA import Is consistent with aminoacylation data. For
example, in sunflower mitochondria, which import tRNA "" . the scryl-tRNA
synthetase activity is indistinguishable fi'ore the cytosolic achvilv, wherea', in
plant nfitochondria that do not import tRNA" the cylosolic and reltochondrutl
SerRS activities are clearly different.
We would like to try and obtain direct evidence lor the role of the aaRS in tRNA
impor! mto mitochondria. Rice mitochondria do not 1report tRNA'"', whereas
wheat mnochondrla do [I]. To verify if wheat histidyl-tRNA synthetase is
controlling this import, we are analysing transgemc rice plants expressing wheat
HisRSs.
If we succeed in transfen'ing tRNA import specificity from one plant to
another, we hope to widen our research to include studms on the maport
mechanism itself and the possibihties for deliberate modification of
reitochondriat gene expression.
[1] Kumar el al., Mol. Gen. Genet., 252, (1996) 404.
[2] Tarassov et al., EMBO J., 14, (1995) 3461.
[31 Dietrich et al., Plant J., 10, (1996) 913.

"Dept. of Biochemistry& bChemometncs,Dept. of OrgamcChemxstry,

Umeh Umversity,Umeh, Sweden

Signal pepttdes are essentml N-terminal extensions in export proteins, and

usually have a positively charged N-terminus, a hydrophobic central core of
certain length, and a C-terminal cleavage region. They interact in a
consecutive manner with different accessory proteins during the secretion
process. Potential patterns or periodicity in the amino acid sequence were
searched, using multivariate techniques (PLS, PCA), for a large number of
signal peptides from Mollicutes (mycoplasmas), other Gram-positive
bacteria, and Escherichia colL
Mollicutes signal peptides were significantly different from the E. coli and
Gram-positive ones by their N-terminal charge, peptide length, and unique
periodicities of side chain hydrophobicity and volume. Their hpoprotein
signal peptides were longer than for any other bacteria. Statistically
significant differences were also recorded between the other bacterial
peptide groups. Among the Bacillus signal peptides the typical amino amd
patterns were more related withm several groups of conserved, secreted
enzymes than for peptides from the same bacterial species. In E. coh signal
peptides from proteins routed for the various cellular destinations revealed
several significant and compartment-specific sequence patterns; a resolution
not evident by other methods. This was substantiated from a large number
of signal peptide secretion mutants for an E. coli periplasmic space protein.
It is proposed that the differences unravelled in amino acid patterns and
side-chain properties are related to the secondary structure sidedness and
topology of the signal peptides, and important for specific interactions
during the secretion process.

(We/8.3/146)

Crystallisation of the Alu domain of the mammalian

signal recognition particle
C. Stehlin~, O. Weichenrieder#,K. Stmb* & S. Cusack#
#EuropeaJ~MoleeMarB~ologvLaboratot3; Grenoble Outstation, France
* Department of Cell BIolo~; Universityof Geneva, Swazerhmd

The mammalian signal recognition particle (SRP) is an I1S cytoplasmic

ribonucleoprotein that plays an essential role in targeting of secretory and
membrane protein to the rough endoplasmic reticulum (RER). It comprises a
300 nucleotide SRP-RNA to which are bound 6 proteins: SRP9, SRPI4,
SRPI9, SRP54, SRP68 and SRP72. The Alu domain consists of the protein
heterodimer SRP 9/14 and the 5" and 3' part of the SRP RNA known as the
Alu sequences. Previously. the functional identification and analysis of a
minimal RNA folding domain, called SA86 [l] ~as reported.
We have reconstituted a ternary complex comprising human SRP9, SRPI4
and various in vin.o transcribed RNAs. Different crystal forms have been
obtained, the best of which diffracts to 4.5 A resolution.
[ I] Wemhenrleder& al.. RNA, 3 (1997) 1262

Abstracts FEBS'99

s313

13.4 Growth factor receptors

(We/13.4/148)

Interaction of soluble p75 NrRectodomain in sensory

and sympathetic tissues from embryonic chicken
P. Amieux ~and M. BothwelP

(We/13.4/149)

"Department of Physwlogy &Btophystcs, University of Washington

Seattle, Washington 98195 USA

The low affinity neurotrophin receptor (p75 ~ ) was the first member cloned
of a large superfamily of receptors containing a series of cysteine-rich
extraceUular repeat domains. Signaling via this family of receptors can elicit
both survival and death responses in target cells depending upon the context.
Members of the p75S~rt/TNF receptor superfamily occur in both soluble and
membrane-associated forms. Soluble p75 NTRcan be isolated from human
urine, plasma, and amniotic fluid, and its production also appears to be
developmentally regulated [1,2]. In order to address the hypothesis that
soluble forms of the p75 NrR may modulate cell signaling, we have used the
human p75 NTRectodomain as an affinity probe in frozen sections of embryonic
chicken [3]. We observe highly specific staining of sensory and sympathetic
tissues and skeletal muscle precursors, all of which normally express p75 NTR
holoreceptor. We hypothesize that soluble forms of the p75 NrR may interact
with endogenous p75 NrR at the cell surface and modulate cell signaling.
[1] DiStefano, P.S. and Johnson, E.M. (1988) PNAS 85,270.
[2] Zupan, A.A. et al. (1989) J. Biol. Chem. 264, 11714.
[3] Chapman, B.S. et al. (1996) J. Neurochem. 66, 1707.

Sex/plexins: a wide family of cell surface receptors

mediating cell-cell interactions
S. Artigiani, L. Tamagnone, P. Angelini, P.M. Comoglio.
Institute for Cancer Resarch, Umversity of Torino. Candiolo (TO), Italy

We have identifed a family of human genes, encoding cell surface

proteins with homology to the extracellular domain of the HGF/SFReceptor tyrosine kinase: these were named SEX,, SEP, O C T and N O V [1].
Mouse homologs of these proteins were independently found and named
"plexins"[2]. We now describe four novel human members of the large
SEX/plexin gene family. Intriguingly, the extracellular domain of
Sex/plexin receptors also shares similarity with semaphorins, a wide
family of soluble and membrane bound ligands. It was recently shown that
-in Drosophila- plexinA interacts with semaphorin-I [3].
In mammalians, the expression of a subset of the SEX/plexin genes
is restricted to nervous tissues in early developmental stages. In human
cell lines, Sex/plexin family members have distinctive expression patterns:
i.e. some of them are widely expressed in epithelial and mesenchymal
cells, while others are restricted to specific cell types.
We observed that Sex/plexin3 protein can both mediate calciumdependent homophilic cell interactions and convey repelling cues to
adjacent cells in culture,
The large cytoplasmic domain of the Sex/plexin receptors is
strikingly conserved among family members and in evolution. It includes
a number of potential tyrosine phosphorylation sites. Although, it does not
contain a bona fide tyrosine kinase domain, it may be tyrosine
phosphorylated by associated kinases.
[l] Maestrini E. et al. Proc. Natl Acad Sci. USA 93 (1996), p.674.
[2] Kameyama T. et al. Bioch. Bioph. Res Comm. 226 (1996), p.396.
[3] Winberg M. et al. Cell 95 (1998), p.903.

(We/13.4/150)

Slap, a negative regulator of mitogenesis

P. Bello, G. Manes, S. Roche
CRBM. CNRS UPR 1086, 34293 Montpellier, France

(We/13.4/151)

Specificity of the proteins of the Grb7 family in

growth factor signaling
V. Brrrziat, A. Kasus-Jacobi, D. Perdereau, J. Girard,
A.-F. Burnol
CNRS UPR 1524, 9 rue Jules Hetzel, 92190 Meudon, France

Slap (Src-like adaptor protein) is a widely expressed adaptor molecule

containing an SH2 and SH3 domain which bear a striking homology to
those of src, followed by a unique c-terminal domain and was originally
shown to interact with the tyrosine kinase receptor, Eck [1]. We have
recently reported its expression in NIH 3T3 fibroblasts and its ability to
interact with the activated PDGF receptor in an SH2-dependent manner [2].
In contrast to other known adaptor proteins such as Shc and Grb2, Slap
appears to inhibit mitogenesis ; we were unable to produce stable Slap
overexpressing fibroblast cell lines and microinjection of Slap into quiescent
fibroblasts inhibited both PDGF- and serum-induced DNA synthesis.
Furthermore, microinjection of Slap antibodies potentiated the cells
response to sub-optimal doses of growth factor and serum. Slap may act by
inhibiting/antagonising Src-dependant/Ras-independant pathway(s) as micainjection of fibroblasts with c-Myc but not with Fos, could overcome the
apparent G1 >S block. Further supportive evidence was obtained when Slap
was transfected into fibroblasts transformed by various oncogenes ; stable
Slap transfectants were obtained from v-Src transformed fibroblasts but not
from cells transformed by v-Raf, V12Ras and V12Rho nor the activated
tyrosine kinases, v-Abl and SrcY527F. Also, c-Src overexpression could not
overcome the Slap GI>S block in microinjection experiments and Slap was
unable to reverse the transformed phenotype of v-Src transformed
fibroblasts. Biochemical analyses of these cell lines has afforded some
insights into the growth inhibitory effects of Slap and will be presented.
Collectively, the results propose that Slap negatively regulates the Src
signaling pathway initiated by growth factors and this influence resides at
the level and/or activity of Slap.

1. Pandey, A., et al, 1995. J. Biol. Chem., 270(33), 19201-19204

2. Roche, S.,. et al, 1998. Current Biology, 8(17), 975-978

Growth factors act through specific transmembrane receptors,

belonging to the receptor tyrosine kinase (RTK) family. After ligand
stimulation RTK recruit various SH2-containing signaling proteins. Among
them, a new family of adapters is now emerging, comprising Grb7, Grbl0
and Grbl4. These Grbs proteins are characterized by a succession of
interaction domains: poly-proline, pleckstrin-homology and SH2. They also
display a newly defined domain, named PIR or BPS, implicated in the
binding to the insulin receptor (IR) [I; 2].
To get insight into the respective implication of Grb7 and Grbl4 in
growth factor signaling, we studied their interaction with different RTKs
(EGFR, FGFR, Ret and IR), by using various approaches (two-hybrid,
GST-pull down, immunoprecipitations). Our results show that, although a
better interaction was observed with the IR, Grb7 is able to significantly
interact with all RTKs tested. By comparison, Grbl4 is likely to display a
specific interaction with the IR: the association of Grbl4 with the IR was 30
to 100 fold higher than with the other RTKs.
Then we used deletion mutants to determine the binding domains of the
Grbs proteins. The SH2 domain of Grb7 is responsible for the interaction
with all RTKs. The Grb7 PIR interacts with the IR using sensitive techniques
like the two-hybrid system, but clearly the SH2 domain is also the major 1R
binding domain of Grb7. By contrast, the SH2 domain of Grbl4 is
dispensable for the interaction with the IR, which is dependent on the Grbl4
PIR domain.
In conclusion, these experiments argue in favor of a specific role of
Grbl4 in insulin signal transduction, and the PIR seems to be the determinant
of this specificity. The respective importance of the PIR and SH2 domains in
the binding of Grbs to RTKs is thus likely to be a clue for the specificity of
these interactions, and thus for a role of the different Grbs in growth factor
signaling.
[1] Kasus-Jacobi et al., J. Biol. Chem., 273, (1998) 26026.
[21 He et al., J. Biol. Chem., 273, (1998) 6860.

s314

(We/13.4/152)

Abstracts FEBS'99

Meiosis resumption ofXenopus oocytes induced by activation of

IGF-I receptor specifically requires calcium mobilization
F.Chesnel', P.Benquet', C.Daguzan b, M.Moreaub, F.Tiaho ~, D.Boujard"
~L'PRES-Ar026('.VRS-Untvers~tbde Rennes 1-35042 Rennes cbdex France

(We/13.4/153)

UPRES EA 1033 Biologie du Ddveloppement,

bL31R5547 CVRS-Lbtverslt~ Paul Sabane-31062 7oulouse codex, France.

In Xenopus postvitellogenic oocytes, resumption of meiosis (also called

maturation) induced by insulin or IOF-I is mediated by the IGF type I
receptor. This mitogenic effect specifically involves the Ras-dependent
activation of MAPK cascade and leads to MPF activation and all subsequent
events allowing meiosis resumption of the oocytes from prophase I to
metaphase II. However, the signalling pathways upstream of or parallel to the
activation of Ras are not fully elucidated yet.
In several studies, an inhibition of insulin-induced meiosis resumption has been
observed when oocytes were incubated in 2 5 mM potassium-containing
medium, which has not been explained so far. Electrophysiological recordings
performed in the laboratory showed that resting membrane potential of
oocytes incubated in K-containing medium was around -80 mV (vs -30 mV in
K'-free medium). Such a low membrane potential in K--containing medium
could prevent membrane calcium channels opening and therefore block insulininduced meiosis, if any calcium influx through these channels was required
during that process. Using the photoprotein aequorin, we then measured
intraoocyte calcium variations following exposure to insulin or progesterone
(the natural inducer of meiosis resumption in Xenopus oocytes). We observed
with both inducers a long-lasting increase of intracellular calcium from 2-3
hours to 10 hours post-stimulation. In addition, insulin specifically triggered a
short-lasting calcium increase starting a few minutes after the stimulation and
which decreased back to the control level within 2 hours.
In order to determine whether this intracellular calcium mobilization was
required and its origin, oocytes were incubated during stimulation in calciumflee medium (containing EGTA), in presence of calcium channel blockers
(Ni z~, verapamil) or in presence of an inhibitor of calcium release from internal
stores (TMB-8) Results showed that the intracellular calcium increase was
required for insulin but not for progesterone to induce resumption of meiosis
and that both extracellular as well as calcium released from endoplasmic
reticulum contributed to this increase. The mechanisms by which IGF-1
receptor triggers an increase of intracellular calcium as well as the role of this
cation in facilitating the mitogenic effect of insulin are currently under
investigation
(We/13.4/154)

S 14-95, a new J A K / S T A T inhibitor from a Peniciilium sp.

G. Erkel*, M. Saul*, T. Anke*, H. Anke* & O. Sterner*"
* 1BWF. Paul-Ehrlich-Str.23. 67663 Kaiserslautern, Germany
** Division o f Orgamc Chemistry 2, Universtty o f Lund, Sweden

A variety of cytokines, growth factors and hormones trigger

phosphorylation of latent cytoplasmatic transcription factors termed STATs.
The S T A T s become phosphorylated on tyrosine residues by one or more
members of the JAK family of proteine tyrosine kinases, then assemble in
dimeric or oligomeric form, enter the nucleus and regulate transcription of
many genes by binding to specific DNA sequences. Beside the NF-r~B
signalling pathway components of the JAK-STAT pathway play an
important role in the stimulus-dependent, transcriptional activation of many
inilammatory genes (e.g. NOS II, ICAM-1). Therefore compounds
interfering specifically with the JAK/STAT pathway could have an
application as antiinflammatory drugs.
In order to search for new inhibitors of the JAK/STAT pathway, IFNy/IFN-cx inducible reporter gene vectors were constructed and 500 mycelial
cultures ofbasidiomycetes, ascomycetes and fungi imperfecti were examined
for the production of compounds which inhibit the stimulus dependent
expression of the reporter gene (SEAP) in
HeLa $3 cells. Cultures of a Penicillium
o
o
species were found to produce a new
inhibitor of the IFN-~, dependent signalling
S 14-95
pathway in HeLa $3 cells. The new
| o
compound S14-95 inhibited the IF'N-?"
dependent expression of the reporter gene
AcO~"
"~
in HeLa $3 cells with an IC~0 value of 5
gg/ml. The NF-rJ3 mediated expression of
the reporter gene was inhibited 50 % at a
ten times higher concentration. No influence on AP-I and glucocorticoid
receptor dependent SEAP expression could be observed up to 50 p_g/ml.
The compound showed no antibacterial or antifungal activities. Weak
cytotoxic activities could be observed starling fi'om 50 I.tg/ml. The mode of
action
of
S 14-95
is
currently
under
investigation.

Mitogenic effect of Nerve Growth Factor for human breast

cancer cells.
S. Descamps 0X. Lebourhis, B. Boilly, H. Hondermarck.
Untverstt~ de Ltlle 1, 59655 Vdleneuve d'A~cq, France

Nerve growth factor (NGF), the prototypic member of the

neurotrophin familly, is well known for its stimulation of differentiation,
survival and regeneration of sympathetic neurons. We have demonstrated
that NGF is able to stimulate breast cancer cell proliferation, but has no
effect on the growth of normal breast epithelial cells [1]. This activation
induces recruitment of the GO cells to reenter the cycle and a reduction of the
G1 phase duration. We have shown by western blot and
immunocytochemistry that breast cancer cell lines as well as normal ceils
exhibit both types of NGF receptors : TrkA and P75. NGF stimulation of
proliferation induces an increase in tyrosine kinase activity of TrkA and an
activation of the mitogen-activatcd protein kinascs (MAPK). This activation
is required for NGF mitogenic effect as demonstrated by use of the specific
inhibitors K-252a and PD98059. The functionality of both PI3 kinase and
PKC is also necessary to obtain NGF mitogenic effect whereas Src activation
does not seem to be involved. NGF naturally exists in mammary gland [2, 3]
and so our results suggest that NGF is implicated in the growth of human
breast tumors. A real time quantitative RT-PCR study is in progress and will
allow to define the expression level of NGF and its receptors in the different
cell lines as well as in breast cancers.
[1] Descamps, Set al., J. Biol. Chem., 273, (1998) 16659
[2] Murphy, RA et al., Proc. Natl. Acad. Sci. U.S.A., 74, (1997) 2330
[3] Grueters, A e t al., Pediatr. Res., 19, (1985) 934

(We/13.4/155)

Identification of a region in the activin molecule that is

essential for initiation of signal transduction
W.H. Fischer, M. Park, S. Koerber, W. VaJe
The Satk lnsnttae, The Clayton Foundatton Laboratorte~for Pept~de
Btology, L~zJotla, CA 92037, USA

Activin A is a pLuripotent hormone/growth factor. Its activities

include hormonal actions such as FSH release from the pituitary, a role in
erythropoiesis, in mesoderm reduction as well as in neuronal survival and
development. Activin, which is a member of the TGF-[3 superfamily of
growth and differentiation factors, signals through two types of receptors.
These receptors are single transmembrane proteins which possess an
intracelluLar serine/threonine kmase domain. Initial binding occurs to the type
II receptor (ActRII). The type I receptor is recruited into the complex and
phosphorylated. This leads to an activation of the type receptor's kinase
domain, and signal is propagated by phosphorylation of intracellular targets.
We have investigated the involvement of predicted surface regions of
the activin molecule in receptor binding and signaling interactmns. A threedimensional model of activin was developed based on the X-ray structure of
TGF-[32. Based on this model we have designed a series of mutant proteins in
which these regions are deleted while leaving the overall topology of the
molecule intact. The mutant proteins were generated by expression in a
baculovirus/insect cell system and tested for their biological activity as well as
for their ability to bind to the type I1 receptor. Mutants wtth deletions in the
N-terminal and central portion of the molecule exhibited biological activities
and binding affinities that were reduced 3-10-fold. A mutant with a deletion
in the C-terminal finger of the activin molecule was approx. 100-fold less
active than the native protein. This mutant also exhibited the lowest affimty
for the type II receptor. We therefore propose that this region is prominently
involved in signaling interactions.
The mutagenesis data were used to develop a model for the activinActRI1 receptor complex. The three-dimensional modcl of activm and the Xray structure of the ligand binding domain of the ActRll [l] were subjected to
semi-automatic docking procedures. The candidate models were inspected for
their compatibility with the results of this mutagenesis study. The predictions
of these models are currently being tested by further investigations.
[1]
Greenwald J. et al., Nature Struct. Biol. 6, (1999) 18.

Abstracts FEBS'99

(We/13.4/156)

E x p r e s s i o n of NGFR in Mouse Sertoli ceils

producing recombinant rat Androgen-binding protein

(We/13.4/157)

Phospho-tyrosine independent interaction of

a putative signal transducer with HGF-receptor

A.Oerard,a, G.L. Hammond,b, K. Hess,c, H. Oerard,~

S. Grisendi a, T. Crepaldi a, B. Chambraud b and P.M. Comoglioa

a, c E24OI "G6.n611qtle et Interactions cellulaires en Reproduchon "

D6part Histologle-Embtyologle, et BiochimJe, Facult6 de M6decine, Nancy,
France, b LR('(" Cancer Research I,aboratorie% London , Ontario, ('anada

alnst for Cancer Research. Umverstty of Torino, 10060 Candiolo (TO),ltaly

blNSERM U488 and Colldge de France, 94276 Bwetre Cddex, France

Background.:
Nerve growth factor (NGFR) has been localized in Sertoli cells, in its
apical part facing the lumen and in the basal part [1]. B-NGF is produced
by germ ceils and stimulates androgen-binding protein (ABP) mRNA
[2] The expression of NGFR mRNA and protein showed maximal levels
at stages VII and VIII of the seminiferous cycle when ABP secretion is
increased at maximal level.
ABP is thought to have a paracrine role within the seminiferous tubule on
germ ceils and an autocrine role as well[3]
Aim and d e ~ n
Our aim was to check whether the presence of germ cells or the presence
of ABP was responsible for the induction of NGFR in Sertoli cells.
The experimental design was to compare, using immunocytochemical
procedures, the Sertoli cell NGFR expression in vitro in the absence of
germ cells in 1) mouse Sertoli cells non-producing ABP, 2) Sertoli cells
producing ABP under FSH stimulation, 3) rat ABP-transfected mouse
Sertoli cells producing recombinant rat ABP.
Non testicular Chinese hamster ovary cells (CHO) producing recombinant
rat ABP were used as controls.
Results:
There was significant difference between the undetectable levels of NGFR
in Sertoli cells producing noABP, a low level in FSH-stimulated Sertoli
cells, while Sertoli cells producing rat ABP showed significantly high
levels ofNGFR. CHO cells did not exhibited any labeling.
The NGFR level was positively correlated with the amount of the
recombinant protein in the culture medium.
From these results, we conclude that NGFR is expressed in Sertoli cells in
the complete absence of germ cells. The expression of NGFR needs the
presence of ABP. High concentration ofintracytoplasmic ABP in Sertoli
cells is correlated with high NGFR expression even in the absence of FSH
stimulation.
[11 Parvinen et al, J Celt Biol, 1992,117, 629;
[2] Lonnerberg et al, Biol Reprod, 1992, 47, 381;
[3] Gerard, J Steroid Biochem Mol Blot, 1995, 53, 533.

(We/13.4/158)

s315

Apoptosis in angiogenie endothelial cells

Helen Hutchings, Jean PlouEt,
IPBS, UPR 9062, t 18 rte de Narbonne, 31062 Toulouse cedex, France

During angiogenesis, endothelial cells must leave pre-established

vessels, gain an angiogenic phenotype, proliferate and migrate towards the
source of an angiogenic signal. The endothelial ceils involved in this
phenomenon are exposed to a provisionnal extracellular matrix which
should act as a hostile environment and induce apoptosis. These cells must
therefore be protected against the apoptotic signals that a different
extracellular environment could induce. The aim of our work is to determine
the factors involved and the differences in protection against apoptosis
between angiogenic and non-angiogenic endothelial cells.
To model in vitro the angiogenic and non-angiogenic endothelial cell
phenotypes, we cloned from retinal capillaries non angiogenic (BREC/0)
and angiogenic (BREC/V) endothelial cell strains on the basis of their
ability to form tubes in 3D cultures upon VEGF exposure. BREC/O and
BREC/V expressed similar amounts of VEGF and VEGF receptors and
exogenously added VEGF induced a similar mitogenic and chemotactic
effect on BREC/O and BREC/V. In 3D cultures, only BREC/V could form
tube-like structures in response to VEGF exposure.
Annexin V and propidium iodide staining has demonstrated that the
angiogenic (BREC/V) cells are more sensitive to serum withdrawal induced
apoptosis than their non-angiogenic counterparts. The level of apoptosis in
only the angiogenic cells can be reduced VEGF (501ag/ml).This protection
from apoptosis can also be observed with P1GF, a VEGF homolog which
binds only to the VEGFR1 receptor, which implies a role for the VEGFRI
signaling pathway. Western blots show that Bcl2 is up-regulated in the
angiogenic (BREC/V) cells.
It therefore appears that VEGF is important not only for the
proliferation and migration of endothelial cells in angiogenesis, but also for
the survival of these cells by prevention of apoptosis during their transit on a
provisional extracellular matrix.

The Hepmocyte Growth Factor (HGF) receptor, encoded by the M E T proto-oncogene, regulates various cellular responses including cell
proliferation, morphology, motility, invasion and survival. Tyrosine
kinase receptor signaling occurs through the recruitment of signalling
molecules to the receptor upon receptor autophosphorylation on specific
tyrosine residues. However. other proteins can interact with the receptor
before its activation and the formation of the signaling complex with SH2
and PTB proteins. They may include proteins with functions in the control
of receptor localization and activity, scaffold proteins required to
assemble components of specific signal transduction cascades, or
signaling molecules that are released from the receptor after activation.
We used the interaction trap system to isolate target proteins that interact
with the not-activated intracellular portion of the HGF-receptor.
We isolated and cloned from a HeLa library the cDNA encoding an
isoform of the previously identified FKBP-associated protein, FAP48.
The interaction of this variant FKBP-associated protein, designated
FAP68. with the not-activated HGF-R has been mapped in the C-terminal
tail of the receptor, downstream the multifunctinal docking site y1349_
yt356. In vivo and in vitro experiments show that the interaction
specifically occurs with the not-activated receptor, suggesting a
mechanism by which FAP68 is released from the receptor after activation
by its ligand. This interaction may unveil a novel mechanism of signal
trasduction activation.

s316

Abstracts FEB S'99

(We/13.4/160) Isocratic RP-HPLC in the analysis of hGH variants

G. Karlsson, P. Gellerfors, A. Persson, B. Nor~n,
P.O. Edlund, C. Sandberg, S. Birnbaum

(We/13.4/161)

*INSERM U 427, 4 Av. de l'Observatoire, 75270 PARIS; ~ IRSC,

UPR 9044, 7 r. G. Moquet, 94801 Villejuif" ~ Lab. Biologie
Cellulaire, Av. G "~de Gaulle, Univ. Paris-Xll, 94010 Cr~teil

Pharmacia & Upjohn, S-112 87 Stockholm. Sweden

Human growth hormone (hGH) consists of a single polypeptide

chain containing 191 amino acid residues and two disulfide
bridges. Reversed-phase high-performance liquid chromatography
(RP-HPLC) was utilized for the separation of recombinant hGH
variants on a C18 silica column at 55 C using an isocratic mobile
phase which contained 27% 1-propanol in a 25 mM potassium
phosphate buffer, pH 6.5. The flow rate was 1.0 mL/min and
detection was carried out by measuring the absorbance at 220 nm.
[n order of increasing retention times the eluted peaks were
characterized to contain: Peak .4, di-oxy Met14/Met125 hGH; B,
Met125 sulfoxide hGH; C, des-Phel hGH; D, deamidated form
(Asn149-->Asp149 or Asn152--->Asp152) of hGH: E, the main peak
ofhGH, and two unidentified peaks ofhGH forms were ehited after
the main peak. Both the oxidized and deamidated forms were
characterized by tryptic mapping and mass spectrometry, The desPhel form of hGH was taken from a fraction that was obtained
from a hydrophobic interaction chromatography (HIC) method,
based on Pavlu and Gellerfors [1]; this fraction was injected and
showed the same retention time as peak C. The resolution between
the deamidated peak and the main peak was 1.1 at pH 6.5 and 0.9 at
pH 7.5. Oxidation was induced by hydrogen peroxide and
deamidation was carried out by heat treatment at alkaline pH.
Compared to the European Pharmacopoeia RP-HPLC method of
hGH analysis [2], this new method gives two additional peaks and
a 50% reduction in the analysis time.

Protein antagonists of angiogenin binding to ribonuclease

inhibitor
M. Moenner~, M. Chauvi~reh, P. Chevaillierc, J. Badet~

A non-enzymatic assay based on the interaction of 125I-angiogenin with

placental RNase inhibitor (RI) was developed to detect pancreatic-type
RNases and potential modulators of their action. Angiogenin, eosinophilderived neurotoxin, eosinophil cationic protein, bovine pancreatic and seminal
RNases, all members of the RNase A superfamily, competed to various
extents for angiogenin binding to RI. Highly basic proteins including the
homopolypeptides poly(Arg), poly(Lys) and poly(Om), core histones,
spermatid-specific S1 protein and the protamines HP3 and Z3 were also
potent inhibitors of angiogenin binding to RI. A minimum size of poly(Arg)
and poly(Lys) was required for efficient inhibition. The inhibition likely
resulted from direct association of the basic proteins with the acidic inhibitor,
as R1 bound to poly(Lys) and protamines while ~2~I-angiogenin did not.
Antagonists of the angiogenin-RI interaction are potential regulators of either
angiogenin-triggered angiogenesis and/or intracellular RI function, depending
on their preferential target.

l. B. Pavlu and P. Gellerfors. Bioseparation 3 (1993) 257.

2. European Pharmacopoeia, 3rd ed., Supplement 1999.
Convention on the Elaboration of a European Pharmacopoeia,
Strasbourg, Monography 0951.

(We/13.4/162) Characterization of the GDNF/Ret anti-apoptotic pathway

B. Mograbi, R. Bocciardi, D. Farahi far,
I. Bourget, T. Juhel & B. Rossi

(We/13.4/163)

Shc-mediated negative regulation on EGF signaling

M. Nagasawa, M. El-Shemerly, and Y. Nagamine
Friedrich Miescher Institute, P.O. box 2543, CH-4002 Basel, Switzerland

INSERM Umtd 364, b2wultd de M~decine Pasteur, 06107 Nice, France

After growth factor binding to the RTK, ShcA protein is tyrosine

phosphorylated directly or indirectly by RTK and consequently forms
Glial cell line-Derived Neurotrophic Factor (GDNF) is a potent survival
factor for several types of neuronal cells. It acts through a multicomponent
receptor composed ofa ligand-binding GPI linked protein (GDNFR-ct) and
the transmembrane Tyrosine Kinase (TK) Ret. Germline mutations
impairing either the Ret TK activity or its expression to the cell surface are
associated with the development of the Hirschsprung disease (HSCR), a
frequent congenital malformation characterized by the absence of enteric
ganglia in the distal gut. Recent observations in ret knockout mice indicate
that the HSCR disease may result from the aberrant conumtment of a cell
death program in the precursors of the enteric nervous system. However,
the intracellular mechanisms used by GDNF/Ret to rescue these cells from
apoptosis are not known yet.
We demonstrate herein that the expression and the activation of a functional
Ret TK in the human neuroblastoma SK-N-MC cells are essential for the
anti-apoptotic signaling pathway initiated by GDNF. Indeed, SK-N-MC
cells expressing the wdd-type but not a kinase deficient form of Ret
(carrying an inactivating HSCR mutation in the TK domain, H13) are
rescued by GDNF from the apoptosis induced with different effeetors. We
also found that the GDNF induced Phosphatldyl Inositol-3 kinase (PI3K)
plays a crucial role in this process. Further analysis of two cytosolic PI-3K
targets showed that the activation of the serine/threonine kinase Akt (also
known as Protein Kinase B) but not of the MAP kinase cascade is essential
for the GDNF/Ret survival signaling. In conclusion, we demonstrate that the
GDNF/Ret~PI-3K-~Akt pathway plays a crucial role in promoting cell
survival. Moreover, our findings allow to better understand the mechanisms
by which the "loss of function" mutations affecting Ret may lead to the
clinical HRSC phenotype.

a ShcA-Grb2-Sos ternary complex on the plasma membrane.

Membrane-bound Sos then activates Ras by catalyzing GDP/GTP
exchange and leads to activation of the Ras/ERK signaling pathway
that is one of the major early events for growth factor-induced cell
proliferation and differentiation. It is believed that this process plays
an important role in growth factor-induced Ras/ERK signaling
pathway. However, it has remained unclear whether ShcA is a major
component to activate the Ras/ERK signaling pathway because Grb2Sos complex also can bind directly to receptor tyrosine kinase.
To investigate whether ShcA is a major component in growth
factor-induced Ras/ERK signaling pathway, we obtained mouse
p46 ShcA, p52 ShcA and p66ShcA eDNA, and examined how ShcA
overexpression affects EGF-, PDGF-, FGF-2- and TPA-induced
Ras/ERK

signaling.

We

found

that

ShcA

overexpression

dramatically decreased EGF-induced ERK-mediated gene expression,

ERK activity and ERK nuclear translocation, but not on PDGF. FGF2 and TPA stimulation. These results suggest that ShcA acts as a
negative regulator in EGF-induced Ras/ERK signaling.

Abstracts FEBS'99

(We/13.4/164)

Angiogenin: expression in the human placenta

N. Pavlov a, E. Hatzi b, M. Moenner a, Y. Bassaglia c, J. BadeP

s3 1 7

(We/13.4/165)

~[NSERM Unit~ 427- Universit~ Paris V,, France

~Laboratory of Biological chemistry - University of loannina, Greece
Unit~ CNRS UPRESA 7053 - Universitd Paris XII, France

Angiogenin is one of the most potent inducer of blood vessel

formation in angiogenesis assays [1]. The 14-kDa protein has 35% aminoacid sequence identity with human pancreatic ribonuclease. It also displays
a ribonucleolytic activity. Although the structural characteristics of
angiogenin have been extensively studied [2], its physiological role and its
mechanisms of action are puzzling. The human placenta constitutes a
relevant model of physiological angiogenesis, easily accessible, presenting
pathological situations of angiogenesis [3]. It is also a model of pseudomalignant development characterized by a limited invasivity of fetal cells
in maternal tissue. Moreover, at the molecular level, the choice of placenta
is justified by the human specificity of our reagents and by the presence of
the natural inhibitor of angiogenin, Placental Ribonuclease Inhibitor, a
tight-binding inhibitor of angiogenin that abolishes all its known
properties. By using specific polyclonal antibody prepared in our
laboratory against human angiogenin, the molecule was located by
photonic and electronic microscopy in placenta. Expression of angiogenin
in the human placenta was confirmed by in situ hybridization using a
digoxygenin-labelled eDNA probe, and by detecting the protein in medium
conditioned by trophoblastic cells using an ELISA. Localization of
angiogenin and its mRNA in the syncytiotrophoblast, in some
mesenchymal cells, in the vascular endothelium, and in the walls of blood
vessels suggests a paracrine and/or autocrine role of the molecule acting on
vascular and tissue homeostasis.
References:
[1] Fen ct al., Biochemistry,24, (1985) 5480.
[2] Badet, ComprehensiveVascular Biology and Pathology - An Encyclopedic
Reference, A. Bikfalvi led), Springer,Heidelberg(1999).
[3] Crosset al., Science,266, (1994) 1508.
Electronic microscopywas carriedout in the laboratoryof Pr. DohnGlitz (UCLA,USA).
Supportedby grants: ARC (n6831), Fondationde France, Fondationpour la Recherche
M6dicale, France.
(Wed13.4/166)

Expression pattern of IGF-1 and its receptor

during X e n o p u s laevis development
L. Richard-Parpaillon, L. Grolgmo, F. Chesnel, D. Boujard
I/PRES-A 6026 CNRS. l ,'rove;wit# de Rettne.~ 1, 35042 RENNf.S( "edex

The IGF-I receptor (IGF-1R) has important functions during normal

development[l]. In mouse, it mediates all the IGF-1 and part of the IGF-2
actions. However, recent knock-out experiments suggest that the insulin
receptor (InsR) might also be implicated during embryogenesis through
an activation by IGF-2 [2]. In order to study the role of IGF-IR during
embryogenesis in X e n o p u s laevls, we have cloned these two receptors
and a partial IGF-2 cDNA in this species. By Northern blot and m s.au
hybridization, we have determined the expression pattern of IGF-1R,
InsR and IGF-1 in xenopus embryos. Transcripts of 1GF-1R and InsR are
weakly detected during early embryogenesis. At the end of neurolation
and during organogenesis, they become largely restricted to regions of
ecto- and mesodermic origins: encephalon, otic and optic vesicles,
somites and gills, lnsR transcripts are specifically detected m the
pronephros. The distribution of IGF-I transcripts is similar to the one of
IGF-1R mRNA. Nevertheless, IGF-1 mRNA appeared later, suggesting
that another ligand, like IGF-2, might be implicated at earlier stages. To
better understand the function of IGF-1R during development, we have
begun to induce ectoplc expression of IGF-1R and IGF-1 by
microinjection of plasmid or m vtlro transcribed mRNA in two cells
embryos.
[1] D'Ereole, A.J. et al., Growth and growth disorders, 25, (1996), 573.
[2] Louvi, A.et al., Dev.Biol., 189, (1997), 33.

Sphingosine 1-phosphate activates the MAPK cascade via

a Gi-eoupled receptor signalling pathway.
S. Rakhit, A.M.Conway, N. Pyne & S. Pyne
Dept. of Physiology & Pharmacology, University of Strathclyde, 27
Taylor Street, Glasgow, Scotland

Our previous studies have shown that in guinea pig airway smooth
muscle (ASM), the lysolipid sphingosine-1 phosphate (SIP) potentiated
platelet derived growth factor (PDGF) induced DNA synthesis and so
acted as a co-mitogen. This is consistent with our observation that SIP
increased p42/p44 mitogen activated protein kinase (MAPK) which is
linked to DNA synthesis. Futhermore, the SIP dependent activation of
MAPK is sensitive to pertussis toxin (used to inactivate inhibitory Gproteins or Gi). This suggests that SIP acts through an extracellular
receptor coupled to Gi. Recently a family of G-protein coupled
receptors (GPCRs) activated by SIP and related lipids, known as EDG
(endothelial differentiation gene family including EDG1, EDG2/vzgl,
ARG16/H218 and EDG3) have been cloned.
We now demonstrate by RT-PCR using specific primers that ASM
contain EDG1 transcripts. Further experiments to characterise the
mechanism by which SIP activates MAPK are also presented. S1P
dependent activation of MAPK was blocked by PP1 (a c-Src tyrosine
kinase selective inhibitor) and to wortmannin and LY294002 (which are
structurally unrelated phosphoinositide 3-kinase or PI3K inhibitors.
The former observation was confirmed when S 1P was found to increase
cellular c-Src activity (as measured by phosphorylation of acid
denatured enolase in c-Src immunoprecipitates) in a pertussis sensitive
manner. A role for PI3K was also confirmed as SIP increased PI3K
activity in Grb-2 (guanine nucleotide releasing binding protein)
immunoprecipitates in a pertussis sensitive manner. This is significant
as Grb-2 is an intermediate in the MAPK cascade. Additionally SIP
stimulation tyrosine phosphorylation of a 100 kD protein bound to Grb2 and this was sensitive to pertussis toxin.
Thus our findings are consistent with a model where SIP binds cell
surface EDG1 to activate the MAPK cascade. Other studies have
shown that lysophosphatidic acid (LPA) binds EDG2 to activate c-Src,
PI3K, tyrosine phosphorylated plO0 and MAPK. This suggests a
common signalling pathway used by EDG receptors activated by
different lysolipids.

(We/13.4/167)

APPLICATIONOF THE SELEX TECHNOLOGY TO THE RET ONCOGENE

Rizzo, C., Libri, D. and de Franciscls V.
Centro di End. e Onc. Sperimentale CNR 80131 Napoll, I
Centre de Genetique Moleculair, Gif sur Yvette, France

Germline mutations of the ret gene, a receptor tyrosine kinase,

cause the multiple endocrine neoplasia (MEN) type 2A and 2B
and familial medullary thyroid carcinoma syndromes. Missense
mutations at one of five cysteines residues, clustered in the extracytoplasmic domain, are the most frequent causative genetic
events of FMTC and MEN2A syndromes. Mutation at codon 634
is the causal event in 85% of MEN2A families and 30% of
FMTC. These mutations convert Ret into a dominant
transforming gene, and cause constitutive activation of its
intrinsic tyrosine kinase activity. The major aim of this project
was to identify a molecular antagonist that could inactivate Ret
activity by binding its extracellular domain, where the activating
mutations responsible for the MEN2A syndrome are located. To
this aim we used the SELEX procedure. This is an in vitro
technology which allows the identification of short
oligonucleotide ligands by their high binding activity to a target
protein. We used a recombinant protein fused to the extracellular
domain of Ret as target to isolate specific aptamers from a
degenerated SELEX library. We prepared a randomized library of
2~
35 nucleotides to provide I x I 0 different sequence possibilities.
By using a filter-binding assay, a starting pool of modified
RNase-resistant oligoribonucleotides has been allowed to interact
with the Ret protein. The RNA molecules possessing high
binding affinity for the extra-cellular domain of Ret have been
amplified by RT-PCR, and the resultant molecules subjected to
multiple rounds of selection. To eliminate those aptamers, which
may react either to peptide residues fused, but unrelated, to Ret or
to other reagents, that depend on the specific procedure utilized
(i.e. filter-binding), we, thus, performed rounds of counterselection.
In conclusion, we developed an RNA pool which is enriched in
aptamers that bind in vitro the extracellular domain of Ret.

s318

(We/13.4/168)

Abstracts

A suppressor of the Abl kinase in chronic

myelogenous leukemia
T. Shishido, T. Akagl, T. Ouchi, M. Georgescu, and H.
Hanafusa
Osaka Blosciencelnstattte. Osaka 565 0874, Japan

More than 90% of the patients with chronic myelogenous leukemia

possesses the Philadelphia chromosome which directs the synthesis of the
Bcr-Abl protein. In the tumor cells, the kinase activity of the Bcr-Abl
protein is elevated and shown to be essential in the transformation. On the
other hand, the kinase activity of the cellular counterpart c-Abl is tightly
suppressed in untransformed cells because of a trans-acting inhibitor
molecule. In the previous study, we found that the complex of the kinasedeficient c-Sin and c-Cbl acts as a suppressor of the c-Abl kinase. When
we tested whether this complex also acts as a suppressor of the Bcr-Abl
protein kinase, we found that the suppression is much limited to this
kinase. This result could explain the mechanism of the activation of the
Bcr-Abl kinase. We wall also report the results of studies on the role of
this suppressor complex in the Bcr-Abl induced transformation. This
study will thus elucidate the initial and the essential step of tumorigenesis
in chronic myelogenous leukemia.

(We/13.4/170)

Anisomycin uses multiple mechamsms to inhibit

neuronal differentiation m PC I2 phaeochromocytoma ceils
Befita Tor6csik and J6zsef Szeber6nyi
Department of Biology
Umversity Medical School of Pecs, Hungar~

Treatment of PCI2 cells with nerx c growth factor (NGF)

stimulates extracellular signal-regulated kmases (ERKs), c-Jun Nterminal kinases (.INKs) and induces neuronal differentiation.
While the pivotal role of ERKs in NGF-mdttced morphological
differentiation is well established, the comrlbution of.INKs is less
clear. The role of the JNK-pathway m PC12 cells was analyzed by
using anisomycin, a protein synthesis inhibitor that acuvates JNKs.
Non-toxic contrentrat~ons of anisomycin ~ere found to stimulate
.INKs, expression of the early-response genes c-jun, c-os and
zit268, and to inhibit NGF-induced neurite formatzon. These
effects of anisomycm appear to be mediated by the generation of
reaclive oxygen species (ROS), which m turn act through both
TrkA,'Ras-dependent and -independent signaling pathways

(We/13.4/169)

FEBS'99

Hormonal regulation of VEGF and its receptors: roles

in progression of prostatic adenocarcinoma.
S. Sordello, J. Plou~t
[PBS, UPR 9062, 118 rte de Narbonne, 31062 Toulouse cedex, France.

The progressive growth of a malignant tumour requires an adequate

blood supply, which is provided by newly formed vessels. There exists a
relationship between the vascularization and the invasivity of prostatic
adenocarcinoma. Since VEGF (Vascular Endothelial Growth Factor) has a
crucial function in tumour angiogenesis. VEGF concentration was increased
in prostatic adenocarcinoma as compared to benign hyperplasia. Two
tyrosine kinases have been identified as VEGF receptors: VEGFRI (Fit-l)
and VEGFR2 (KDR/FIk-I). In our laboratory, we took advantage of antiidiotypic strategy to obtain circulating agonists specific for VEGF-R2,
which stimulated endothelial cell proliferation. We studied its role in
prostatic adenocarcinoma.
Immortalised prostatic epithelial cells (PNTI) express VEGF.
Dihydrotestosterone (DHT) up-regulated VEGF at mRNA and protein
levels. Immnnoneutralisation of VEGF increased proliferation of PNTI
cells, in contrast VEGF decreased it as did the VEGFRI specific agonist:
PIGF (Placenta growth factor). This result suggests autocrine pathway for
VEGF which is mediated by VEGFR1 and thereafter inhibits proliferation.
We studied VEGF hormonal regulation in viva. After castration
VEGF decreased and then increased together with the weight of prostate
gland when the rat received testosterone. No regrowth of the prostate gland
was observed with the VEGFR2 agonist alone, but testosterone and VEGFR2 agonist had synergetic effect on prostatic angiogenesis.
We demonstrated that VEGFR2 activation increased tumour growth
and vascularization of prostatic adenocarcinoma in nude mice. We
conjugated the VEGFR2 antibody with a toxin to target angiogenic
endothelial cells. This immuntoxin induced the regression of tumour
development.
Our results demonstrated that the VEGF system is involved in
prostatic adenocarcinoma progression, and the angiogenic conversion of
endothelial prostatic cells requires VEGFR2 activation and hormonal
impregnation. We demonstrated that VEGFR2 is a possible target in a anti
angiogenic therapeutic.
(We/13.4/171)

INDEPENDENT ACTIVATION OF PHOSPHATIDYLINOSITOL

3-K1NASE- AND RAS-DEPENDENT PATHWAYS BY INSULIN.
T. Tsakiridis, E. Tsiani, C. Whiteside, and G.P. Downey.
Division of Clinical Science. Department of Medicine, University of
Toronto, 1 King's College, Toronto, Ontario, Canada, M5S IA8

Insulin stimulation of cells activates a complex signaling pathway

which is initiated by phosphorylation of the adaptor molecules Insulin
Receptor Substrates ORS) and the Src and collagen homologues (Shc).
Phosphorylated IRS binds and activates phosphatidylinositol 3-kinase (PI
3-kinase) which leads to the activation of downstream protein kinases such
as the ribosomal p70 $6 kinase (p70S% and the protein kinase B (PKB,
also known as Akt). Phosphorylated Shc binds the small adaptor Grb2
which, through the exchange factor Sos, activates the small G protein Ras.
Activated Ras then initiates a protein kinase cascade which leads to
activation of the Mitogen-activated protein kinases (MAPK) Extracellular
signal regulated kinase (Erk) and p38 MAPK, which are involved in
regulation ofgene expression and proliferation. A number of studies in the
recent years have suggested that Ras interacts with and leads to activation
PI 3-kinase. Other reports have suggested that PI 3-kinase activity is
required for Ras activation. In this study we investigated the importance of
PI 3-kinase - Ras interactions in the signaling pathway of insulin, in
differentiated L6 skeletal muscle cells (myotubes). Disassembly of the
actin network of myotubes with the structurally unrelated agents
cytochalasin D (1 ~tM) or latrunculin B (1 ~tM), inhibited the interaction
of Shc with Grb2 and abolished insulin activation of Ras. Additionally,
these treatments abrogated the ability of insulin to activate both Erk and
p38 MAPK. However, actin disassembly did not inhibit insulin induced
interaction of IRS-lwith PI 3-kinase nor the activation of PI 3-kinase and
its downstream target p70 s6k. On the other hand, inhibition of PI 3-kinase
activity with 100 nM wortmannin, which inhibits insulin-stimulation of
glucose transport and the insulin-induced actin re-organization in
myotubes, did not affect the stimulation of either Erk or p38 MAPK by
insulin We conclude that the the IRS - PI 3-kinase - p70 s6k and the Shc Grb2/Sos - Ras - - Erk/p38 MAPK pathways are independently regulated
by insulin in muscle cells and that PI 3-kinase - Ras interactions are not
required for the propagation of the insulin signaling pathway.

Abstracts FEBS'99

(We/13.4/172)

HB-GAM and Midkine induced expressions in the PI9

cell differentiation processes
M.Vigny,N. Brunet-De Carx albo. B.Sounou. D Ratllal~
INSERM U440. IF'M. 17 rue du fer fi moulm . 75005 Pans. France

Heparin-Binding Growth-Associated Molecule HB-GAM (also named

Pleiotrophin) and Midkine (MK) are developmentally-regulated heparinbinding proteins with putative functions in cell grov,lh and differentiation.
MK is highly expressed during early embryogenesis by all three germ layers
[1,2]. In contrast. HB-GAM was found to be expressed later during
development, for example at the perinatal period of the rat development In
addition, this expression is restricted to the developing central nervous
system, the muscle and the embryonic cartilage and forming bone [3A].
P19 teratocarcinoma cells present a number of characteristics which
make them particularly' valuable to study the cellular and molecular events
associated with the early development [51. Retinoic acid (RAy treatment of
P19 cells effectively induces the cells to differentiate into neurons, astroglia
and microglia, cell types which normally derived from the neuroectoderm.
P19 cells exposed to dimcthyl sulfoxide (DMSO) differentiate into
endodermal and mesodermal derivatives.
We investigate whether HB-GAM and MK were differently expressed
during the differentiation processes of the P19 cells. We report that MK is
already" expressed in control P19 cells. However RA and DMSO treatments
induced a modulation of the MK expression. More interestingly.. HB-GAM
was not expressed in the untreated P19 cells and was differently induced by
the two differentiating agents. In addition, the expression of the ,\-~)'ndecan
(the HB-GAM neuronal receptor [4]) roughly follo~cd that of ttB-GAM in
the 2 differentiation pathways suggesting that the biological effect of HBGAM could be mediated in the tyro cases by this receptor. "[his anal3 sis and
the
cellular
localization
of
these
proteins
(analyzed
by
immunoc.~ tochemistry) strougly suggest an invoh ement of these molecules
in molecular mechanisms b y ~ hich P19 cells differentiate.
I Cockshutt. A M etal De,, D,,l~amlc~ (1994}200 198-211.
2 Kadomatsu Kctal JCellBiol (1990) 110607-616
? Drexfus.Jeta] hlt.JDex Bi01.(1908)42 189-198
4 Nolo. Relal NeutosClLett.(1Oq5) IOI 39-42.
5 \lcBumey. MtX' Int .I De\ Biol. ( 1993137 135-140

(We/13.4/174)

Tyrosine Kinase Inhibitors in Chalcone

Derivatives and Their Inhibitory Mechanism
Er-Bin Yang, Yung-Jian Guo and Peter Mack
Dept. o f Exp. Surg., SGH, Singapore 169608.

Abstract: In our previous study, it was found that butein, one of

chalcone derivatives, was a specific tyrosine kinase inhibitor [1, 2]
In this work, chalcone and seven chalcone derivatives were used to
analyze the relationship between the structure of these chemicals and
their inhibition of tyrosine kinase activity Three of these chalcone
derivatives, including butein, marein, and phloretin, were found to
have an ability to specifically inhibit epidermal growth factor
receptor (EGFR) tyrosine kinase activity by the determination of
ELISA method The inhibition of EGF-induced EGFR tyrosine
phosphorylation was also observed by the preincubation of human
hepatoma HepG2 cells with butein, while marein and phloretin were
not found to inhibit the phosphorylation The molecular docking by
computational method revealed that butein, marein, and phloretin
were fixed into ATP binding pocket of EGFR by hydrogen bonds
and hydrophobic interaction Comparing the energy interaction when
the binding of butein, marein and phloretin to EGFR, it was found
that the complex of butein-EGFR was the most stable, whereas the
stability of marein-EGFR and phloretin-EGFR was second and third,
respectively. It was corresponding to the inhibition of these chalcone
derivatives to EGFR tyrosine kinase by ELISA determination

References
[l]. Yang, EB, etal. BBRC, 245, (1998) 435
[2] Yang, EB, et aL BBRC, 224, (1996) 309.

s319

(We/13.4/173)

Role of receptor tyrosines in G-CSF-R signaling

Alister C. Ward a. Louise Smith b. John P. de Koning a.
Yvette van Aesch', and Ivo P. Touw "'b
~Erasmus Untverstty, Rotterdam, The Netherlands,
eDr Dante/den Hoed Cancer Center, Rotterdam, The Netherlands.

Granulocyte colony-stimulating factor (G-CSF) regulates neutrophil

production through activation of its cognate receptor, the G-CSF-R.
Previous studies vdth deletion mutants have shown that the membraneproximal cytoplasmic domain of the receptor is sufficient for mitogenic
signaling, while the membrane-distal domain is required for
differentiation signaling. However. the function of the four cytoplasmic
tyrosines of the G-CSF-R in the control of proliferation, differentiation
and survival has remained unclear. Here we investigated the role of these
tyrnsines by expressing a tyrosine "null'" mutant and single tyrosine "add
back" mutants in maturation-competent myeloid 32D cells. Clones
expressing the "null'" mutant showed only minimal proliferation and
differentiation, with survival also reduced at low G-CSF concentrations.
Analysis of clones expressing the "add-back'" mutants revealed that
multiple tyrosines contribute to proliferation, differentiation and survival
signals from the G-CSF-R. Analysis of signaling pathways downstream
of these tyrosines suggested a positive role for STAT3 activation in both
differentiation and survival signaling, while SHP-2. Grb2 and Shc appear
important lbr proliferation signaling. In addition, we show that a
tyrosine-independent "'differentiation domain" in the membrane-distal
region of the G-CSF-R appears necessary but not sufficient for mediating
neutrophilic differentiation in these cells.

(Wed13.4/175)

Distinct Calcium-dependent Pathways of E(; FR Transacti~ation

and PYK2 Tyrosine Phosphorylation in PCI2 Cells
E. Zwick, N. Prenzel, C. Wallasch, H. Daub and A, Ullrich
Department o/~tole~uhu" Bmlo~v. Mar-PtanckImtitut [ur Btochcmle.
4m Klop[ersptit: L~'4 82152 Marttn.srted Germato"

Recently. we have demonstrated thai in PCI2 cells activation of the

Ras/extracellular signal-regulated (ERK) kinase pathway in response to
membrane dcpolarizahon or bradykinin is mediated by calciuln-dependent
transactivation of the epidennal grox~th Factor receplor (EGFR). t[ere ,ac
adress the question whether Ca2+-calmodulin-dependent protein kinase (CaM
kinase) has a role in tim EGFR transactc~ation signal. Using cotnpounds x,&ich
selectively interfere with either CaM kinase activity or cahnodulin function,
we show that KCl-lnediated membrane depolarization-, but not bradykinintriggered signals involve CaM kinase [:unction upstream of tile EGFR. While
both. depolarization-induced calcium mllux and bradykinin stimulation of
PCI2 cells ,,,,ere found to induce c'-~s transcription through EGFR ach,~ation.
tile former signal is CaM kinase-dcpendent and the latter was shown to be
independent. Since PYK2 is also acti'~ated upon elevation of intracellular
calciuln. ~e investigated the potential in,,olvenrent of tills cytoplasnnc
tyrosinc kinasc in EGFR lransacti~ation. Interestingly, x~e observed that
inhibinon of CaM kmase aclivity in PCI2 cells abrogated tyrosme
phosphorylation of PYK2 upon KCI but not bradykinin treatment.
Nc'~ertheless, PYK2 activation ill response to both stimuli appeared to be
mediated b) pathways parallel to EGFR transactivation, as FGFR inhibition
neither affected PYK2 tyrosine phosphor.,,lation nor did tile expression of a
dominant-negative PYK2 mutant interfere '.,,ith EGFR u'ansactlvation upon
membrane depolarization or bradykimn stilnulatioll. Our data dclnonstrate the
existence of tv, n distinct calcium-dependent inechanism, leading either to
EGFR-inediatcd ERK acti'~ation or to PYK2 tyrosinc phosphor~lation. Both
pathway,s either in concert or independeotl~ migbt contribute to the
definition of biological responses in neuronal cell t'vpes.

s320

Abstracts FEBS'99

15.1 Growth regulator and signalling in plants

(We/15.1/176)

Mechanism of kinetin formation

J.Barciszewski a, G.Siboska b, S.I.S.Rattan b and B.F.C.Clark b
~lnstltute of Bioorganic Chemistry,Noskowsklego12.61-704 Poznafi.
Poland and bUniversityofAarhus, GustavWieds Vej 10, DK-8000Aarhus
C, Denmark.

Kinetin (K) was isolated from DNA as an artifactual rearrangement product c

the autoclaving process and later on its cytokinin activity has been establishet
Recently new data appeared which show that K occurs in cellular DNA as tlproduct of oxidative, secondary modification reaction of DNA [1-3]. Varim
biological effects produced by this hormone in vitro and in vivo made kineti
scientifically interesting and commercially attractive as an ingredient of man
beauty cosmetics [4]. The crucial evidence for a presence of 6-furfuryladenir
in natural products came from the mass spectrometric analysis which showe
molecular signal of 215 m/e for free base or 287 and 359 for trimethylsil:
derivatives. Furfural is precursor of K formed during hydroxyl radical oxidatic
of the C5' of deoxyribose and spontaneous cleavage of the DNA backbone wil
b-elimination. Once furfural is formed in the vicinity of DNA, it can efficientl
react with the exocyclic amino groups of DNA components and can form tl"
Schiff base with adenine residues, and possibly also cytosine moietie
Furthermore dehydration and reduction of the intermediate lead to formation c
kinetin at the level of DNA. This bulky substituent probably induces sonconstraints and should be repaired in DNA by repairing enzyme. We ha~
started to isolate kinetin binding protein from wheat germ. To do it we prepare
affinity chromatography column in which kinetin riboside, oxidised wit
sodium periodate was bound to aminohexylsepharose (AH-Sepharose). Putatix
kinetin binding protein of wheat germ was isolated.
References: 1. Barciszewski, J., Siboska G.E., Pedersen, B.O., Clark, B.F.(
and Rattan. S.I.S. (1996) Evidence for the presence of kinetin in DNA and ce
extracts, FEBS Letters 393, 197-200; 2. Barciszewski, J., Siboska G.lZ
Pedersen, B.O., Clark, B.F.C. and Rattan, S.I.S. (1997) A mechanism for the i
vitro formation of N6-furfuryladenine, kinetin as a secondary oxidative dama~
product of DNA. FEBS Letters 414, 457-460; 3. Barciszewski, J., Siboska G.E
Pedersen, B.O., Clark, B.F.C. and Rattan, S.I.S. (1997) Furfural, a precursor
the cytokinin hormone kinetin, and base propenals are formed by hydrox:
radicaI damage of DNA, Biochem. Biophys. Res. Commun. 238, 317-319;,
Barciszewski, J., Siboska, G., Rattan. S.I.S. and Clark, B.F.C (1999) Kinetfl
40 years after, submitted.

(We/15.1/178)

Ion currents in Nod Factor signaling

F Bouteau".A. Kurkdjian~',A.M. Pennaruna. D CorneY,M
Convert". O Dellis~. J P Rona~. U. Bousquet"
"Electropkq~iologie des Membranes ~'mversit(" D. Dtderot. F- 7.5251
Part.~ cedex 05 andtLS'l ] (' .V 1 S~ F- 91198 G!f-suJ~) vetW ('t, dex.

(We/15.1/177)

The stimulation of generative development

of winter plants by zearalenone and its derivatives.
J.Biesaga - Ko~cielniak, A.Skoczowski,F.Dubert, l.Marcifiska, M.Filek
Department of Plant Physiology Polish Academy of Sciences
30-239 Krakrw, Podluzna 3

Zearalenone (ZEN), an estrogenic substance, was first isolated from extracts

of fungus Gibberella zea (Fusarium graminearum). Its chemical structure
was determined as 6-(10 - hydroxy- 6-okso-trans-l-undecenyl)-B risolic
acid lactone. Endogenous zearalenone has also been found in many other
plant species, e.g. wheat, corn, rice, cotton, carrot, celery, race, onion, etc.
Its seems also that zearalenone may be essential for the control of the
generative development of winter rape and other plants species.
Also in the author's own investigations it has been shown that zearalenone
applied in various concentrations, stimulates the generative development of
winter crops, which manifested by increased number of heading plants and
shortening of their vegetative phase. The aim of experiment was comparison
the action of zearalenone with its derivatives (~ and 13 zearalanol,
zearalanon, c~ and 13zearalenol ).
Among used derivatives of ZEN mostly effective were et and 13
zearalanol stimulating the heading 98 and 90% plants respectively after 7days long vernalization whereas plants treated with ZEN flowered in 70/'0.
The examined derivatives accelerated also the heading of plants by 17 days
in comparison with ZEN.
However the inhibition of generative development of winter plants
brought about !3 zearalenol and zearalanol, which resulted in heading about
40 % plants. The time of heading oftbese plants amounted to above 70 days
delaying heading for 10 days. Even these less active compounds stimulated
plant generative development in comparison with controlled non-treated
plants which developed only vegetatively.
The study is supported by the Polish Committee for Scientific Research
(KBN) grant No P06A 033.12.

(We/15.1/179)

Kinase activity of SRK, a receptor-like kinase

involved in self-pollen recognition, in Brassica oleracea.
D.Cabrillac~, J.L. Girmton a, J M Cock a, C Dumas ~, T Gaude'~
Laboratotre de Reproduction et Ddveloppement des Plantes,
ENS Lyon, 69364 Lyon Cedex 07, France.

Nod factor [NodRm-lV (Ac,S)], isolated from the bacterium Rhizobiom

nwhloti, induces a well known depolarization in M e d i c a g o sa/ivtt (cv Sitel)
root hairs [1]. The analysis of this membrane response shows that anion
channels and H+-ATPase currents are involved in young root hairs
displaying no inwardly rectifying potassium channels. The early Nod factorinduced depolarization is due to the inhibition of the H+-pump and to the
increase of the conductance of inwardly rectifying anion channels. A
calcium ionophore (A23187) induces the same type of response as that of
Nod factor, whereas the inhibition of Ca 2+ influx (using a Ca
-9+ channel
inhibitor, La 3+) and the substitution of Ca 2+ by Mg 2+ partially prevents
the depolarization Anion channels are also involved in the long term
rectification of the membrane potential (Em), after the depolarization
indttced by Nod factor, A23187, and vanadate. The H+-pump was involved
in the rapid repolarization processes (except for vanadate) All our results
are consistent with a key role of anion channels and the H+-pump in Nod
factor-induced electrical membrane responses in root hairs They are in
agreement with the initial signaling events recently involving Ca 2+ influx
described for Nod effect [2] and also corroborate the recent data obtained
fi)r Nod induced ion fluxes by selective microelectrodes [3]
[ I] Kurkdjian A, Plant Physiol. 107 (1995) 783
[2] Pingret et al Plant Cell 10 (1998) 659
[3] Felle HH et al, Plant J 13 (1998) 455

Self-Incompatibility (SI) in Brassica oleracea is a process which blocks selfpollen growth on the stigma (the surface of the pistil receptive to pollen).
This process is controlled by a single, highly polymorphic locus, the S locus
(S for Self-incompatibility), and self-pollen is rejected when the same S
alleles are expressed both in the stigma and pollen. In the stigma, the S locus
encodes a membrane spanning receptor-like ldnase, SRK (for S-locus
Receptor kinase) which is thought to be involved in self pollen recognition.
The kinase domain of SRK exhibits a Ser/Thr ldnase activity in vitro when
expressed in F~coli, and plants defective for this receptor have been shown
to be self-compatible. Current models propose that SRK interacts with a
self-pollen borne signal, not yet identified, and Iriggers the SI response via a
dimerisation process [1].
In order to obtain more information about the mode of action of SRK
during self-pollination, we expressed different forms of the SRK protein in a
membraneous environment, using the baculovims-insect cell system. In
microsomes, recombinant forms of SRK were found to autophosphorylate
on serine and threonine residues. Surprisingly, this phospborylation was
constitutive, as it happened in the absence of pollen or stigma extracts.
Phosphorylation was shown to occur in trans which strongly suggests the
existence of homo-oligomers of recombinant SRK proteins in insect cells. To
investigate the physiological relevance of these results, in planta studies are
in progress in our laboratory.
[1] McCormick S., Cur. Op. Plant. Biol. 1, (1998) 18

Abstracts FEBS'99

(We/15.1/180)

s321

Identification and characterisation of novel 14-3-3 binding

proteins in Arabidopsis cells

(We/15.1/181)

alnstitut de Btotechnologie des Plantes, UniversitdParisXT,,91405 Orsay, Franc~

bLaboratoirede Physiologte Vdg~tale, Universtt~de Tours, 37000 Tours, France

MRC Protein Pho.ff~hor3,1at~on Unit, Department of Bioc'hemistt~;

MSI/IVTB Complex. UniveJw~tvof Dundee. Dundee, DD I 5EIL U K.

14-3-3 is a family of ubiquitous, homo-/hctcrodimcric proteins in

eukaryotes known to regulate a divcrsc set of cellular processcs [1, 2].
14-3-3s interact with their target by binding to specific sequences, which
in most cases includc a phosphorylatcd residue.
In plants, 14-3-3 proteins are involved in the regulation of the activity of
diverse enzymes such as nitrate reductase, the plasma membrane HATPase, sucrose-phosphate synthase and a calcium-dependent protein
kinase [3]. 14-3-3s have also been identified as binding to a number of
plant proteins [3]. However, many 14-3-3's targets remain probably still
unidentified and little is known about how the phosphorylationdependent interactions between 14-3-3s and their targets are regulated.
In order to characterise novel 14-3-3 binding phosphoproteins from plant,
a 14-3-3 affinity strategy for purifying such proteins in sufficient quantity
for identification by amino acid sequencing has been developed in our
laboratory. Utilisation of this procedure has allowed us to identity, new
partners of 14-3-3s from Arabidopsis cell, such as protein kinases,
proteins involved in plant metabolism, protein synthesis, and vesicular
transport. The identity of some of these proteins has been confirmed by
using specific antibodies in Western blot experiments. Moreover, farWestern overlays performed with digoxygenin (DIG)-labelled 14-3-3s
showed that all of these proteins interact directly with 14-3-3s.
Future work will involve further study of the interaction between 14-3-3s
and their newly identified targets and to assess the regulatory effect that
14-3-3s may have on the activity of these enzymes.
[1] Ferl R.J., Annu. Rev. Plant Physiol. Plant Mol. Biol., 47, (1996), 4973.
[2] Aitken A., Trends Cell Biol., 6, (1996), 341-347.
[3] Moorhead G. et al., Plant J., in press.
(We/15.1/182) Analysis of histological and biochemical alterations induced
by water-soaking in melon fruits

C., du Chateneta, A., Latch6b, J-C., Pechb, R., ganjevaB,A., Grazianaa.

aCNRS/UPS UMR n5546 and bENSAT Pole de Biotechnologie Vdgdtale,
31326 CASTANET, France.

Water-soaking is a physiological disorder characterized by a

glassy texture of the flesh that greatly depreciates the commercial
quality of early-season Charentais cantaloupe melons. Although it is
recognized that the genotype and a number of physiological and
environmental factors play a role in the development of the syndrome,
the intimate mechanisms responsible for water soaking remain
unknown. In the present work, physiological, molecular and
ultrastructural analysis have been combined to examine relationship
between watersoaking, ethylene and calcium. Experiments were carried
out using melon fruit (Cucumis melo L. cv. Talma) at same stages of
ripening. NMR analysis, a non invasive method, and physiological
parameters were used to evaluate stages of ripening and watersoaking.
We have shown, using NMIL that the water mobility of tissues
exhibiting the disorder was increased. In addition, the alteration of the
cell wall and the presence of large intercellular spaces was correlated
with a severe depletion of cell wall calcium. Water soaking developped
at the late stages of development of the fruit, but no correlation was
found with ethylene biosynthesis and fruit in which ethylene action was
blocked by 1-MCP developped the disorder. The two genes encoding
key-enzymes involved in ethylene biosynthesis (ACS and ACO) and the
synthesis of CaM protein remained identical in response to watersoaking. In contrast, a specific CaM-BP disappeared in water-soaking
tissues. This CaM-BP may be marker or determinant of this
physiological disorder.

Phosphoinositide-specific PLC signalling in C4 plants

S. Coursola,N. Giglioli-Gnivarc'hb,A. Tr&noli~s~,J. Widala, J.-N. Pierre

V. Cotelle, N. Mortice, C. MacKintosh

In leaves of C4 species, the primary CO 2 fixation is carried out by the C 4

phosphoenolpyruvate carboxylase (C4 PEPC) isoform which catalyses the
[3-carboxylation of phosphoenolpyruvate by HCO 3 in the presence of a
divalent cation. C4 PEPC is subjected to a light-dependent, reversible,
regulatory phosphorylation [1 ]. This process involves activation of a complex
light-signal transduction cascade that upregulates the activity of a
Ca 2-independent C4 PEPC kinase, and thereby increases the phosphorylation
state of the photosynthesis-related PEPC isoform. A cellular approach, based
on the isolation of mesophyll cell protoplasts from the Ca plant Digitaria
sanguinalis has been used previously [2]. The pharmacological implication of
a tonoplast inositol- 1,4,5-trisphosphate (Ins(1,4,5)P3)-gated (TMB-8 sensitive)
charmel has led us to investigate the involvement of the phosphoinositide
signal transduction pathway in light-mediated C4 PEPC phosphorylation
cascade. Phosphoinositide-specific phospholipase C (PI-PLC) antagonists
(neomycin and U-73122) were shown to markedly inhibit in situ increases in
the apparent phosphorylation state of the C4 PEPC and the activity of its
C4 PEPC kinase in light plus weak base (NH4Cl)-treated protoplasts. A
PI-PLC activity was detected in D. sanguinalis protoplasts. This enzyme
showed an optimal phosphatidylinositol-4,5-bisphosphate-hydrolysingactivity
at physiological calcium concentrations, and was inhibited by neomycin
and U-73122. Light plus NH4CI, which promoted a transient increase in
Ins(1,4,5)P3 content, also triggered increases in PI-PLC activity. Along these
lines, D. sanguinalis protoplasts contain a Ca2+-dependent PI-PLC which
displays immunological (monoclonal anti-bovine brain PLC-61) similarities
with the mammalian PLC-~51. These data provide evidence for the
involvement of a functional phosphoinositide signal-transduction system in
the regulation ofC 4 PEPC phosphorylation in C4 plants.
[1] Vidal and Chollet, TIPS, 2, (1997) 230.
[2] Giglioli-Guivarc'h et al., Plant Cell, 8, (1996) 573.
(We/15.1/183)

Cellular localizationand spatial control of a tobacco SNARE

DannyGcelen,BarbaraLeyman,and MichaelIL Blan

Laboratory of Plant Physiology and Biophysics, University of
London, Wye College, Wye, Kent, TN25 5.4tt, U.K.

A key feature in vesicle fusion processes is the assembly and disassembly of

protein complexes controlled by NSF and SNAPs (NSF is Nethylmaleimide-sensifivefactor; SNAP is soluable NSF attachment protein).
The core of the complex consists of three different SNARE proteins
(SNARE is SNAP receptor), that bind through conserved coiled coil protein
domains. SNARE proteins share sequence identity constituting a
supeffamily of coiled coil proteins including syntaxins. We recently isolated
a syntaxin from Nicotiana tabacom (NtSyrl) with high sequence similarity
to the plasma membrane localized syntaxins SynlA from human and
Ssol/Sso2 from yeast. Antiserum raised against a soluble fragment of
NtSyrl binds to a 34 kd protein band in Western blots. NtSyrl is tightly
associated with membranes and copurifies with a plasma membrane enriched
fraction. Immuno-gold labeling of thin sections of leaf and root tissues
showed the presence of antigen in the plasma membrane, as well as in a
vesicular compartment of the cytosol, different from the Golgi or
endoplasmie reticolum The spatial distribution of NtSyrl was determined
by fractionation and light microscopy. NtSyrl is present throughout the
plant. The level of expression was highest in the roots and to a lesser degree
in the stems. NtSyrl protein and RNA levels are transiently upregulated by
the hormone abscisic acid (ABA), but are not influenced by auxin or
gibberillic acid. The control of expression by ABA was apparent in most of
the tested tissues, except for the roots. In line with the responsiveness to
ABA, NtSyrl expression was also induced by drought, salt stress and
wounding.

s322

(We/15.1/184)

Abstracts FEBS'99

Prenylated proteins regulate the alkaloid biosynthesis in

suspension cells of Catharanthus roseus
N. G i l t , r a t e ' h , k Ton'm,M. T h i ~ P. ~
ard P. ~

(We/15.1/185)

The lectin kinase receptors family ofArabidopsis thaliana

C. Herv6, C. Riou, V. Pacquit, P. Dabos, and B. Lescure
LBMRPM
UMR 215 CNRS-LVRA, BP27 31326 Castanet Tolosan cedex, France.

Laboratotre de phystologte v~gdtale. UFR Scienceset Techniques, Pare de

Grandmont. 37200 Tours. France.
Catharanthus roseus produces several valuable therapeutic terpenic indole

alkaloids such as ajmalicine, vineristine or vinblastine. In the C20 strain cell

suspensions, the production of monoterpenic indole alkaloids is enhanced by
auxin depletion from the culture medium [1]. Slyictosidine is the first precursor
of alkaloids and results from the condensation of seculeganin and tryptamine.
Previously:, mevalonate was supposed to be a precursor of secologanin. But, it
has been recently established that a new pathway initiated by pyruvate,
produced the secologanin used in alkaloid biosynthesis [2]. Moreover, the
inhibition of mevalonate production in cell suspension inhibited the alkaloid
production, but when these cells were supplied by exogenous 14C mevalorlate,
alkaloid production was restored and we observed that prenylated proteins and
sterols were labeled contrary to alkaloids [3]. These data suggest that mevalonic
acid or its derivatives could be necessary for the regulation of alkaloid
biosynthesis. Thus, we studied the influence of prenylated proteins in the
activation process of alkaloid biosynthesis. We have shown that 1)
prenyltransferase inhibitors such as S-perillyl alcohol or manumieine A
inhibited in a dose dependant manner the production of alkaloids, 2) the
addition of secologanin in cells pretreated by these drugs reversed totally the
inhibition of alkaloid production, and 3) the analysis of total protein extracts of
C. roseus cells labeled with 14C mevalonate shown two prenylated proteins of
20 kD and 55 kD potentially involved in the alkaloid biosynthesis activation
process. Thus, prenylated proteins were identified as part of the transduction
chain from the "auxin depletion" signal to the activation of the pyruvate/GAP
pathway leading to the production of secologanin.
In order to investigate the prenyltransferase involved in C. roseus alkaloid
biosynthesis, we used an RT-PCR strategy with degenerated primers deduced
from sequence alignment of famesyltransferases cloned from plants (Niconana
and Pea), rat and human. A cDNA of expected length of 600 pb had been
amplified. Nature of this eDNA will be checked by sequencing.

An Arabidopsis cDNA clone [1] that defines a new class of plant

serine/threonine receptor kinases was found to be one member of a small

family of four genes (lecRK-al-a4) clustered at the same locus on
chromosome 3. In the Columbia ecotype only the lecRK-al and perhaps the
lecRK-a3 genes are functional, since lecRK-a2 is disrupted by a Ty-copta
retroelement and lecRK-a4 contains a frameshitt mutation. Structural analysis
of the lecRK-al and lecRK-a3 deduced amino-acid sequences suggests that
the lectin domain is unlikely to be involved in binding monosaccharides but
could interact with complex glyeans and/or with hydrophobic ligands such as
auxins. Immunodetection of lecRK gene-products in plasma membranes
purified by free flow electrophoresis showed that the IecRK proteins are
probably highly glycosylated integral plasma membrane components [2]
Transgenic A.thaliana plants expressing antisense lecRK-a DNA sequences
show numerous severe morphological abnormalities, suggesting that these
genes play a fundamental role in plant development. Yeast two hybrid system
is used to explore the lecRK-a transduction pathway
[1] Herve et al., J. Mol. Bio1.,258, (1996) 778
[2] Herve et al., PMB, in press, (1999)

[1] Arvyet al., Biochem, 76, (1994)410

[2] Continet al, FEBS Lett., 434, (1998)413.
[3] Imbaultet al., Physiol Plant, 98, (1996)803

(We/15.1/186)

LeSBT1, a Subtilisin-Like Protease from Tomato

I. Janzik, N. Amrhein and A. Schaller
Institute of Plant Sciences, Swiss Federal Institute of Technology
(ETH), Universitdltstrasse 2, CH-8092Zffrlch

Mammalian prohormone convertases (PCs) which belong to the

subtilase superfamily of serine proteases play an important role in the
maturation of peptide hormones, neuropeptides, growth factors, and
receptor proteins. We are interested in subtilases from higher plants and
in their potential role in peptide hormone signaling. Here we report on
the cloning, the overexpression, and the characterization of LeSBTI, a
subtilase from tomato plants.
A probe was generated by PCR based on conserved regions in
mammalian PCs and was used to screen a tomato eDNA library. A fulllength eDNA designated LeSBTI was isolated. The deduced amino
acid sequence of LeSBT1 revealed structural features typical fur
eukaryotic subtilases, i.e. a signal peptide for targeting of the nascent
protein chain to the secretory pathway, a prodomain which may assist
in protein folding, and the catalytic domain comprising the catalytic
triad. Northern blot analysis demonstrated tissue specific expression of
LeSBT 1 in tomato plants.
Overexpression of LeSBT1 in the baculovirus/insect cell system
yielded two forms of the enzyme which accumulated within the cells
and in the medium, respectively. As shown by N-terminal sequence
analysis, the intracellular form lacked the signal peptide while the
signal peptide as well as the prodomain were found to be processed in
the secreted form. LeSBT1 was purified to homogeneity from the
medium and was characterized biochemically. When assayed with
glucagon as the substrate, LeSBTI showed strong preference for Gln
residues in the P1 position of the substrate. However, pH-dependent
autocatalytic processing was observed resulting in an activation of the
enzyme accompanied by reduced substrate specificity. No activity was
detected with short peptide substrates indicating that LeSBT1 requires a
minimum of three amino acids N-terminal of the processing site.

(We/15.1/187)

Circadian clock regulated phytochrome gene expression

L. Kozma-Bognad, A. Hallb, E. Adam',A. J. Millarb ,F. Nagy~
aBiol. Res. Center, HungarianAcademyof Sciences, Szeged, Hungary,
bDepartmentof Biological Sciences,WarwickUniversity,Coventry,LrK

Phytochrome is the best characterised photoreceptor regulating a wide range

of photomorphogenetic responses, such as seed germination, flowering and
senescence in higher plants. The phytochrome apoprotein is encoded by a
small multigene family; in Arabidopsis five genes (PHYA-E) have been
identified.
Physiological and biochemical processes in plants, like in many other
organisms, exhibit endogenous rhythms with periodicity close to 24h
(circadian). These rhythms are regulated by one or more endogenous
oscillators (biological clocks). The biological clock is synchronised to
periodic environmental changes (e.g. day/night cycles) by specific stimuli,
among them the most important is the light. PHYA and B are known to be
involved in setting/synchronising the clock by transducing the light signal to
the oscillator.
Now, here we report another relationship between the phytochrome and the
clock.
The temporal expression pattern of the tobacco PHYB1 gene was analysed in
transgenic tobacco seedlings carrying the PHYB1 promoter fused to the
firefly lucfferase reporter gene. Under 12/12 light/dark cycles the expression
exhibited a clear circadian rhythm with a maximum level just after dawn and
with a minimum level just after dusk. The rhythm persisted under continuous
light or continuous dark conditions as well, with a pattern and period similar
to that described for the expression of the gene encoding the chlorophyll a/b
binding protein (CAB). However, unlike CAB expression the PHYB1 rhythm
was not dampened in continuous dark.
Similar results were obtained in Arabidopsis transformed with the
Arabidopsis PHYB promoter fused to the luciferase reporter gene.
Furthermore, using 5' deletion derivatives of the PHYB1 promoter fused to
the luciferase gene, we showed that a 450bp fragment of the 5" untranslated,
flanking region is sufficient for circadian regulation.
Our results clearly demonstrate that expression of PHYB is regulated by the
endogenous biological clock.

Abstracts FEBS'99

(Weg15.1/188)

s323

Cell suspension culture from immature embryos of wheat

influenced by various volumes of vessels
I. Marcifiska, J. Biesaga-Kogcielniak, M. Filek

(We/15.1/189)

Institute of Experimental Botany, Academy of Sczences of the C2ech

Repubhc. Rozvojov6 135, 165 02 Praha 6, Czech Repubhc

Department of Plant Physiology of Polish Academy of Sciences,

30-239 Krak6w, uL Podluzna 3, Poland

The effect of vessels' volumes (100, 200 and 300 ml) on the growth
characteristics of a suspension culture was determined. The suspensions were
obtained from callus initiated from immature embryos of three winter wheat
cultivars. Several culture parameters (settled and packed cell volume, fresh
and dry weight and cells viability) were monitored during a 30-day period.
Also some factors of culture medium as: pH, conductivity and osmolarity
were evaluated. The significant correlation between settled and packed cell
volume and fresh and dry weight was found for all examined cultivars.
Moreover, the viability of suspensions correlated with its fresh and dry weight
and the medium conductivity correlated with its osmolarity. The kinetic of
growth in two out of three studied suspensions was similar. The most visible
changes in the majority of parameters were noted between 10th and 16th days
of culture. The conductivity and osmolarity increased from 10th up to 30 th
days of culture. Viability reached the maximum in the 10th day of culture.
Generally, the volume of culture vessel did not influenced significantly the
growth of the wheat suspension culture.
This study was supported by The Polish Commtttee for Scientific Research, Grant No. 5 PO6A
021 15

(We/15.1/190)

cAMP, cGMP and cytosolic calcium are mediators of signal

transduction in trausgenic tobacco and oat protoplasts
O. Molchan ~, S. Sokolovski a, I. Volotovski a M. Knight b "
lnsntute of Photo&ology, 220072 Minsk. Belarus.
Untversttv of Oxford, Oxjord OX1 3RB. UK

A number of chemical or physical stimuli are mediated by transient increase

in the [Ca2+]cyt in plants. There is evidence that cyclic nucleotides (cAMP,
cGMP) are also operating in plant cell transduction. However, occurrence of
the cyclic mononucleotides systems in higher plants and communication
between these mediators and [Ca 2+ ]oylin higher plants remain to be unclear.
The presented data show that membrane-permeable derivatives of cGMP and
cAMP and different modulators of cyclic mononucleotides systems induce a
large transient increases in [Ca 2+ ]cytof aequorm expressed transgenic tobacco
protoplasts in Ca2+-free medium suggesting that this increase is due to ion
release from intracellular Ca 2+ stores. Calcium-channel blockers verapamil
and CdC12 suppressed cyclic nucleotide-mediated [Ca2+]cyt increases. These
results indicate the significant proportion of the [Ca 2+ ]cytresponses occurs via
the activation of calcium channels. The influences of verapamil and CdCI2 on
cAMP-mediated effect were distinguished from those on cGMP-mediated
effect
Simultaneously, we detected by RIA that red light irradiation and membranepermeable derivatives of cGMP and cAMP induce CaZ+-uptake in etiolated
protoplasts. This data also indicate that red light reduce the level of
endogenous cAMP in plant tissue. Additionally, the plant growth regulators
IAA, N A A and GA3 were shown to induce increases in cAMP endogenous
level.
This suggests that potential communication point for crosstalk among signal
transduction pathways employing as second messengers as cyclic nucleotides
and Ca 2+ in plants exist.

The Role of Phosphoinositide Cycle in Phytohormone

Signal Transduction
Jan Martinec, Jan Petr~igek, Ivana Machd~kovfi, Eva Za21malov/t

The phosphoinositide cycle is one of the major signalling systems thought

to operate in plant cells. Auxins together with cytokinins are the key
phytohormones in a regulation of the development of plant cells, especially
when cultivated in vitro. The role of phosphoinositide cycle in the transduction
of both auxin and cytokiniu signals was studied in in vitro cultivated tobacco
cells.
The cell suspension culture of the auxin-dependent and cytokininindependent VBI-0 strain, derived from the stem pith ofNmotiana tabacum L.,
cv. Virginia Bright Italia, is used as experimental material. This tobacco cell
strain possesses some specific properties: stable filamentous phenotype with
time-separated phases of cell division and elongation, the high spontaneous
friability and standard growth at standard cultivation conditions. Therefore
such cell suspension culture may well serve as a model for both physiological
and biochemical studies.
The aim of present work is identification and characterisation of major
components of auxin signalling cascade: membrane-bound auxin-binding site
(putative receptor) and elements of phosphoinositide cycle - phospholipase C
(PLC) and D-myo-inositol-l,4,5-trisphosphate receptor (InsP3-R) in VBI-0
strain. The activity of these structures is monitored in relation to the growth
cycle of the cell strain.
Acknowledgement: Grant Agency of
No. 206/98/1510, supported this work.

(We/15.1/191)

the

Czech

Republic,

project

Transient modification of endogenous cytokinin

concentration in sunflower immature zygotic embryo.
J. Molinier, G. Halme
lnstitut de Biologic Mol~culaire des Plantes, CNRS-ULP
12 rue, du Gdn,~ral Zimmer 67084 Strasbourg Cedex, France

Somatic embryos or shoots can be induced in vitro on immature zygotic

embryos (EZI) of sunflower (Helianthus annuus L.) depending on the sucrose
concentration of the culture medium. On a medium containing 12% sucrose
somatic embryos are induced while on a medium containing 3% sucrose shoots
are formed. Both processes orignate from the identical group of cells localized
in the crown, and are induced by the action of a cytokinin, the BAP, which is
the sole phytohormone in the culture medium [1], [2]. The essential role that
cytokinin plays in the induction of cell division gives us the possibility to study
more precisely the effect on cultured immature zygotic embryo. Using the ipt
gene, involved in cytokinin biosynthesis, under the control of a constitutive or
an inducible promoter, we have studied the effects of a transient modification
of the endogenous cytokinin concentration on the morphogenic response. This
study permits us to better understand the role that cytokinins play in the
induction of in vitro morphogenesis.
[1] Bronner et al., Can. J. Bot., 72, (1994) 239.
[2] Jeannin et al., Plant Cell Rep., 15, (1995) 200.

s324

(We/15.1/192)

Abstracts FEBS'99

Auxin- and light-regulated signalling elements expressed

during wheat somatic embryogenesis
A. Nato ~, C. Fresneau a, J. de Buvser a, N. lreloursalimovaa,
D. Lavergne', M. Mirshahi b, G'. Ducreux~, Y. Henry~
a ~niversit~ de Parts Sud Bat. 360, 91405 Orsay, France.
lNSER34-CRB des Cordeliers, 75720Paris, France.

The modulation of signal transduction elements : G-proteins, NDPK and

arrestin-like was assessed during the induction of somatic embryogenesis in
wheat, under various different auxin and light conditions, via an immunological
approach, enzyme activity measurements and [ct-32p]GTP-binding assays [ 1].
Cell proliferation induced by 2,4-D stimulated the appearance of a second
NDPK (32 kDa) isoform, without any significant change in the measured
NDPK activity, which is mostly attributable to the classical 16-18 kDa form.
The 32 kDa polypeptide was detected only under light conditions.
Moreover, the induction of embryogenic capacity by 2,4-D was characterised
both by the over-expression of the classical 40 kDa Arrestin-like form and the
appearance of an additional arrestin-like protein of 29kDa in the soluble and
microsomal fraction. The 40 kDa Arrestin-like soluble form was unaffected by
light stimuli. The 29 kDa form, which appeared ailer 3 weeks o f in vitro culture
ofembryogenic tissue, was found to be light-regulated.
From embryogenic cultures, additional small G-proteins were detected by
probing nitrocellulose blots with [ot-32p]GTP : they consisted of a set of soluble
proteins ranging from 22 to 48 kDa, and a polypeptide of 20 kDa, which was
mostly detected in the microsomal fraction from light-grown cultures.
Evidence has been obtained for the involvement of plant G-proteins, arrestinlike and NDPK molecules in cellular responses to auxin and light.
Their widespread tissue distribution and their modulated expression under these
two stimuli support the view that they play critical roles in signalling cascades
participating in plant development.
[ I ] Nato et al (1998) Plant PhysioL, 113, 801

(We/15.1/193)

Signalling pathways and DNA methylation vs. somaclonal

variation in embryogenie in vitro cultures ofoil palm
A. Rival a, A. Nato b, D. Lavergne b, J.L. Verdeil a, J. Tregear ~
"CIRAD-CP/1RD. GeneTrop, P. 0. Box # 5045, 34032 Montpellier, France.
b LMVE. Universitd de Paris Sud. Bat. 360, 91405 Orsay, France.

The phenotypic fidelity of oil palm (Elaeis guineensis Jacq.) clonal plantlets
depends on the type of embryogenic callus lines used for micropropagation [1].
Regenerants originating from Nodular Compact Calli (NCC) have been shown
to exhibit t h e , mantled )> variant phenotype, which shows changes in the floral
architecture, at an average level of 5%, whereas this rate has been found to be
100% for plantlets regenerated from Fast Growing Calli (FGC). In order to
characterise epigenetic mechanisms underlying oil palm somaclonal variation,
global levels of genomie DNA methylation and regulation of signalling
pathways were investigated in both types of embryogeulc cam.
Levels of global DNA methylation were estimated by HPLC quantification of
5mdC after enzymatic hydrolysis of genomic DNA. The Sssl Methylase
Accepting Assay was also used. We measured a significant hypomethylation in
FGC (23.2 % vs. 18.7%) as compared with NCC from the same origin.
The expression of major signalling pathway elements, together with NDPK, a
key enzyme of cellular energy transfer, were analysed through enzyme assay and
Western blots using specific antibodies [2]. In FGC cells, an over-expression of
elements from the MAPK (ras and ERK) and PKC pathways was demonstrated.
In addition, the expression of G-proteins, NDPK and PLC were detected at
comparable levels in both types of plant material. The presence of two very
distinct isoforms of PEPC which were specific to FGC suggest a role for PEPC
as a target protein for proteolytic events that may occur in this highly
proliferative cell line.
Our studies of the differential activation of plant signalling pathways and
genomic DNA methylation in normal and variant cells is part of an integrative
approach to investigating the responses of plants to tissue culture, which is
considered to constitute an environmental stress.
Since it was previously demonstrated [3] that no significant amounts of
cytokinins could be detected in FGC whereas they accumulated in NCC, the
possible role of cytokimns in the MAPK-type phosphorylation signalling
cascades and the subsequent modulation of genome expression potentially
leading to somaclonal variation phenomena is discussed.
[1] Rival (1999) In : Somatic Emb. in Woody Plants, 6. Jain S.M. (ed), Kluwer Acad Publ
[2] Nato et al (1998) Plant Physiol., 113, 801
[3] Besse et al (1992) J Exp Bot., 252, 98.

(We/15.1/194)

ExtraceUular perception ofABA inA. thaliana cells.

J P Rona~. E. Jcannelle~. D Cornel~. F. Bardatb. O Dellis~. B Sottat'
and E. MigmiaO.
"]~lectropf[~:~tologte de~ Ilentbrane.~. I'mv D Dtderot and ~7"l~vstolo,4te
(ht D~l'eh)ppement de~ Phmtes. ~ntv P. et.t[ Curie. F-75005 Parts

Impmtant progress has been made ill characterization of the ABA

signalling components using genetic and molecular approaches [1].
However. we do not know, yet, the modalities of ABA perception.
Conflictual results concerning the site of ABA perception have been
published The prevalent conclusion is that since ABA controls many
responses, different perception sites for ABA might exist
In order to establish the cellular localisation of the ABA perception
sites in ,4t'ahldop.w.~ thtthana suspension ceils, we developped two
physiological tests based upon the capacity of impermeant ABA-BSA
conjugate to mimic permeant free ABA effects
We show that purified ABA-BSA COrljugate is able to trigger R A B I 8
gene expression and that this response is strictly due to the natural (+)-ABA
enantiomer The rate of R A B I 8 gene expression was independent on the
level of ABA uptake by the cells Using the voltage-clamp technique [2] we
shov, that ABA-BSA, as did ABA, evokes a membrane depolarization and
activates time- and voltage-dependent outward rectifying currents (ORC).
We demonstrate that those ORC are due to a K- effiux as assessed by
tail currents and specific inhibition by both tetraethylammonium (TEA) and
Ba: These observations provide evidence in favour of an extracellular
localisation fbr ABA perception [3]
[ 1] Leung et al, Physiol Plant Mol Biol, 49, (1998) 199
[2] Bouteau et al, Physiol Plant, 98, (1996) 97.
[3] Jeannette et al., Plant J., In press, (1999).

(We/15.1/195) The/so oncogene from Agrobacterium tumefaeiens and

the roIB family
Julien Schmidt and L6on Otten
htstttut de Btologle Mol~culatre ties Phtnte~/CNRS-Umvet ~itd Lout~' P(tstcnr,
12, rue du G#n~ral Ztmmer. 67084 Strasbourg Cedex, France

Agrobacterium tumeJLtciens strain AB2/73 is a limited-host-range

strain isolated from Lippia canescens. Previous studies showed a lack of
homology between its T-DNA and other T-DNAs from well known
strains [ 1].
We have isolated a T-DNA fiom AB2/73 by using a heterologous
border sequence as a probe. Thin T-DNA was sequenced and contains an
ORF that we called lso for Lippiu strain oncogene. This gene induces nondifferentiating tumors on a hmited number of hosts and is able to induce
callus on Nicotiana rustica leaf fragments on medium without hormones
when transferred by a Ti plasmid from a wide-host-range strain. The Iso
gene may therefore be considered as an oncogene [2].
Part of the predicted Lso protein is weakly homolozous to other
Agrobacterium oncoproteins encoded by genes rolB, rolBTR~, rolC, orJ73,
orfl4, c', d, e, 5, 6a, 6b, and 3'. We have called this group of genes the rolB
family.
In order to study the relationships between the various members of
the rolB family and the action of the lso gene, we have placed them under the
control of the CaMV 2x35S promoter.
BY2 tobacco cell lines were transformed with the lso gene. Under
standard conditions, such lines grow like control lines. However, at reduced
auxin concentation, they grow better than controls.
Studies of transgentc tobacco lines carrying lso are underway.
Primary regenerants show a particulary pbenotype snnilar to those induced
by the rolC gene. Gratiing experiments will enable us to study a possible
distance effect of the lso gene.

References:
[1] Unger et al., J. Bacteriol., 164, (1985) 723.
[2] Otten and Schmidt, M.P.M.I., l l, (1998) 335.

Abstracts FEBS'99

(We/15.1/196)

The influence of Rhizobium bacteria on field bean callus

culture.
E. Skrzypek, F. Dubert
The Franciszek Grrskt Departmentof Plant Physiology,Polish Academy
of Sciences,Podlu~m 3, 30-239 Kxakrw, Poland

The Rhizobium-legnme symbiosis is characterised by numerous interactions

between plant and bacteria. This complex co-operation depends on molecular
signals from both partners. At first it starts by exchange the signals during the
recognition. Possibly root lectins are produced not only in roots, may be the
same substances are produced also in other plant organs.
The aim of the present investigation was to test the field bean epicotyle
tissue culture for its capability to associations with Rhizobium bacteria. We
examined the strains of Rhizobium leguinosarum and methods of its
application to plant cell cultures as influencing the growth rate and vitality of
callus. In addition, we studied the possibility of Rhizobia to protect field bean
callus before toxic products of polyphenolic oxidation and ability of callus to
organogenesis in presence of the bacteria.
Callus was induced on Murashige & Skoog agar medium. Explants
were cultured on continuous light 180 mW/m2s-1 or in darkness. Rhizobium
leguminosarum biovar viceace strains: Fa65, BA1, P, AS were pre-cultured on
the yeast extract-mannitol medium and then were used as inoculant. After 8
weeks co-culture the mass of callus and rate of blackening (0- black, necrotic,
1- brown, 2- brown/green, 3- green) were measured. The Rhizobia strains were
co-cultured on three ways: a) they inoculated the nutrient medium at the
beginning or after 2, 4 or 6 week of the callus culture, b) bacterial filtrate was
added to the nutrient and covered with culture medium (double-layer
medium), e) 5-days-old bacteria cells were autoclaved and then added to the
medium.
The fact of signal exchange between bacteria and callus cells was
proven. Callus inoculated after 4 weeks culture reached highest increase of
mass (25-32%) and for the longest time maintain green colour as compared to
the control tissue The callus inoculated with bacteria strain Fa65 exhibited
highest viability (rate of blackening: 1.88). After diffusion of metabolic
compounds from the bacteria through the double-layer medium calluses which
were cultured on the top of the upper medium layer retained green colour, but
their fresh masses were about 50% smaller than control. Similar effect on the
callus darkening was observed on medium containing killed bacteria, however
the callus growth was comparative with control. About 50",4 tissues cultured in
darkness and inoculated with bacteria formed numerous root hair and roots.

s325

(We/15.1/197)

ACC metabolism in the cambial region of Scots pine

(Pinus sylvestris L.)
Alia Tiltu and Leif Eklund,
Dept. of Engineering and Natural Sciences, Viixjo University,
S-351 95 Vaxjo, Sweden.

The plant hormone ethylene has been shown to reduce wood formation,
specific changes in genetic expression and to elicit many physiological
responses. The purpose of this work is to describe the enzyme activities
involved in the biosynthesis and regulation of the ethylene precursor 1aminocyclopropane-l-carboxylic acid (ACC) during dormancy and growth
in the cambial region of Scots pine. Ethylene, ACC and its conjugates were
determined in cambial region of one-year old shoots of 10 year-old pine
trees growing in South Sweden. HPLC and quantitative analysis on GS-MS
were used to measure the amount of ACC and ACC cor(jugates after
purification. The analysis of ACC oxidase, ACC synthase and ACC Nmalonyltransferase activity was performed in crude protein extract and
ethylene evolution was detected using GC.
ACC synthase catalyses the conversion of S-adenosyl-L-methionine (SAM)
to ACC and 5'-methylthioadenosine (MTA) and plays a key role in
regulating the ethylene production. An oxidative enzyme, ACC oxidase,
carries out the conversion of ACC to ethylene. The N-malonylation of ACC
is provided by the malonyltransferase enzyme, preventing overproduction of
ethylene. Ethylene in the camblal region exhibits a seasonal variation with a
maximal concentration in July. The levels of ACC in the cambial region are
very low, compared to ACC conjugates. The level of ACC conjugates does
not display seasonal variation and is constant around the year. ACC
synthase and ACC N-malonyltransferase are the key enzymes providing the
regulatory mechanism for limiting ethylene production.

s326

Abstracts FEBS'99

3.2 Cell cycle and cancer

(We/3.2/198)

Morphological, biochemkal and immunohistochemicai aspects

of clear cell renal carcinoma and renal transitional carcinoma
I.Aschie, M Aschie - Manescu, N. Rosoiu,
Faculty o f Medicine, "Ovidius"Umverslty Constant&Romania

We tried to discriminate ambiguous renal carcinomas by using

histopathological, biochemical and immunohistochemical methods.
The immunohistochemical study included the application of
monoclonal antibody 2 - 2, lgG~ subclass (MoAb 2 - 2) on all slides.
We used an indirect immunoperoxidase technique on frozen tumour
tissues. International studies show a high specificity ofMoAb 2 - 2 for
tumoral cells of renal carcinoma (97%), their metastasis (70%),
transitional cell carcinoma (70%) and for some epithelial cells of
proximal tubules of the normal kidney. MoAb 2 - 2 does not stain the
epithelial cells on the non-renal cells carcinomas The biochemical
investigation included: concentration in blood of ureea, creatinine, uric
acid, glucose, Na-, K-, hemoglobin, VSH etc.
We detected 42 Grawitz tumours (20 clear cell type, 8 granular and
papillary type and 14 poor differentiated type), 5 Wilms tumours, 2
epidermoid carcinomas and 14 transitional cell carcinomas. Reactivity
of MoAb 2 - 2 for renal cell carcinoma tissues was 95%, except
nephroblastoma which revealed negative reaction Reactivity for
transitional cell carcinoma was 72%
The application of MoAb 2 - 2 may be useful for establish the
histological type of renal carcinomas in case of ambiguous diagnosis.

(We/3.2/200)

Localization of extracellular matrix components

durin~tumoral invasion.
C. Catussea,M. Polet~, P. Dehan,C. Corauxa,J.M.Foidag~,
R Bimmbauta.
"L~Sk3O,M U514.51092 Reb~L~F~a tce-J'lJ3772 2~t-Ttb~r~, B-40gOLi~ge,Belg~ te.

During tumoral progression, extracellular matrix and especially basal

membrane (BM) appears to be dynamic structures that are not only degraded
but also deposited around tumoral clusters. Type IV collagen heterotrimer,
constitued by combination of six chains (ctl-~6), is the major structural
protein of basement membrane. Type VII collagen and laminin 5 are
components of cell adhesion complexes which link the BM to the underlying
connective tissue. In this study we examined by immunohistochemistry the
localization of three chains of type IV collagen (ctl, 0t3 and o~5), type VII
collagen and laminin 5 at different stages of bronchopulmonary cancers.
In normal tissues, o~I(IV) chain was detected in all BMs (bronchial, vascular,
alveolar and glandular), ~5(IV) was only present in vascular BM, laminin 5
and type VI1 collagen were co-localized in bronchial and glandular BM
whereas ~3(IV) immunolabelling was totally absent from normal bronchi. In
well-differentiated carcinomas of pre-invasive cancers, cO(IV) chain staining
was found in some neosynthetized BMs laying at the interface between
tumoral cells and stromal compartment as a disrupted and irregular labelling.
&l(IV) chain showed strong reactivity in all BM. As c~3(IV)chain, laminin 5
and type VII collagen were detected in neosynthetized BM. In invasive
cancers, &3(IV) chain, laminin 5 and type VII collagen staining was
markedly attenuated, cO(IV) chain distribution was restricted to stromal
compartment giving clues of BM degradation.
These data showing a neosynthesis of o~3(IV) chain around some welldifferentiated tumoral clusters at pre-invasive stage of bronchopulmonary
cancer and a disappearance of this chain in invasive cancer, clearly suggest
an important role of this chain in tumor invasion.
C. Catusse is a doctoral fellow of the ARERS (Association Rdgionale pour
t'Enseignement etla Recherche Scientifique).

(We/3.2/199)

Histological diagnosis corelated to the immunohistochemical

results in gastro - intestinal LMNH
M.Aschie - Manescu, I.Aschie, N. gosoiu,
Faculty o f Medicine, "Ovia~us" University 8. 700 Constanta, Romama

We tried to discriminate the morphological types of gastro- intestinal LMNH

based on clinical, morphopathologieal and immunohistoehemical criterias.
Immunohistochemical studies using peroxidase--antiperoxidase method were
performed on paraffin sections of the tumour. A panel of monoclonal
antibodies was used to characterise the tumoral cells: CD-20, L-26-(-) for
tumoral B cells. CD-3-(-) for tumoral T cells and (+) for reaetional T cells;
CD68, PGMI-(+++) for tumoral maerophages; EMA, E29-(-) for tumoral
tissue and (+) for normal glandular epithelium. Monoclonal Mouse AntiHuman epithelial Membrane Antigen (DAKO-EMA, E29) is widely used for
the detection of epithelial neoplasm, particular when of glandular origin. There
is a strong reaction even for the epithelial neoplasm unlabeled for cytokeratine.
Monoelonal Mouse Anti-Human Maerophage, CD68, PGMI stains
macropbages in a wide variety of human tissues. All digestive LMNH B type
had reacted with CD20,L26 and, being diffuse type, the level of reactive T cells
and maerophages was low. On contrary, the follicular types contains malignant
cells, reactive T cells and macrophages at high level. In diffuse eentroblasticcentroeitic lymphomas we observed, using immunohistoehemical methods, a
decreased number or even the absence of the cells. The follicular centroblastiecentrocitic lymphoma is a special morphological type of malignant lymphoma,
with a more favorable prognosis than diffuse centroblastic-centrocitic type. The
most important differential diagnosis of follicular lymphomas is with reactive
follicular hyperplasia. Differential diagnosis of large cell malignant LMNH is
difficult and we must consider reticular Hodgkin disease and metastasis of
undifferentiated carcinoma. That is why, in our study we used Monoclonal
Mouse Anti Human Macrophages, CD68(DAKO-CD 68) to reveal two types
of histoeitary lymphomas with pale and abundant eitoplasm, with vacuolar
nuclei, E-shaped, with many mitoses. On contrary, the immunoblastic
sarcomas contains intense basophilic tumoral cells, with/without visible
vacuoles, but without non-specific esterasis l i k e histoeites have.
Immunohistochemical techniques were very useful in correlation of tumoral
fenotype and clinical evolution, therapeutical response and prognosis.

(We/3.2/201) Net, a negative Ras-switchable TCF, mediates

repression through CtBP and de-acetylation.
P. Criqui-Filipe, S.-M Malta, B. Wasylyk

Instltut de G#n~tique et de Biologie Moleculaire et Cellulaire, 1 Rue

Laurent Fries, BP 163, 67404 IIIkirch cedex, France.

Signalling cascades are integrated at the transcriptional

level by the interplay between factors such as the ternary
complex factors (TCFs) that interact with SRF and the serum
response element (SRE) of the fos promoter. Net is a negative
TCF that is switched to a positive regulator by the Ras signal.
To understand the mechanisms of repression by Net, we used
a yeast dual hybrid screen to identify factors that interact
with its inhibitory domain. We isolated mCtBP1, the murine
homologue of huCtBP1, a factor implicated in negative
regulation of transformation by E1A plus Ras. We show that
mCtBP l interacts strongly with Net both in vitro and in vivo.
The CtBP interaction domain of Net, the C1D, mediates
repression independently of the previously identified negative
element, the NID. The CID inhibits by recruiting the corepressor mCtBPI and requires de-acetylation, whereas the
NID apparently represses by other mechanisms. Finally, we
provide evidence that CtBP and de-acetylation repress the cfos SRE in low serum when it is inactive, but not in high
serum when it is active. These results provide insights into
the cross-talk between pathways that inhibit and stimulate
transformation at the level of Net, a regulator of gene
expression.

Abstracts FEBS'99

(We/3.2/202)

Canine Cathepsin B in Normal and Pathologic Conditions.

M Drobni~-Kogorok~, A Nemec". P Zrimgek ~, I Kri/~ajb,
J Kos bx P Juntes", Z Pavlica ~,
~l ~termarv ['acuity; ( nwersitv of Ljub[ja~a ~D:'pt of Btochem and Mol
Btol Jozef Stefan lnsntute. LjuhOana "Krka Vovo mesto, Slovema

It is well known that uncontrolled cJ~teine proteinase activity have been

implicated in the development of variety of human diseases including
muscular dystrophy, rheumatoid arthritis, osteoporosis, Alzheimer's disease,
glomemlonephritis, lupus erithematosus as well as cancer
Our recent studies indicated the elevated levels of cathepsin B activity in
canine mammary gland tumours The possibility of proteolytic as well as
immunohistocbemical detection of the cathepsin B suggests us to select
cathepsin B as a tumour marker for our studies of canine mammary gland
tumours. Polyclonal antibodies against human cathepsins appeared to be only
partially suitable for immunohistochemical studies of animal tissues
Antibodies against human cathepsin B reacted well in human and monkey
liver tissue, while in the liver of dog the reaction was considered unspecific,
suggesting that only specific antibodies against canine enzymes can be used
in diagnostic ofinvasiveness of the canine mammary gland tumours.
Therefore canine cathepsin B was isolated from healthy dog liver using a
modification of affinity chromatography on the semicarbazone of Gly-Pheglycinal linked to Saepharose 4B [1]. SDS.PAGE revealed two proteir "
bands with molecular weight of 29000 Da and 42000 Da On the basis of Nterminal sequences it is evident that they belong to a precursor and processed
form & t h e enzyme On the basis of sequences alignment the greatest extent
of homology with bovine enzyme was established The specific antibodies
against dog cathepsin B were rised in rabbits and used in
immunohistochemmical studies of canine mammary' gland turnouts and
gingival tissue from patients with chronic peridontitis
[l]Rich DH et all, Biochem J, 235, (1986), 731.

s327

(We/3.2/203)
Oncostatin M induces cell-cycle arrest in human glioma cells
through inhibition of p27 K'p~degradation
M. Friedrich, R. Laumann,F. StOgbauer,E. B. Ringelstein, and H. Halfter
Chnicof Neurology, Universityof Muenster, 48149Muenster,Germany

Oncostatin M (OSM) belongs to the IL-6 family of cytokines and transduces

its biological signals through the common receptor subunit gpl30 by
activation of the Janus kinases (Jaks) and members of the 'signal transducer
and activator of transcription' (STAT)-family. We have shown that OSM
inhibits proliferation and induces differentiation of human glioma cell-lines to
an astrocytic phenotype [1]. OSM activates components of the Jak/STATpathway as well as tyrosine phosphatase SHP-2 and mitogen activated-kinase
2 (MAPK2) [2].
FACS analysis of OSM treated glioma ceils revealed arrest in Gl-phase of the
cell-cycle. For further understanding of the growth inhibitory effects of OSM
we examined the influence of cytokine on components of the cell-cycle.
About 16 hours after OSM treatment the retinoblastoma-like protein p130 is
entirely hypophosphorylated. The hypophosphorylated state of p130 is
dependent on iuhibtion of cyclin-dependent kinase. We analyzed the effect of
OSM on the level of cyclin-dependent kinase inhibitors (Cdki). OSM induced
an increase in p27 ~p~ level within 4 hours. Northern-Blot analysis of p27 ~apl
expression in response to OSM treatment showed no increase in p27 zap~
mRNA level. As shown by metabolic labeling and determination of protein
stability OSM alters the 'half-life' of p27 Kip1by decreasing its phosphorylation
and subsequent degradation. Reduced stability of p27 gapn is known to be
dependent on phosphorylation through Cyclin E-CDK2 [3]. Inhibition of
CDK2 activity by tyrphostin B42 also increased p27 KIp~level. We propose that
OSM induces cell-cycle arrest in human glioma cells through alteration of
v,lpi
CDK2 activity and thereby prolongs 'half-life' of p27 " . We are now further
analyzing the members of the OSM induced signaling pathway leading to
reduced CDK2-activity and thereby to cell-cycle arrest in human glioma cells.
Ill Halfteret al. (1998)GrowthFactors 15, 135.
[2] Hairieret al (1999)submitted
[3] Vlach,J. et al. (1998)EMBOJ. 16, 5334

(We/3.2/204) Role of Crk II in the activation of JNK pathway by

growth factors
S. Girardin, M. Yaniv
Laboratotre de~ Vzrus Oncog~nes, URA 1644 du CNRS. lnstttut Pasteur.
25, rue du Docteur Roux, 75724 Parts. France

The interplay between the actin cytoskeleton and the stressresponse pathway JNK/SAPK has been emerging in the last few years
with the characterisation of the dualistic role of racl and cdc42, two
members of the Rho-family of GTP-binding proteins. Moreover, we
have recently shown in our laboratory that although JNK/SAPK
pathway was down in confluent fibroblasts, such an inhibition could be
abolished by adding a drug that depolymerises actin. Recently, two
proteins of the focal adhesions complexes, pl30Cas and Crk II, have
been implicated in YNK/SAPK activation upstream of rac 1[1 ]. We have
shown that J'NK1 interacts directly with Crk II in vitro and in vivo in
HeLa cells. Moreover, this association is regulated by growth factors but
is insensitive to UVC irradiation. We also show that Crk II major site of
tyrosine-phosphorylation is necessary to regulate Crk II / JNK1
interaction. Thus, we propose a role for Crk 1I in the regulation of
growth factors-induced activation of the JNK/SAPK pathway.
[1] F. Dolfi et al., J. Biol. Chem., Vol. 95, (1998) pp15394

(We/3.2/205)

Genes specifically activated in jun-transformed avian

fibroblasts
M. Hartl, A. Bader, K. Bister
Institute of Biochemistry, University of lnnsbruck,
Peter-Mayr-Str. l a, A-6020 lnnsbruck, Austria

Two genes were identified based on their specific transcriptional activation in

jun-transformed avian fibroblasts, also employing a tetracycline-controlled juninducible system. One of them, BKJ, was found to be activated during jun- or
fos-induced fibroblast transformation [1, 2]. We now show that B K J is a direct

transcriptional target of the AP-I transcription factor components Jan and Fos.
The complete structural organisation of the quail BKJ gene was determined by
nucleotide sequence analysis and transcriptional mapping. The B K J promoter
region contains two authentic AP-1 binding sites. By transactivation of reporter
gene constructs and direct binding of Jan recombinant protein, the proximal
AP-1 element was shown to be essential for B K J promoter activation. The
activation of BKJ in jun-transformed avian fibroblasts was also demonstrated at
the protein level. B K J is a novel gene related to the avian ]3-keratin gene family
whose members display highly specific expression patterns during
embryogenesis and epidermal development. In contrast to BKJ, the other gene,
termed JAC, was found to be specifically induced in jun- but not in fostransformed fibroblasts suggesting that its activation does not require a Jun/Fos
heterodimer. JAC encodes a putative 155 amino acid protein with distinct
sequence motifs characteristic for transcription factors but with no homology to
known sequences. Ectopic activation of these genes in fibroblasts by deregulated
jun may contribute to cell transformation.
[1] Hartl, M., and Bister, K. Proc. Natl. Acad. Sci. USA 92 (1995) 1173111735.
[2] Hartl, M., and Bister, K. Oncogene 17 (1998) 2901-2913.

s328

(We/3.2/206)

Abstracts FEBS'99

PPARs in colonic carcinoma

C. Huin a, H. Schotma, F. Plenat b, M. Daufa a
aEA 2402.Prolif~rateurs de Peroxysoraes, FacultE des Sciences, 54506
Vandoeuvre-les- Nancy Cedex et bLaboratoire d'Anatomie Pathologique,
CHU Nancy-Brabois, 54505 Vandoeuvre-les-Nancy Cedex, France.

PPARs (Peroxisome Proliferator-Activated Receptor) are

transcription factors known to be involved in gene regulation of lipid
metabolism [1]. They are present in colon. Recently, PPAR~t isoform has
been implicated in colon carcinogenesis as well as in colonic cell
differentiation [2]. The expression and role of PPARa isoform in normal
and neoplastic colon have not been stated even though it participate to
hepatocarcinogenesis [3]. No role to PPAR[3 has been devoted in colon.
We studied both transcriptional and translational levels of PPARs in
human normal and neoplastic colon mucosa. Several situations have been
investigated : adult colon, different fetal stages of colon development since
tumor markers are often present during ontogenesis and tumor biopsies. In
order to analyze the transcriptional level of PPARs, we developed a nuclease
S1 protocol based on direct hybridation of PPARs cDNA to the
corresponding mRNA extracted from tissue samples. PPARs translational
level has been studied using rabbit antibodies directed against partial
aminoacids sequences of PPARs. All of these antisera have been developed
in our laboratory.
Our recent results show that PPARs expression varies
according to colon development and colonic cell differentiation. In tumor
biopsies tested both PPAR~ and ~' are present and exhibit an upregulated
expression.
1. Gustafsson, J. A.(1998) Nutr Rev 56, $20.
2. Saez, E., Tontonoz, P., Nelson et al (1998) Nat Med 4, 1058.
3. Peters, J. M., Cattley, R. C. & Gonzalez, F. J.(1997) Carcino-genesis
18, 2029.

(We/3.2/208)

The overall genomic DNA methylation in the primary

human endometrial cancers
K. Postawski~, A. Semczuk~, J.A. Jakowicki~, G. Dirhetmerb,G.Keithb
Dept Gyn Surg Univ School Med., Jaczewsklego 8, 20090 Lublin, Poland
IBMC/CNRS, 15, rue Descartes. 67084 Strasbourg,France,

It has been generally accepted that the overall DNA 5-methyldeoxycytosine

(mSdC) content is decreased in most human neoplasms and it was shown that
oncogenic ras modulates the DNA methylation (M) by influencing the DNA
methyltransferase which is strongly involved in mechanism(s) of this
modification. The aim of the study was to investigate the total M level in
human endometrial cancers (EC) and to compare it to normal endometrinm
(NE) and to the presence of K-ras codon 12 point mutations that are believed
to be the most common and earliest alterations of this gene in the uterine
carcinogenesis. We have analyzed the mSdC versus the deoxycytosine content
of the genomic DNAs isolated from 44 EC and 25 NE samples using enzymatic
digestion into nucleotides, 32p postlabeling, two-dimensional thin-layer
chromatography on cellulose plates and phosphobioimaging quantitation. In
addition RFLP-PCR revealed K-ras gene point mutations in 25% of the
cancers. The overall mSdC of the EC DNAs (3.43-+0.47%) was significantly
higher than that of the proliferative NE (2.94+0.4%, p=0.003) and lower than
that of the secretory endometrium DNAs (3.75-+0.47%, p=0.03). The M level
of neoplasms with mutant K-ras was lower than that of cancers bearing wildtype gene (3.190.31 vs. 3.5 I-+0.048%, p=0.047). Among EC DNAs six were
found to be bypomethylated, eight were hypermethylated, whereas the
remainder 30 had mSdC in the mean range of the NE. M was positively
correlated with the histological tumor grading (r=0.4, p=0.012) being
significantly higher in poor-differentiated (G3) than in lower grade neoplasms
(3.68-+0.6 vs. 3.3-+0.3 %, p=0.025). Lower M seemed to be associated with
diminished tumor invasiveness. The prevalence of K-yas gene alterations that
were absent in hypermethylated EC was related to genome demethylation
(r=0.999, p=0.03). M was here inversely correlated with the histological grade
of the tumor (r=0.56, p=0.001) being highest in GI neoplasms. This could
indicate that K-ras activation of DNA methyltransferase is attributed mainly to
well-differentiated cancer cells. Our results suggest that overall DNA
metbylation is probably a result of neoplastic transformation and could
therefore be used as a prognostic molecular marker of ECs.
This work was supported by the Ligue Nationale contre le Cancer du Haut-Rhin
and Polonium Grant

(We/3.2/207)

Inhibition of Intestinal Carcinogenesis by the Flavonoid

Quercetin
G. Jacobasch, A. Salomon, H. Pforte, S. Florian, K. Schmehl
German Institute of Human Nutrition, 14558 Bergholz Rehbrucke, German)"

Dietary flavonoids are known to act antiproliferative and may play an

important role in cancer chemoprevention. In this study, we analyzed the
anticarcinogenic potency of quercetin in the intestinal tract. The intestinal
carcinogenesis often starts with a primary mutation in the apc-gene.
Therefore, C57BL67-ape-Min -/+ mouse provides a genetically defined
model for inherited and sporadic forms of human intestinal carcinogenesis.
Four to five week old animals were fed with a semisynthetic diet as a control
or supplemented with 14ag/kg body mass pure quercetin or 3.6 ~tg/kg body
mass in the treated groups for about 60 days, respectively.
The mesoscopical counting of the intestinal neoplasias showed that both
quercetin and rutin diet inhibited adenoma development up to 90%. In
comparative studies, rutin preparations absorbed before or after degradation
by eubacteria ramulus failed this anticarcinogenic effect. It was concluded
that the inhibition of carcinogenesis by flavono~ds takes place systemically.
Therefore, flavonoid absorption should happen in the upper
part of the intestinum Both the aglycon and the glycoside can be absorbed.
Rutin is transformed into quercetin after absorption indicating the existence
of 8-glycosidases in the intestinal epithelial cells. Unlike rutin, a high
absorption rate of quereetin is accompanied by a modification into
isorhamnetin in the intestinal wall.
The anticarcinogenic mechanism seems to be the result of both strong
inhibition of proliferation as well as low activation of apoptosis. An
increase of cyclin D1 and cdc 2 as well as a decrease in cyclin A was found.
The overexpression of cyclin D 1 may inhibit the DNA synthesis by binding
to PCNA and the expression of S-phase related genes by binding of cyclin D1
to cdk2. The cyclin DI- cdk2 complex suppresses the cdk2-dependent kinase
activity because it does not exhibit protein kinase activity. An inhibition of
proliferation results from both effects. The activation ofapoptosis by quercetin
is related to the Ape - [3-catenin - E-cadherin system. Quercetin increases the
expression of Ape and E-cadherin leading to a decrease in the nuclear !3catenin level by its translocation into the cytosol and proteolytical degradation
after binding to APC and phosphorylation of this dimer. Furthermore, a higher
part of 13-catenin is bound to E-cadherin at the cell membrane.

(We/3.2/209) Molecular chaperones and tumor supressor p53

Z. Milicevic i, , M. Petrovic b. D. Bogojevicb,.
"Institute of Nuclear Sciences "Vinca", Belgrade, Yugoslavia
blnstitutefor Biological Research, Belgrade, Yugoslavia

Both under normal conditions and in response to stress, hsps are

implicated in protein-protein interactions such as folding,
translocation and prevention of inappropriate protein aggregation.
Many features of the functions, regulation and expression of hsp~
suggest they play a roll in cancer. The increased expression of hsps
in tumours may have implications in other biological processes,
including apaptosis, differentiation and call-cycle progression, p53 is
a short-lived transcription factor that is frequently mutated in tumor
cells. Mutant p53 proteins often assume an aberrant conformation
devoid of tumor suppressor activity and newly capable of binding to
the cognate or inducible hsp70. In this study, we characterize p53
and hsp complexes and their subcellular localization in the
transformed colorectal cancer cells which contain aberrant p53
conformers. These immunological analysis of p53 from whole cell
lysates revealed the additional presence of a broad 70 kDa band and
a 90 kDa band. Anti-p53 antibody DO-7 and CM-1 recognize an
exposed site present in aberrant conformations of p53 protein in
nuclear fraction. Under these conditions p53 resolves as a doublet
which may represent different charged isoforms of full length p53.
The interactions of aberrant p53 protein with cellular proteins are
likely to be important in understanding the biology of the
transformed phenotype. The interaction of hsp70 with mutant p53
may prevent mutant-wt p53 complex formation and thus allow wt
p53 to function unhindered. The possible relationship of hsp
complexation to p53 as a cellular stress response to abnormal protein
is discussed.

Abstracts FEB S' 99

(We/3.2/210)

Characterization of glutathione S-transferases in normal

s329

(We/3.2/211) Histone deacetylases and transcriptional repression by Rb

E. Nicolas~,L. Magnaglu-Jaulinb, A. Harel-Bellanb, and D. Trouche~

human kidney and renal cell carcinoma

J MJmic-Oka, T Slmic, Z Reljlc, D Dragtcewc

aLBME/IBCG/CNRS, 118 route de Narbonne, 31062 7bulouse, France

bCNRS UPR 9079. 7 rue Guy 3docquet, 94800 Vtllejutf France

ln~tttute of B~ochemtxt~v, I~ocult~ ' ~Jj3[e~hctne, ~ )m,er~J(v (~f Belorade,

)'ugoMavta.

The exposure of kidney cells to the toxic metabolites of various origins,

which may have carcinogenic potential, is supposed to play role in the
imtiation of renal cell carcinoma Glutathione S-transferases (GSTs), a
family of detoxification enzymes, play a critical role in protecting the kidney
by catalyzing conjugation of different carcinogens with glutathione.
Glutathione S-transferases were investigated in normal human kidney,
different renal tumors and corresponding kidney tissue adjacent to human
renal tumors. Enzyme purification was performed by using affinity
chromatography and isoelectric chromatofocusing The isolated enzymes
were further characterized by determining their activity towards specific
substrates and kinetic parameters,
Purification of normal kidneys' GSTs by affinity chromatography revealed
the presence of two GST fractions flow-througt GST (26-28%), with
lower affinity for GSH linked to epoxy-activated agarose affinity resin and
GST fraction tightly bound to affinity matrix Further purification of bound
GSTs resulted m a rich profile of different GST isoenzymes with balanced
expression of both anionic and cationic forms The results obtained, suggest
efficient cellular anti-carcinogenic potential in normal kidney However,
kidney tissue adjacent to human renal tumors had substantially less flowthrough GST fraction (1-4%), whereas renal tumors did not express flowthrough GST at all. Isoelectric focussing indicated significantly smaller
number of GST isoenzymes in non-tumor kidney regions when compared to
normal kidney, w~th anionic forms being dominant Isoenzyme pattern of
renal carcinoma tissue differed significantly from both non-tumor adjacent
renal tissue and normal kidney The GST pattern in renal cell carcinoma
showed the predominance of anionic GST forms
Based on the results obtained, it can be concluded that patients with renal
cell carcinoma have defective anti-carcinogenic potential due to the
qualitative changes in GST expression It seems reasonable to assume that
the absence of flow-trough GST fraction as well as small number of cationic
GST forms might be risk factors for the occurrence of renal cell carcinoma

(We/3.2/212)

Non-genomic cell cycle specific antiproliferative effect of

progesterone. E.L.Olsen,a P.C.Endresen b A.Orbo c and
G.Sagera Dept. of Pharmacology,Inst. of Medical Biology, University
of Troms~, N-9037 Tromso, bDept, of Ciin. Pharmacologyand ~Dept.of
Pathology,University Hospital of Tromso, N-9038 Tromso. Norway

The effects of progesterone are conveyed through genomic and non-genomic

mechanisms. It is well-known that progesterone has ability to modulate cell
growth, an effect that has been assumed to be conducted by classical
receptors. Previous studies have shown that progesterone has a dosedependent antiproliferative effect in C4-1 cells [1] which represent a
transformed cell-line derived from a carcinoma of the uterine cervix and
which are devoid of <<classicab>progesterone and estrogen receptors [2].
These results were supportive for a non-genomic mechanism beyond the
antiproliferative effect of progesterone. The present study was conveyed to
determine whether the effect of progesterone was cell cycle specific and if
the effects could be explained by a cross reaction with androgen receptors
or to determine whether the presence of progesterone caused expression of
its own receptors. The cells were cultured as described previously [1] with
the exception that phenyl red was left out. Flowcytometric DNA content
analysis was performed after ethanol fixation and propidium iodide staining.
The steroid receptors were assayed by immunohistochemistry. The
antiproliferative effect of progesterone was reproducible. The present study
showed a half-maximal inhibition at about 10 gg/ml and that progesterone
caused an increased cell fraction in GI phase. Androgen receptors were not
detected and progesterone was not able to induce its own receptor. The
present study indicates that progesterone has a growth inhibitory effect
conveyed by a specific non-genomic mechanism of action which arrests the
C4-1 cells in the Gi-phase.
References: [1] A.Orbo et al., Int. J. Oncol., 5, (1994) 619. [2] A.Orbo et al.,
Anticancer Res., 15, (1995) 1905.

The product of the retinoblastoma susceptibility gene, the Rb protein, is a

key regulator of cell cycle progression in mammalian cells. In its active
hypophosphorylated form, Kb prevents cells to progress towards S phase.
During a normal cell cycle, Rb is inactivated by cyclin-dependent kinases.
One of the major targets of Rb is the E2F transcription factor, which
activates at the G1/S transition the expression of proteins required for S
phase progression. During G1, Rb contacts directly and inactivates the E2F
activation domain. Moreover, the E2F/RB complex binds E2F sites and
represses transcription of E2F-regulated genes. This repression is believed
to be crucial for the proper control of cell growth. We have previously
shown that transcriptional repression by Rb involves the histone deacetylase
HDAC1. HDACI is the catalytic subunit of complexes that are able to
modify chromatin through the deacetylation of the histones N-terminal tails,
thereby repressing transcription. We are presently charactefising at the
molecular level the histone deacetylase complex recruited by Rb to repress
E2F activity. In particular, we are testing the involvement of RbAp48, a
protein which has been shown to contact Rb and which belongs to many
histone deacetylase complexes.

(We/3.2/213)

Analysis of size variation of CAG/CTG repeat during

testicular tumorigenesis
Y. One a, F. Bulle ~, E. Rajpert-De Meyts b, G. Guellaen a
~INSERM Unit-99. th~pttal Henri Mender, 94010 Cretetl, France,
~Copenhagen UniversiQ' Ho~pttal, Rigshospttalet, Copenhugen, Denmark

Genomic instabilities of trinueleotide repeat sequences have been observed in

several types of human cancer [1 ]. Recently, it has been described that several
human tumor cell lines, as well as some tumor samples, exhibited expansion of
trinucleotide CAG/CTG revealed by rapid expansion detection [2]. Since we
identified 65 CAG/CTG containing genes which are expressed in human testis
[3,4], we investigated whether 33 of these genes might exhibit variation in their
repeat numbers in testis tumor. Using specific oligonucleotides, teaming the
CAG/CTG repeat, we performed RT-PCR on total RNA extracted from 3
seminomas, 3 teratocarcinomas, and 4 combined tumors with their surrounding
normal parts obtained from patients undergoing radical orchidectomy. Three
testicular RNAs, obtained from men without testicular cancer, were added as
control. The specific PCR products were separated on 8% acrylamide gel, and
the specific bands were revealed using a 3zp-labeled (CAGh4 oligonucleotide as
probe. Out of the 33 analyzed genes, 10 displayed variations of size and/or
number of specific bands. One of them has additional bands in the seminoma
group, another gene has additional bands in the combined tumor group, and two
genes have additional bands in all three types of tumor as compared to the
controls. The 6 remaining clones displayed more complex patterns. These
results argue lbr a specific alteration of mRNA transcribed from genes
containing CAG/CTG during testicular tumorigenesis. We are now investigating
in more detail the mechanisms involved in the modifications of these genes
occurring during testicular tumorigenesis.
[1] Wooster el al., Nat Genet, 6,(1994), 152.
[2] King et al., Cancer Res, 57, (1997), 209.
[3] Bulle et at.. Genome Res, 7,(1997), 705.
[4] Pawlak et al., Mamm Genome, 9, (1998), 745.

s330

(We/3.2/214)

Abstracts FEBS'99

Glutathione system and human breast cancer proliferation

M. Perquin a, T. Oster ~, A. Maul b, N. Froment c, D. Bagrel a

(We/3.2/215)

~Laboratotre de Btochtmte M~tabohque et Cellulaire & ~D~pt. StatJstique

et Traitement Informattque des Donn~es, University of Metz, France
~CHR Metz-Thtonville, H6pttal Bon-Secours. Metz, France

BACKGROUND: Breast cancer is still the leading cause of cancer deaths

in French women, with a relatively high risk for recurrence in a metastatic
disease. Many environmental and genetic factors are determinant properties
for each individual breast tumour, in particular with respect to growth rate and
response to the treatment. Pertinent diagnosis still urges the need for
prognostic markers in order to design adapted therapeutic strategies. As a
major intracellular detoxification system, glutathione (GSH) and its associated
enzymes (glutathione S-transferases - GST, peroxidase - GPX, and reductase
- GR) have been reported to contribute to tumour resistance. Moreover, GSH
is well known to be involved in cell growth and differentiation processes.
Therefore, the GSH-related system could be of importance as a marker of the
intrinsic protective and proliferous capacities of each tnmour tissue and could
therefore be associated with poor prognosis. METHODS: In order to identify
how GSH system could be correlated with commonly considered prognostic
and clinicopathologic factors, we studied glutathione content and associate
enzyme activities in a population of 49 women, aged 24 to 84 (median:
53 years), with breast cancer. For each patient, peritumoral tissue was used as
an internal control. Statistical analyses were performed by using MannWhitney test and multiple linear regression. RESULTS: Despite important
interindividual differences were observed in the biochemical data, tumour
samples showed significantly higher GSH content. Also, the activities of total
GST and pi-class GST Pl-1 were increased as well as that of the GR, but no
significant difference appeared in the tumour with respect to peroxidase
activities. In contrast, higher tumoral GPX activity was strongly correlated
with accumulation of cells in S phase (R2=34%, p=0.0116) and G2+M phases
(R2=80%, p<0.0001). On the other hand, tumoral/non-tumoral GPX ratio
increased in a similar way, but also when cathepsin D, a protease associated
with tumour invasion and metastatic development, was detected in the tumour
(R2=28%, p<0.001). CONCLUSIONS: The proliferous activity of breast
tumours, known to be inducible by various hormones and growth factors,
could also depend to some extent on protective, and especially antioxidative,
mechanisms linked to GSH.

(We/3.2/216) ESTABLISII~dENT OF A PANEL OF STABLE CELL LINES FOR

TETRACYCLINE-INDUCIBLEEXPRESSION OF TUMOR
SUPPRESSOR CANDIDATE GENES.
A. Protopopov, V. Kashuba, G. Winberg, E. Zabarovsky
MTC, Karolinska Instttute, Stockholm, Sweden

The region on human chromosome 3p21.3 harboring a putative renal

cell carcinoma and lung cancer tumor suppressor gene (TSG) was
previously defined by allelotyping and recently refined by mapping of
homozygous deletions. We report the construction of a combined 3.6
Mb PFGE and Fiber-FISH map covering this critical region (named
AP20). Thus far, 2 new genes represented by partial or fulMength
cDNAs were isolated, characterized, and precisely positioned on the
contig. Four previously cloned genes, namely MLH1, integrin A4L,
ACAA, MyD88, MOBP, cardiac tetrodotoxin-insensitive voltagedependent sodium channel gene and GLB1 were positioned (Allikmets
et al., 1996, Protopopov et at., 1996, Kashuba et al., 1997). To test
candidate TSGs, we are planing to introduce those genes and ESTs
under the control of inducible, tetracycline regulated promoter.
We have modified the original tet-regulated expression system of
Gossen & Bujard (1992). In short, the tTA protein was expressed from
a stable provirus in the cells (see the Table of cell lines transformed) and
not only integrating, but also episomal variants of the response unit
were created.
KRC/Y
RCC
ACC-LC5 SCLC
H1607
SCLC
GLC20
SCLC
U2020
SCLC
A549
NSCLC
HONE1-2 NPC
LNCaP
prostatecarcinoma
SCC15
oral squamous cell carcinoma
HeLa
cervix, adenocardnoma
RHEK-1 ] epidermal keratinocytes
BCLL
mouse fibrosarcoma

loss of one allele of HSA3

600kb homozig, deletion at AP20
loss of one allele 3p24-p21
700kb homozi9, deletionat 3p21
homozyg deletion at 3p
HSA3 can suppress tumorgentoty
LOH assigned to 3p21.3
homozig, deletion at WC3 7
3 regions of homozig deletions
control line
control hne
control line
control hne

These functional test and high resolution maps will prove crucial in the
identification of the RCC and lung cancer TSG(s).

p53 protein detection in normal and neoplastic endometrium

M. B13'~t. A. Semczuk:*. M W6jcik ~. W. M. Krajewska 1.

K. Postav, skiz. J. A. JakovAcki:
Department of Cytobmchenustry. Umversltyof L6dz, and -'Second
Department of GynecologicalSurgery_.Lubhn School of Medicine. Poland

The aim of the study was to investigate p53 protein expression by the
Western blotting technique (estimated by Integrated Optical Density1OD) in normal (n=13) and neoplastic (n=40) human endometrial
tissues as well as in a case of uterine carcinosarcoma and in a specimen
of the botryoid sarcoma of the uterine cervix, p53 protein levels were
correlated with patients' age as well as with conventionally used
clinicopathological features of the endometrial neoplasm. A
statisticallly significant difference was noted in p53 levels in the
nuclear, but not the cytoplasmatic, fraction between the normal
endometria and endometrial cancer tissues (p<0.0001). In the
neoplastic endometria, nuclear p53 protein expression was higher than
in cytoplasmatic fraction, and the difference was significant (p<0.05).
Higher nuclear p53 protein levels correlated with advanced histological
grading of endometrioid endometrial carcinomas, but no relationship
was noted between p53 protein expression and patients" age, clinical
stage, histological type or depth of myometrial invasion. A case of
uterine carcinosarcoma and a specimen of a botryoid sarcoma of the
uterine cervix expressed nuclear p53 oncoprotein (57 IOD and 89 IOD,
respectively). In conclusion, we found a statistically higher nuclear p53
levels in malignant as compared to normal human endometfial
specimens by the Western blotting technique. Although there were no
significant differences between p53 expression and clinicopathological
features of the neoplasm (except poor histological grading), further
studies are necessaly to evaluate the influence of p53
nuclear/c3oplasmatic levels on the clinical outcome of Polish patients
suffering from endometrial cancer.

(We/3.2/217)

Usefulness of transgenic mice in genotoxicity studies:

e.g. of 3-methylcholanthrene
B Rihn*, MC Boron*, C Coulais*, W Baranowski, G Kelth
*INRS, BP 27, 54501 Vandoeuvre C~dex, France,
IBMC/CNRS, 15 rue Descartes, 67084, Strasbourg, France.

Transgenic mice represent a unique paradigm in tissue specifictty and

mutagenic potential studying of chemicals.
As 3-methylcholanthrene (3MC) was found mutagenie in bacteria, clastogenic
in bone marrow, and reduces DNA adducts m animals, we were interested in
determining if that xenobiotic provokes (t) cell proliferation, (u)
transcripnonal activity"changes, (u?) DNA adducts and (ip) mutations in livers
of transgenic Big BlueT M mice carrying the L/Z phage shuttle vector.
Big BlueTM C57/B1 male mice were treated with a single IP dose of
80 mg/kg 3MC and the experiments were terminated after 1, 3, 6, 14 and 30
days. Cell prohferaUon was checked by BrdU labeling and
immunohistochemical detectmn. The maximal increase of the mitotic index
was e~denced after 3 days (2,9 times the control value, p<0.01). The relative
nucleus area, reflecting the transcriptional activity, was also the highest in the
treated group after 3 days: 1.87 times the control value on average (10<0.01).
Four major DNA adducts determined according to the 32p-posdabeLing
method were evidenced in Liver DNA of treated mice 3 days after the
treatment: the spot intensities increased in a time-dependent manner. The
mutant frequency of liver DNA was the highest after 14 days: 20.3_+2.9 in
the treated vs 7.6+2.7 in the control mice (p<0.01). Sequencing of the k/at1
mutant plaques showed mainly G---~T and C--'~A transversions in contrast to
the control ones.
In conclusion 3MC was found to induce m mice livers ftrstly gene
transcription and a slight increase of cell proliferation, followed by a parallel
formation of DNA adducts and mutations. Our study shows how transgenic
models could allow to evaluate in mt~ mechanistically simultaneous
endpomts.

Abstracts FEBS'99

(We/3.2/218)

A fluorescence-based assay for the h u m a n tumor

suppressor Fhit protein
P. Rotllhn, A.C Asensio, S. Oaknin.
Depto. Bioqulmica, Universidadde La Laguna, Tenerife, Spain

The FHIT gene is a putative human tumor suppressor located at a fi'agi

site in chromosome 3, encoding Fhit protein. A recent investigation of catalyt
properties of Fhit as a GST-Fhit fusion protein indicated that Fhit protei
displays diadenosine triphosphatase activity resembling the already knox~
Ap3Aase [1]. This enzyme (EC 3.1.6.29), cleaving Ap3A to AMP and ADP, w~
discovered over two decades ago [2] but its cellular functions still remain to 1:
clarified. Radioehemical-chromatographic methods involving [3H]Ap3A c
FIPLC using Ap3A are usually chosen to estimate Fhit enzyme activity [1,3].
We report here the results of a study addressed to investigate the abllil
of unconjugated Fhit protein [3] to recognize the fluorogenic Ap3A analog d
ethenoadenosine triphosphate, e-(Ap3A), as a substrate. Cleavage of e-(Ap3.~
to c-AMP and e-ADP is associated with a 9-fold increase of fluorescen
emission (~305mrt, ;%m410nm), this allowing reaction rates to be monitored I:
continuous fluorimetric assays. Fhit protein cleaves 6-(Ap3A) according t
hyperbolic kinetics with K~ of 2.0 o.M and V~, 0.56 relative to V~, measure
for Ap3A. Hydrolysis of e-(Ap3A) occurs in the absence of added divalel
cations but is stimulated by Mg2+, Mn2+ and Ca2 (maximal effect at 0.5-1 mM
inhibited by Znz* (ICs0 about 15 rtM) and is tmaffected by F- (up to lmM
These results closely match those obtained using [~H]ApaA as substmte [1,3
indicating the suitability of this fluorimetric approach. Fluorimetric assa3
demonstrate that e-(Ap4A) is a poor substrate and that diethylpyrocarbonate an
suramin inhibit Fhit enzyme. Suramin, an antiparasitic and anticancer drug, is
strong inhibitor of Fhit enzyme (IC50 2 BM), however the degree of inhibition
inversely co-related to serum albumin concentration in the assay mixture.
In conclusion, the fluorescence-based assay here presented allow
precise and rapid low-cost estimations of Fhit enzymatic activity avoiding tl:
use of expensive radiolabeled substrates and time-consuming chromatograpE
procedures currently in use.
[1] Barnes et al., Biochemistry, 35, (1996) 11529.
[2] Lohat6n et al. Biochem. Biophys. Res. Commun., 67, (1975) 279.
[3] Pace et al., Proc. Natl. Acad. Sci. USA, 95, (1998) 5484.

s331

(We/3.2/219)

Estrogen receptors and transcription cofactors: in vitro

interaction and expression in target cells.
S. Thenot, S. Bonnet, A. Boulahtouf, C. Royer and
V. Cavailles. INSERM U148, Montpellier, France.

Hormonal regulation of gene activity is mediated by nuclear receptors

acting as ligand-activated transcription factors, intcrmediary factors
interacting with their activation functions arc required to mediate
transcriptional stimulation. We have analysed the in vitro interaction of
human TIFI~ (Transcription Intermediary Factor 1) with ER~t and
demonstrated that a short region containing a leucine charged domain is
sufficient to interact with ERct in a ligand-dependant manner. We then
investigated how DNA binding influenced the in vitro interaction
between ERct and hTIFlot. We then used measurements of fluorescence
anisotropy to define the equilibrium binding parameters of the
ERtx/hTIFhx interaction (dissociation constant was 2"10"7M). We also
developped a protein-protein-DNA-assay allowing the quantification of
T1F/ER/ERE complexes formation in the presence of ligand.
In order to approach the molecular basis of the tissue-specific agonist
activity of antiestrogens, we then compared, at the mRNA level, the
expression of various transcriptional coactivators or corepressors of
estrogen receptors in different breast and endometrial human cell lines.
We showed that for SRC-1, CBP, hTIFlot, RIP140, N-Cor and SMRT,
no significant differences in the expression level were observed between
breast and endometrial cells. For hTIFlct, both isoforms were also
detected at similar levels in all the cells tested. By contrast,
overexpression of AIB 1 mRNA was observed in MCF7 cells but not in
other breast or endometrial cells irrespective of their ER-status. Finally
we demonstrated that RIP140 mRNA is directly induced by estrogens in
ER-positive MCF7 breast cancer cell lines but not in Ishikawa
endometrial ceils. Together these results indicate that some differences
exist between breast and endometrial cancer cell lines at the level of
estrogen receptor cofactor expression.

s332

Abstracts FEBS'99

6.4 Molecular recognition in transcription: tissue specificity

(We/3.2/220)

Characterization of a human hormone-sensitive lipase

testis -specific promoter
R. Blaisel, J. Grober2, P. RouetI, G. Tavemierl, D. Daegelen3
and D. Langinl .

(We/3.2/221)

i MGC Deoartment of Cell Biology Erasmus Umverslty Rotterdam.

The Netherlands; : lnsntut ffur ~olecularblologw und Tumo~forschung.
Philipps-Untversttfit Marburg, Germany

l L\S~RM L31- Youlo~tse. 2 EVSt~AAA Dgon. 3 IVSER~4 U129 Par~s, t:~-ance

The testicular isoform of hormone-sensitive lipase (HSLtes) is

encoded by a testis-specific exon and 9 exons common to the testis and
adipocyte isoforms. In mouse, HSLtes mRNA appeared during
spermiogenesis in round spermatids. Two constructs containing 1.4 kb
and 0.5 kb of the human HSLtes gene 5'-flanking region cloned upstream
of the chloramphenicol actyl transferase (CAT) gene were microinjected
into mouse oocytes. A very low CAT activity was detected in transgenic
female tissues and in transgenic male non gonadal tissues. Testis of 60days old transgenic mice showed high CAT activity whereas low CAT
activity was detected in testis of 25-days old transgenic mice. These data
suggest that the first 0.5 kb of the HSLtes promoter is sufficient to direct
expression in post meiotic germ cells in testis.
Cell transfection experiments showed that CREMx, a testis-specific
transcriptional activator, does not transactivate the HSLtes promoter.
Using gel retardation assays, four testis-specific binding regions (TSBR)
were identified in the HSL~s promoter using testis and liver nuclear
extracts. The testis-specific protein binding on TSBR4 was selectively
competed by a probe containing a SRY/Sox protein DNA recognition
site. Sox5 and Sox6/Sox-LZ which are expressed in post-meiotic germ
cells, bound TSBR4. Moreover, binding of the high mobility domain of
Sox5 induced a bend within TSBR4.
Together, our results showed that 0.5 Kb of the human
promoter contain cis-acting elements essential for the testis specificity of
HSL and bind Sox proteins.

Functional analysis of the transcrintion factors Sp3 and Sp4

R.J.P. Bouwman ~, H. G611ner~, G. ~uske", F.G. Grosveld ~and
S. Philipsen ~

Sp3 and Sp4 are members of the Spl (Specific protein 1) family of transcription
factors. Spl, Sp3, and Sp4 are closely related and share similar functional
domains. Spl and Sp4 are mainly known as transcriptional activators, whereas
Sp3 can also repress transcription via a repressor domain that occurs in this
family member only. The three zinc finger motifs of Sp 1, Sp3 and Sp4 bind GT
and GC boxes with identical affinities. These binding sites are found in
numerous enhancer and promoter regions of tissue specific and ubiquitous genes
(e.g. in the 13-globin LCR). Like Spl, Sp3 is ubiquitously expressed, whereas
Sp4 is abundant in adult brain but barely detectable in other tissues. To
investigate the functional properties of Sp3 and Sp4, knockout mice were
generated by homologous recombination.
Targeted deletion of the zinc finger region of Sp4 revealed that this transcription
factor is required for normal marine growth, viability and male sexual behaviour
[1]. This is largely confirmed by the results obtained from our Sp4 knockout
mice.
Sp3 appears to be indispensable for neonatal survival, since Sp3 knockout mice
die soon after birth. Caesarean sections at 18.5 days of gestation (E18.5) revealed
that they are not able to breathe and survive only for 20 minutes. This breathing
inability might be due to a general delay in development during the last day(s)
of gestation. The Sp3-/- neonates are approximately 30% smaller than their
littcrmates, but otherwise there are no gross morphological abnormalities.
Currently, we are looking for target genes of Sp3 that might explain the
phenotype. For this purpose, mRNA from Sp3-/- and +/+ ES cells and total
embryos has been used to screen cDNA arrays. Also, differentially expressed
total embryo mRNA has been selectively amplified, using the SABRE protocol
[2].
[1] Supp et al., Dev.Biol., 176, (1996) 284.
[2] Lavery et al., Proc.Natl.Acad.Sci.U.S.A., 95, (1997) 6831.

(We/3.2/222)

Cloning of the human and rhesus monkey 5' flanking

region of the dihydropyrimidine dehydrogenase gene.
ESR Collie-Duguid, SJ Johnston, G Watson, RH Powrie, HL
McLeod. ~)Ttverscty o f Aberdeen. Aberdeen. Scotland. (.'Ix'.

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate limiting enzyme

in the catabolism of uracil and thymine to lS-alanine and [3-aminoisobutyrate,
respectively. DPD also inactivates over 80% of an administered dose of the
chemotherapeutic agent, 5-fluorouracil (5FU). DPD deficiency is associated
with neurological problems in pediatric patients and toxicity in response to 5FU
therapy in cancer patients. The D P Y D gene encodes a 105 KDa DPD protein.
Variant D P Y D alleles identified to date do not account for the large variation
(8-21 fold) in DPD activity observed in healthy subjects. In addition, DPD
activity and mRNA levels are differentially regulated in human tissues, with
highest expression in liver. Although DPD enzyme activity has been well
studied, very little is known about the molecular mechanisms controlling
expression of the D P Y D gene. We have cloned 1.85 kb of sequence upstream
of exon 1 of the human D P Y D gene (-1751 to +103, ntmabered from
transcription start site). Rapid amplification of genomic DNA ends (RAGE)
was utilised in human genomic DNA to obtain 570 bp of sequence upstream of
the D P Y D translation start site, using exon 1 specific primers. The 3' end was
homologous to the partial D P Y D 5' UTR (86 bp) reported previously and the
first 5 coding bases of the DPYD gene. A further 1.28 kb was obtained with a
second round of RAGE using primers designed at the 5' end of the 570 bp
sequence. The sequence of the 1.85 kb D P Y D fi'agment was confirmed in
genomic DNA from 3 individuals using PCR and automated fluorescence
sequencing. This region of the D P Y D gene was shown to be a novel sequence
by comparison with the Genbank and EMBL databases. This putative D P Y D
promoter region contains 5 T A T S boxes, 3 CCAAT elements and consensus
binding sites for SP-1, NF-1, E2A, AP-2, AP-3 and other transcription factors.
Consensus binding sites for HNF-4 and C/EBP, transcription factors that
regulate hepatic-specific gene transcription, were also identified. A 5' flanking
region (-1751 to +75) of the rhesus monkey D P Y D gene was also sequenced
using the human PCR primers. The human and monkey nucleotide sequences
had 96% identity and a region spanning -427 to +75 had 100% identity,
indicating that this region may have functional importance. Cis- and transacting factors regulating transcription of this clinically important enzyme are
now being further characterised.

(We/3.2/223)

The role of transcription factor Phox2b in the expression of the

x3cholinergic-nicotinic subunit in the autonomic nervous system
A. Floraa,R.Benfantea,S.Terzanoa,C.Goridisb,F. Clementia,D.Fomasaria
aCNR Cell. MoL Pharmacol. Ctr., Dept. Med. Pharmacol., Univ. of Milan"
blnst. Biol. Develop. Marsedle, CNRS-INSERM, Univ. de la Mediterrande

Nicotinic acetylcholine receptors are ligand-gated ion channels expressed at

the neuromuscular junction, in autonomic ganglia and in several areas of the
vertebrate CNS. Like their muscular counterpart, neuronal-type nicotinic
receptors have a pentameric stoichiometry and are composed of tigandbinding-subunits and structural subunits, which are encoded by a large
family of genes homologous to those encoding the muscle isoforms. In the
autonomic nervous system, the u3, 1~4and ct5 subunits form the ganglionictype nicotinic receptor, whose activation is responsible for the fast excitatory
potential in postganglionic neurons, with the consequent initiation of the
post-synaptic spike. Because of the fundamental role of the a3 subunit in
ganglionic transmission, the comprehension of the mechanism that govern its
transcription may help to obtain new insights into the functions of the
autonomic nervous system. Recent evidence suggest that specific families of
homeodomain transcription factors control the generation and the survival of
distinct neuronal phenotypes. Among them, Phox2a and Phox2b seem to
play a fundamental role in the specification of the autonomic nervous system.
We demonstrate that Phox2b, but not Phox2a, is able to activate the
transcription of the ~3 nicotinic subunit gene through a direct stimulation of
the activity of its minimal promoter, that is 60 bp long. The molecular
mechanisms underlying this transcriptional activation will be discussed.

Abstracts FEBS'99

(We/3.2/224)

ESSENTIAL ROLE OF IRF-3 TRANSCRIPTION FACTOR

IN THE ACTIVATION OF RANTES EXPRESSION:
IMPLICATIONS FOR HIV PATHOGENESIS.
Hlseott .I., Heylbroeck C., G6nin P., Mamane Y., and Lin
R. McGill AIDS Center, Lady Davis Research Institute,
McGill University Montreal, Canada, H3T 1E2.

HIV replicates more efficiently in activated cells and viral levels consistently
increase when the immune system of infected individuals is activated by
exogenous stimuli such as opportunistic infections. This increase in the rate of
viral replication is associatedwith cellular activation and expression of HIV
inducing cytokines/chemokines, as well as an acceleration m the course of
HIV-induced disease. The effects of HIV stimulating cytokines are
counterbalanced bythose that suppress HIV replication, inhibitory cvtokines
include IFNet, IFN13, IL-10 and the chemokmes RANTES, M'IP][-et, and
MIPl-13. Activation of the Interferon Regulatory Factors (IRF) in turn are
critical mediators of cytokine gene transcnptlon,Recent studies have focused
on the 55kDa IRF-3gene product as a direct transcriptional regulator of type I
interferon (IFNct and IFNI5) activation in response to virus infection. Infection
by many types of viruses (HIV, paramyxovirus and herpes) induces
phosphorylation of IRF-3 on specific C-terminal serine residues and permits
cytoplasmic to nuclear translocation of IRF-3, activation of DNA binding and
transactivation potential, and association with the CBP/p300 co-activator.
Phosphorylation also facilitates dimerization of IRF-3 proteins. We previousiy
generated constitutively active [IRF-3(5D)] and dominant negative (AN) forms
of IRF-3 tlaat control IFNI5 and IFNct gene expression. In an effort to
characterize the range of immunoregulatory genes controlled by IRF-3, we
now demonstrate that endogenous human RANTES gene transcription is
directly induced in tetracycline-inducible IRF-3(5D) expressing cells or virusinfected cells. We also show that a dominant-negative IRF-3 mutant inhibits
virus-induced expression of the RANTES promoter. Specific mutagenesis of
overlapping ISRE-like sites located between nt -123 and -96 in the RANTES
promoter reduces virus-induced and IRF-3 dependent activation. T h e
realization that IRF-3 regulates CC-chemokine RANTES suggests that it may
play a role in HIV-1 patho.genesis, particularly with regard to chemokine
activation and virus multiplication. The influence of IRF-3 activation and
RANTES expression on HIV-1 replication is currently being investigated in
HIV-infectedmyeloid cells stably transduced with different forms of IRF-3 by
retroviral gene transfer.

(We/3.2/226)

IGF-II, PI 3-kinase, N F - ~ B and iNOS define a

c o m m o n m y o g e n i c signalling p a t h w a y
P. Kaliman, J. Canicio, X. Testar, M. Palacfn, A. Zorzano
Departament de BioquDnica i Biologia Molecular. Faculto!de Biologia.
Untversitat de Barcelona. Avda. Diagonal645. 08028 Barcelona.Spare

Differentiation of rat skeletal muscle L6E9 cells with insulin-hke growth

factor-ll (IGF-II) transiently induced both inducible nitric oxide synthase
(iNOS) expression and nuclear factor-~zB (NF-xB) DNA-binding activity;
these events, which were maximal at differentiation day 1, correlated in time
with the stimulation by IGF-II of nitric oxide (NO) production. Both IGF-IIinduced iNOS expression and NO production were blocked by NF-xB
inhibitors such as pyrrolidme dithiocarbamate and sodium salicylate. Terminal
IGF-II-induced differentiation parameters (cell fusion and expression of
skeletal muscle proteins, i.e. myosin heavy chain, GLUT4 and caveolin 3)
were dependent on NF-KB and iNOS activation as they were completely
blocked by NF-)B or NOS inhibitors. Similar results were observed in
human myoblast primary cultures in which NF-KB inhibitors blocked IGFII-mduced myotube formation.
Regarding the mechanisms that lead to NF-)cB and INOS activation during
myogenesis, increased expression of both IKB-cx and p65 NF-)zB subunit
was observed after 12 h of IGF-II-treatment. Thereafter, IGF-II induced a
marked decrease m total cell content of l)~B-ct and in the amount of I~:B-ct
associated with p65. Moreover, the phospbatidylinositol 3-kinase (PI 3kinase) inhibitor LY294002 -a well charactenzed myogenic inhibitor- totally
blocked the ability of IGF-II to induce NF-KB activation, iNOS expression
or NO producti~n. This correlated with the mhibinon by LY294002 of both
the IGF-ll-induced degradation of IKB-ct and the decrease in the amount of
cx-l)~B complcxed to p65 NF-KB.
Data prcsentcd here define a model in which IGF-II stimulates a series of
events involved in myogenesis: (i) PI 3-kinase activation; (n) ct-I)<B
degradation; (in) NF-)B activation; (iv) iNOS expression and (v) NO
production. The NOS substrate L-Arginine (L-Arg) was able to mimick 1GFII-mduced myogenesis and this effect was blocked by either NOS or Pl 3kinasc inhibition. However, PI 3-kinase inhibition d~d not block the L-Arginduced NO production. This suggests that there are other signals derived
from a basal PI 3-kinasc activity that act together with NO to induce
differentiation and that NO is a necessai T but not a sufficient signal to initiate
the myogenic program.

s333

(We/3.2/225)

Transcriptional control of mouse 13 enolase gene

expression in d e v e l o p i n g striated muscle
C. Jabet, J-D.Rouzeau, K. Brodolin, N. Lamand~, M. Lazar
CNRS-UPR 9065, Coll~ge de France, Paris

The gene encoding the 13 subunit of the glycolytic enzyme enolase is

expressed only in striated muscle. Previous studies in rodents showed that this
gene is likely to be the target of multiple regulatory signals, modulating its
expression in the context of distinct myogemc programs (in cardiac, epaxial
and hypaxial muscles), in specific muscle fiber-types, and according to the
physiological status of heart or limb muscles [ 1 and references herein]. The [5
enolase gene therefore appears as an attractive candidate for investigating the
multistep regulation of gene expression in developing and adult muscle. In
addition, ex vivo, like few other muscle-specific genes, the 13enolase gene is
already active in dividing myoblasts, an increase m its transcriptional activity
accompanying terminal differentiation of myoblasts into mvotubes [2]. We
have cloned and sequenced the mouse 13enolase gene and 5'-flanking region
[3] and undertaken studies to identify the regulatory sequences, and cognate
trans-acting factors, which are responsible for its specific pattern of
expression ex vivo, using the C2.7 myogenic cell line as a model system, as
well as in vivo, during mouse ontogenesis.
A DNase-I hypersensitivity analysis of chromatin in nuclei from myoblasts,
myotubes and non-myogenic ceils allowed to locate upstream and intragenic
potential control regions. A single myotube-specific hypersensitive site was
found, mapping into the 3' end of the first intron which encompasses a
muscle-specific transcriptional enhancer. Further functional analyses, and in
vitro studies of DNA-protein interactions, showed that most of the enhancer
activity can be attributed to aca. 60 bp sequence (bp +520 to +577), comprising
adjacent binding sites for transcription factors of the myocyte enhancer factor2 (MEF2) and myogenic basic helix-loop-helix (m-bHLH) families. Disruption
of the MEF2 or m-bHLH binding sites by substitutive site-directed mutagenesis
showed that the corresponding trans-acting factors are major players in the
up:regulation of mouse 13 enolase gene expression during terminal
differentiation of myogenic ceils ex vivo. Full activity of the intronic enhancer
also requires the integrity of binding sites for factors of the nuclear factor-I
(NF-I) and Spl families, located into a 120 bp proximal promoter region. As
a first step to assess the functional role of these protein-binding sites in vivo,
11 lines of transgenic mice were produced in which nls-LacZ was placed
under the control of the wild-type enolase fragment from bp - 118 to +672 : no
transgene expression was obtained at any developmental stage. However,
extending the 5'-flanking region to bp -1775 was sufficient to observe (at
11.5 dpc)muscle-specific LacZ expression in myotomes but not in cardiac
muscle. Later stages of myogenesis and other putative regulatory sequences
are under study using transgenic founder analysis.
[1] Keller A e t al., Amer J Physiol, 38, (1995) H1843; [2] Lamand6 N et
al, Mol Reprod Dev, 41, (1995) 306; [3] GenBank Database accession
numbers X61600 and X60464.
(We/3.2/227)

Lactoferrin - a new transcriptional factor with a lot

of unique biological functions
T. Kanyshkova, D. Semenov, V. Buneva, G. Nevinsky
Novosibirsk Institute of Bioorganic Chemistry,
Novosibirsk 630090, Russia

Many different unique functions have been attributed to lactoferrin (LF),

including cell growth regulation; DNA and RNA binding, RNAse activity,
and transport into the nucleus, where LF binds specific DNA sequences and
activates transcription. Here we present evidence that in addition to the
above mentioned functions human milk LF binds ATP with a stoichiometry
of 1 mole of nucleotide per mole of the protein and a Kd = 0.3 mM and
possesses two sites for interaction with various specific and nonspecific
DNA. The first site of LF demonstrates a -20-40-fold higher affinity for
single and double stranded specific oligonucleotide (Kdl(SS)=8.0+2.0el0 "8
M and Kdt(ds)=l.l-+0.2ol0 "s M) than for the nonspecific d(pT)t0 and its
duplex (Kdl(ds)=0.4-+0.1 and Kd2(ds)=20.0+5.0 I.tM). At the same time,
while LF binds the second molecule of d(pT)10 with an affinity about 40
times lower than the first one, its affinity for the second molecule of single
stranded specific sequence (Kdz(ss)- 1.0.10 -4 M) is about 1250 times lower
that that for the first one. Thus, it can not be ruled out that the initial
difference of nonspecific d(pT)10 oligonucleotide affinity for two DNAbinding sites may be even more dramatic due to anti-cooperative binding in
the case of the specific oligonucleotide. The ATP-binding site is localized hi
the C-terminal domain of LF (G475-M604),in contrast to the antibacterial and
DNA-binding sites, which are located in the N-terminal domain (GI-R30.
Binding of ATP by LF leads to dissociation of its oligomeric forms and to a
change of the protein's interaction with polysaccharides, proteins and
DNA. The absence of homology of LF with other known transcriptional
factors, allosteric changes of the LF interactions with various ligands, and
polyfunctional properties of LF distinguish it from other known activators
of transcription, and may be very important for untypical functioning of
this protein. Our results taken together with previously observed in
literature suggest the existence of a new class of transcriptional activators,
those secreted in one cell and taken up by a second target cell in which they
are transported to the nucleus and directly activate gene expression.

s334

(We/3.2/228)

Abstracts FEBS'99

Expression of a novel isoform of the EDA gene transcript

in human umbilical cord
K. Kobielak, A. Kobielak, W. H. Trzeciak

(We/3.2/229)

"Department of Cytobiochemistrg, University of Lodz, Poland

~Department of Pathology, Institute o f Polish Mother's Memorial Hospital,
Lodz, Poland

Dep. of Physiological Chemistry Univ. of Medical Science, Swiecickiego

6/60-781, Poznan, Poland

Anhydrotic ectodermal dysplasia (MIM 305100) results from mutations

in the ectodermal dysplasia anhydrotic (EDA) gene.
Originally described gene contains two exons separated by a 200 kbp
intron. Recently, several alternatively spliced isoforms of the gene
transcript were caracterised.
We investigated the EDA gene expression in human umbilical cord, with
the use of RT-PCR and a pair of primers: one specific for exon 1 of the
EDA gene and the other specific for exon 3 or exon 5 of a mouse
homologue of the EDA gene, the Tabby gene.
Amplification of DNA fragments has demonstrated that the EDA gene is
expressed in umbilical cord as a novel form of transcript containing in
comparison to the isoform originally described, at least two additional
exons.
We postulated that the novel isoform of the EDA gene transcript in
umbilical cord might originate from differential splicing of the primary
transcript.
Supported by grants No P05A 059 15 from the State Commitee for
Scientific Research (KBN) Poland.

(We/3.2/230) p53-homologue participates in transcriptional regulation

of the ct2-macro~Jobulin ~ene in letal rat liver
M. Mihailovica, M. P~trovica, ZTMiliccvicb, D Bogoje~ia
a ]nstltute for Btologtcal Research, Belgrade, Yugoslavia, ~ The
Institute of Nuclear Sctenees "16nea', Belgrade, Yugoslavta

Although tetrameric nuclear protein p53 orchestrates a response leading

to the elucidation of several mechanisms by which p53 might exert a
tumor suppression effect, cell-cycle arrest and DNA repair, or
programmed ceil death, wild-type p53 can bind to DNA in a sequencespecific manner [1] as a transcriptional regulator of different genes
Homologue sequence to the consensus p53-DNA-binding site [2] was
espied in the gene of ~2-macroglobulin (ot2MG), the most prominent rat
acute phase protein mainly expressed in the liver. Knowing that the
elevated transcription of the ot2MG gene during acute phase reaction
relies essentially on the increased binding affinity of hepatocyte regulatory
DNA-bmding proteins to the hormone response element (RE) of the
2MG gene located in the promoter proximal region at position -852/12
bp. as well as distinctive feature of the underlying molecular events in fetal
and adult liver, we have assumed that p53 takes part in the regulation ofc~
2MG gene transcription in fetal rat livers Using the livers of adults and
fetuses from untreated and 12h-turpentine treated dams on day 19 of
gestation, we have shown by South-Western analysis that protein with
molecular mass of 53 kD had binding affinity to the RE of ~2MG gene
only from nuclear extracts of prenatal hepatocytes lmmuno-Western
blotting with antibody to p53 identified a nuclear protein from fetal livers
as protein of the same molecular mass and epitopes as transcriptmn factor
p53 Evenmore, we have shown that this protein is in complex with hsp70
and hsp90 On the basis of presented results we have concluded that
homologue of p53 participates in transcriptional regulation of the c~2MG
gene
[l] Kern S E et al. (199l) Science 252, 1708.
[2] El-Deiry W S e t al. (1992) Nat Genet. 1, 45

Androgen receptor in relation to estrogen and progesterone

receptors in female breast cancer
W.M.Krajewska, M.Brys, H.Romanowlcz-Makowska,

The expression of androgen (AR), estrogen (ER) and progesterone (PR)

receptor genes in female breast cancer was assayed by RT-PCR. Freshfrozen samples of 27 primary breast carcinomas i.e. 20 infiltrating ductal
carcinomas and 7 infiltrating lobular carcinomas were obtained from
patients who underwent surgery for breast neoplasms. The patients had not
received anti-tumour treatment before operation. RT-PCR was performed
using RNA PCR Kit ver.2.1 (Takara Shuzo Co., Ltd., Japan) according to
manufacturer. The integrated optical density (IOD) of electroforetically
separated amplification products, in a digitalized picture, was measured
using a video densitometer and Gel-Pro 3.0 software.
The expression of All. was analyzed in the context of ER and PR as well
as tumour grade. Of the breast cancers investigated, 66.7% (18/27) were
found to be AR positive, in 44.4% (12/27) of studied tumours the
expression of all three receptors, AR, ER and PR was seen while in 18.5%
(5/27) only AR was present. In 14.8% (4/27) of the samples neither AR,
ER nor PR expression was observed, these were all grade III turnouts. A
significant correlation between expression of AR and ER (p=0.03) was
stated.
The obtained results suggest that the determination of androgen
receptor, in addition to estrogen and progesterone receptors, has potential
value as a prognostic factor and/or predictor of response to endocrine
therapy.

(We/3.2/231)

The histone deacetylase HDACI does not co-operate

with RB in co-activation of MyoD
A. Polesskaya, R. Ferrmra, P. Robin, A. Harel-Bellan
CNRS UPR 9079. IFC 01, 7 rue Guy Moquet, 94800 Villejuif, France

Muscle cell differentiation provides a convenient model for

investigation of two extremely important events of cell life: exit from the cell
cycle and activation of tissue-specific genes, or differentiation, both of which
are essentially controlled at the transcriptional level. Recently, transcriptional
regulation has been linked to the remodeling of chromatin structure, in
particular through acetylation or deacetylation of core histone tails. The
purpose of the current work is to analyse the implication of histone-modifying
enzymes in muscle terminal differentiation.
The RB protein is known to interact with the histone deacetylase
HDACI to induce cell-proliferation block in GI [1]. On the other hand, there
is evidence of co-activation of MyoD and myogenin, two most important
myogenic proteins, as a result of physical interaction with RB [2].
We have performed transient transfection experiments in RB-deficient
SAOS-2 cells in order to investigate whether co-activation of MyoD by RB
also needs recruitment of HDAC1. Two different approaches were employed:
treatment of cells transfected with RB and MyoD-expressing plasmids with
TSA, a well-known inhibitor of HD-activity, and co-transfection of the same
cells with HDACl-expressing plasmids in various concentrations. In both
cases, there is no evidence to suggest that co-activation of MyoD by RB
requires the HD activity. On the other hand, treatment of differentiating
C2C12 cells with TSA results in inhibition of differentiation and subsequent
death of differentiated cells, suggesting that a histone deacetylase is required
for muscle differentiation.
[1] L. Magnaghi-Jaulin et al., Nature, Vol. 391, 1998, 601.
[2] W. Gu et al., Cell, Vol. 72, 1993, 309.

Abstracts FEB S' 99

(We/3.2/232)

s335

Effects of the downstream and upstream regions on the minimal

promoter of the human c<3 nicotinic subunit gene

(We/3.2/233)

S. Terzano, A. Flora, E. Battaglioli, R. Benfante, F.Clementi, D,

Fornasari.CNR -Cellular and Molecular Pharmacology Center, Dept.

Djabari, JP Ortonne and D Aberdam. 1NSEI~I U385 ,Vice,

France.

Medical Pharmacology. University of Milan

The human gene for the ct3 subunit of the neuronal nicotinic acetylcholine
receptor is controlled by a TATA-Iess, CAAT-less multistart promoter,
similarly to other neuron-specific genes. We've funtionally mapped the ct3
minimal promoter to a 60 bp sequence, 190 bp upstream the start codon.
This sequence is highly GC-rich and contains several putative Spl and AP2
binding sites. The activity of the ct3 minimal promoter was found to be
positively regulated by the adjacent regions, the 190 bp downstream region
(DRR), containing most of the transcription start sites, and the 50 bp
upstream region (URR). When separately analysed, the URR and the DRR
regions activated the ct3 minimal promoter by 1.5-3 fold, both in neuronal
(SKNBE and SY5Y human neuroblastnma cells) and non neuronal (TE671
human rhabdomyosarcoma cells) cell lines. When simultaneously present.
the URR and DRR exhibited a strong and neuron-specific enhancing effect
on the ct3 minimal promoter activity, resulting in 16 fold incrcase in
SKNBE and 6 fold in SY5Y. Furthermore, the DRR sequence was found to
activate the SV40 heterologous promoter, only in the neuronal cell lines,
whereas the URR region resulted ineffective on the SV40 promoter. These
observations seem to suggest a tissue-specific, promoter-specific sinergistic
interaction between the region adjacent to the ct3 minimal promoter. We're
currently investigating the DNA-protein interactions that are relevant to the
activity of the URR and DRR regions.

(We/3.2/234)

Exercise modulates rat antioxidant enzyme m R N A levels

D. Wilson and P. Johnson

Department of Chemistry, Program in Molecular and Cellular Biology and
College of Osteopathic Medtcme, Ohio University, Athens, Ohio 45701, USA

Previous studies have shown that exercise can cause a change in the specific
activities of the antioxidant enzymes glutathione peroxidase (GPX), CuJZn
superoxide dismutase (Cu/Zn SOD) and catalase (CAT) in SHR
(spontaneously hypertensive) rats and WKY (Wistar-Kyoto) rats. In order
to determine if the changes observed in the specific activities of the enzymes
were due to a change in mRNA levels, the levels of mRNA in myocardium
and liver were determined by quantitative ribonuclease protection assay. In
the myocardium of SHR rats, exercise caused a significant decrease in the
mRNA levels of the Cu/Zn SOD mRNA and GPX mRNA and a significant
increase in the CAT mRNA. Exercise caused a significant decrease in the
CAT and GPX mRNA levels of the myocardium of the WKY rats. In the
liver of both the WKY and SHR rats, exercise caused a significant decrease
in the Cu/Zn SOD and GPX mRNA levels. The CAT mRNA levels show a
significant increase due to exercise in the liver in both the WKY and SHR
rats. The changes in the GPX mRNA levels in the myocardium and the liver
of both the SHR and WKY rats and the WKY myocardium CAT mRNA
levels parallel the changes in the antioxidant enzyme specific activity
suggesting that the changes in enzyme activity may be caused by changes in
the mRNA levels. The changes in the Cu/Zn SOD mRNA levels in the
myocardium and liver of the SHR and WILY rats and the CAT mRNA levels
in the myocardium and liver of the SHR rats and the liver of the WKY rats
inversely correlate with the enzyme specific activity changes suggesting a
post-transcriptional control mechanism for these enzymes.

Human LAMA3A Promoter Reveals Cell type-specificity

through AP-1 Binding Sites. T ViroUe, MN Monthouel, Z

Laminin-5, an adhesion ligand of specialized epithelia presents a complex

transcriptional regulation under both physiological and pathological
conditions. We have cloned the 5'-flanking region of the gene coding for the
human ct3A chain ( L A M A 3 A ) and analyzed its promoter activity. Transient
expression of a luciferase reporter gene under the control of serially deleted
5'-flanking sequences revealed that the proximal promoter which contains
three AP-I sites was sufficient for keratinocyte cell type-specific expression.
A single copy of this 204 bp-genomic fragment was able to confer high
keratinocyte-specific expression to TK heterologous promoter. Simultaneous
mutations of the three AP-1 sites cooperatively abated promoter activity and
keratinocyte-specific stimulation of the TK heterologous promoter. Removal
of the sequences located between the AP-I sites does not alter significantly
the cell-specific activity of the promoter, although the distance between the
AP-1 sites seems critical for optimal transactivation in keratinocytes. These
results provide evidences that the AP-1 sites in the promoter play a crucial
and indispensable role in keratinocyte-specific expression of the gene.
Electrophoretic mobility shift assays showed that each AP-1 site formed a
single protein-DNA complex containing Jun/fos in keratinocytes and in
fibroblasts with identical binding affinities. Fos and Jun protein levels are
identical in the two cell types and do not show any difference in overall
phosphnrylation states. We have further shown that oxidation-reduction
modification of AP-1 could not explain the difference in cell-specific
transaetivation and that methylation state of genomic DNA cannot be
responsible for that cell specificity. Therefore, these results suggest the
presence of an epithelial-type specific nuclear protein which might interact
with the AP-1 complexes only when all 3-dimensional requirements
~involving contacts wit the DNA and the AP-1 proteins are satisfied. The
identification of such an epithelial-specific cofactor should further our
understanding of the molecular mechanisms regulating L A M A 3 A gene and
more generally epithelial gene expression.

(We/3.2/235)

Cytokines cause a differentiation stage specific Stat activation pattern in macrophages

Irina Woldman, Birgit Klose. Dagmar Stoiber, Thomas Decker
lnstttute of Microbiology and Genetics, Dr Bohr Gasse 9, A-1030 Vienna

Stat family members are cytosolic proteins that are activated by tyrosine
phosphorylation catalyzed by receptor-associated Jak kinases in response to
a variety of stimuli. Phosphorylated Stats dimerize and translocate to the
nucleus to regulate gene transcription by binding to so called GAS-elements
[1]. The Stat activation occurs in a tissue- and ligand- specific pattern. We
have investigated the Jak-Stat pathway in human and chicken macrophages
in response to interferons before and after differentiaton and find a similar
activation pattern in both cellular differentiation model systems: interferons
cause a stronger activation of Statl in mature macrophages and a decreased
activation of Stat5 as compared to undifferentiated cells. This activation
pattern might constitute one level at which transcriptional activation of gene
expression is regulated in maturing macrophages during an immune
response. We make use of the human cell line U937 that can be
differentiated in vitro by T P A and of myeloid chicken progenitor ceils that
are derived by transformation of bone marrow with the retrovirus TsE26 that
encodes a temperature sensitive oncoprotein p135 ~a~'myb~s [2]. The
proliferation of these cells at 37C and their differentiation at 42C depends
on the presence of the growth and differentiation factor cMGF (chicken
myelomonocytic growth factor). The identity of the chicken Stat homologues
has been determined by use of antibodies that specifically recognize the
respective mammalian Stat. Furthermore we have cloned the coding
sequence for chicken Stat5b by colony hybridization and we have shown that
differentiation stimuli cause the activation of Star5 in the cells described here
[3]. Future experiments will address the mechanism of Stat activation in
macrophages; in collaboration with Matthias Kieslinger and Hartmut Beug
we test currently if Stat5 is required for the differentiation of macrophage
progenitors by overexpression of dominant negative mutant forms of the
protein.
[1] T.Decker et al., Immunobiol. 198. (1997) 99
[2] H.Beug et al., Cell 39, (1984) 579
[3] I.Woldman et al., J. Immunol. 159, (1997) 877

s336

Abstracts FEBS'99

(We/3.2/236)

Age- and tissue-specific expression of mouse aquaporin 4

M1 and M23 mRNAs.
S.M. Zelenin, A. Aperia
! .,,~,TmZa h~,t,,tmet. Stockholm, Sweden

Aquaporin 4 (AQP4) is a water channel expressed in nlammalian

brain, kidney and lung. It has been reported that there are two forms of
AQP4, designated as M1 AQP4 and M23 AQP4, that are transcribed front
two different starts of transcription of the same AQP4 gene and undergo
alternative splicing afterwards. We report here the sequence of M1 and
M23 forms of mouse AQP4 mRNA. The expression of M1 and M23
mRNA was studied in brain, kidney, and hmg in 2-day old and adult mice
with semiquantitative RT PCR. M I mRNA was detected predominantly in
the brain in both groups. A relatively high expression of M23 was found in
lung from 2-day old mice and in kidney from mature animals. In the brain,
M1 mRNA was upregulated and M23 mRNA was downregulated with age.
In the lung both MI and M23 were higher at 2-day then adult mice. We
have previously suggested that lung AQP4 may be important for the lung
water clearance around birth [1]. Our results indicate the existence of
tissue-specific factors that provide differential age-dependent regulation of
two alternative promoter regions in AQP4 gene. Specific patterns of M1
and M23 AQP4 mRNA form expression may reflect distinct roles for each
isoform in different organs and at different developmental stages.
[ 1] References: Yasui M., et al., J. Physiol. (London), 505, (1997) 3.

Abstracts FEBS'99

s337

7.3 tRNA identity and aminoacyl-tRNA synthetases

(We/7.3/237)

Switching the tRNA specificity of an

aminoacyl-tRNA synthetase with a peptide loop transplant
A. Brevet, J. Chen, S. Commans, P. Plateau, S. Blanquet

(Well.3/238)

"UPR9002 IBMC, 15 rue RenO Descartes, 67084 Strasbourg-F

~UPR9004 IGBMC, BP163, 67404 lllktrch-F

Laboratoire de Biochimie, Ecole Polytechnique, 91128 Palalseau, France

At the center of the genetic code, the aminoacyl-tRNA synthetases establish

the relation between each amino acid and the corresponding tRNAs. To insure
the matching between one amino acid and one tRNA, the synthetases often
directly recognize the anticodon triplet in the tRNA sequence (reviewed in
[1]). To achieve such a recognition, the synthetases display small defined
domains capable of specifically interacting with the base triplet. For instance,
Escherichia coli lysyl-, aspartyl- and asparaginyl-tRNA synthetases recognize
the anticodon of their corresponding cognate tRNAs through an OB-folded
extension at the N-terminus of the protein [2]. 3-D models of the association
of tRNA have enabled to detail the mode of binding of the anticodon bases by
this domain. A rigid scaffold of amino acid residues along the 5 g-strands of the
OB-fold recognizes the base U at the center of the three anticodons. Binding of
the adjacent bases is obtained through the conformational change of a flexible
loop joining strands 4 and 5 (L45). As a result, switching of the specificity of
one synthetase could be attempted. Transplantation in lysyl-tRNA synthetase
of the small loop L45 from aspartyl-tRNA synthetase was enough to give
lysyl-tRNA synthetase the specificity for tRNAASp. The engineered
synthetase has become at least 50-times more active with the tRNA Asp
substrate than with the tRNA Lys one. Moreover, in vivo, trans addition of the
gene of the engineered synthetase specifically suppresses a missense
Asp --~ Lys mutation in the g-lactamase gene.
[1] Mcinncl et al. (1995) in tRNA. Structure, biosynthesisand function (D. $611 & U.
RajBhandary, eds), ASM Press, Washington,D.C., 251; Gicg~ et al. (1998)Nucleic
Acids Res. 15, 5017.
[2] CavareIliet aL (1993) Nature 362, 181-184; Onesti et al. (1995)Structure 3; 235-240;
Commans et al. (1995) .Z MoL Bid 253, 100-113; Cusack et al. (1996) EMBO ,I. 15,
6321-6334; Commanset aL (1998)Z Mol BioL 278, 801-813.

(We/7.3/239)

c P 1 Domain is essential to the Editing Function of

Leucyl-tRNA Synthetase from Eseherichia coli
Jian-Feng Chen, En-Duo Wang.

Essential residues in yeast arginyl-tRNA synthetase

G. Eriania, J. Cavare[lib, B. Delagoutteb, D. M o r a s b and J. Gangloft

Arginyl-tRNA synthetase is one of the three class I aminoacyl-tRNA

synthetase which needs its homologous tRNA to catalyse the first step of the
aminoacylation reaction: the activation of arginine by ATP. In order to
understand the structure-function relationships of yeast arginyl-tRNA
synthetase (ArgRS), we have used several biological and physical approaches.
The gene RRS1 encoding ArgRS was first isolated and cloned into a protein
expression vector (pTrc99). The moderate level of ArgRS expression was
improved by co-transformation with pSBET, a vector carrying the E. coli
a r g U g e n e . This gene encodes the rare tRNA4Argdecoding the AGA and AGG
arginine codons rarely used in E c o l i but present at higher frequencies in
eukaryolic coding sequences. The presence of this 'helper' tRNA increases
the level of expression and the fraction of soluble ArgRS in the E. coil cells.
Large amounts of the protein were purified to homogeneity by two
chromatographic steps [1]. Crystals of ArgRS with L-arginine bound to the
active site were then obtained and the crystal structure was solved at 2.75A
resolution [2]. In parallel, we have demonstrated the essential character of the
gene into the yeast cell by the disruption of the genomic copy. Taking
advantage of this mutated strain we have developped a genetic screen which
allowed isolation of ArgRS mutants affected in the essential function(s). We
have selected 26 mutations which inactivate the ArgRS function. The
mutations mapped the important peptides of the protein: consensus motifs
(HIGH- and KMSKS-Iike peptides), arginine binding site and tRNA
interacting domain.
[1] Marie Sissler, Gilbert Eriani, Franck Martin, Richard Gieg6 and Catherine
Florentz. Nucleic Acids Research (1997) Vol 25, 4899-4906.
[2] Jean Cavarelli, BOnOdicte Delagoutte, Gilbert Eriani, Jean Gangloff and
Dino Moras.The EMBO Journal (1998) Vol 17, 5438-5448.

(We/7.3/240)

The identity of the tRNA specific for tyrosine in yeast

P. Fechter, J. Rudinger-Thirion, A. Th6obald-Dietrich, R. Gieg~
UPR 9002/CNRS. 15 Rue Rend Descartes, 67084 Strasbourg cedex, France.

State Key Laboratory of Molecular Biology. Shanghat Institute of

Biochemistry, Academia Sinica, Shanghai, 200031, China.

The high fidelity of genetic information transfer in protein biosynthesis

was maintained by the editing reaction of aminoacyl-tRNA synthetase with
the overall accuracy of about 3 mistakes in 104 amino acids incorporated Iq.
With the editing function, leucyl-tRNA synthetase (LeuRS) can correct its
errors of misaetivating methionine (Met) and isoleucine(Ile).
Some evidence show that the CP1 insert region of isoleucyl-tRNA
synthetase (IIeRS) and valyl-tRNA (ValRS) synthetase can deacylate the ValtRNA and Thr-tRNA, respectively f21. Like IleRS and ValRS, LeuRS has a
large CP1 domain from 127T to 385G.
Here, we have obtained a mutant of LeuRS (LeuRS-(328-368)O) with a
duplication of the peptide fragment from residue 328M and 368P (in the CPI
domain). In the pre-transfer proofreading reaction, the discriminations of Met
and Ile for LeuRS were 3.3x10 "4 and 7.9x10 -4, however, for LeuRS-(328368)0 these values w e r e 8 . 0 x l 0 "4 and 1.7x10"3, respectively and two times
more when compared with those of native LeuRS. Furthermore, when 50 mM
of Met and lie were added to the aminoacylation reaction system (containing
0.1 mM laC-leucine) respectively, the leucylation activity of the mutant
decreased to about 83% and 66% as compared with that in the absence of Met
and lie. In contrast, the leucylation activity of the native LeuRS with the
addition of 50 mM Met and lie was almost the same as that without Met and
Ile. These results show that the insertion mutant of LeuRS discriminates noncognate amino acids weakly in the activation of amino acid and loses its
proofreading function for misactivated amino acids in aminoacylation reaction
when compared with the native enzyme.
[1] S. English et al., Nucleic Acids Res, 14, (1986) 7529.
[2] L. Linet al., Nature, 384, (1996) 33.

The accurate translation of the genetic information is correlated to the

errorless charging of transfer RNAs (tRNAs) by aminoacyl-tRNA
synthetases (aaRSs). The specificity of aminoacylation of tRNAs by aaRSs
is linked to the presence on the tRNA of a reduced number of nucleotides,
called identity set. The search of identity set by the in vitro method is based
on the synthesis of transcripts derived from the natural tRNA sequence,
followed by the measurement of their aminoacylation kinetic parameters
(KM and l%,t ) [1].
We search for the tyrosine identity in yeast which is of peculiar interest
since prokaryotic tRNA TM presents a long variable region whereas
eukaryotic tRNA TM presents a short one. Furthermore, it exists in nature
RNAs folded into tRNA-like structures recognized by TyrRS [2]. However,
production of tRNA ryr transcript is difficult, since the first nucleotides of its
gene are unfavourable for the transcription by the T7 RNA polymerase.
Insertion of a hammerhead ribozyme between a strong T7 promoter and the
tRNA TM sequence allows production of considerable amotmts of wild-type
and mutant transcripts [3]. Kinetic analysis of these molecules demonstrates
the importance of the anticodon G34U35A36, the discriminator base A73, and
the first base-pair CI-G72 for tyrosylation. Expression of this identity is not
sensitive to architectural features of tRNAs. The completness of the
tyrusine identity set was verified by transplantation experiments which
confered tyrosylation activity to the engineered tRNAs.
[ 1] Gieg6 R. et al., Nucleic Acids Res., 26, (1998) 5017.
[2] Florentz C. and Gieg6 R. in tRNA: Structure, Biosynthesis, and
Fonction, eds. Soll D. and RajBhandary U.L. (Am. Soc. Microbiol. Press,
Washington, DC), (1995) 141.
[3] Fechter P. et at., FEBS Letters, 436, (1998) 99.

s338

(We/7.3/241)

Abstracts FEBS'99

Functional and structural role of the N-terminal

domain in yeast Aspartyl-tRNA synthetase
M. Frugier, R. Gieg6

(We/7.3/242)

aUPR 9002 and b9004, IBMC/IGBMC 67000 Strasbourg- France

UPR 9002/CNRS, 15 rue Rend Descartes. 67084 Strasbourg cedex. France

Several deletants in yeast aspartyl-tRNA synthetase, differing by the length

of their N-terminal domain (present only in lower eukaryotes) and the
peptide corresponding to the 70 amino acids of this extra domain have been
cloned and overexpressed in E. coli. We have searched for the role played

by this extra domain that is not necessary for aminoacylation in vitro and in
vivo. Previous experiments [ 1], like the resolution of the crystal structure of

the complex AspRS/tRNAAsp or the free protein were done with proteins
partially or completely deleted of this domain [2, 3]. The capacity of the Nterminal 70 amino acids to recognize RNA was tested and the motif
responsible for RNA binding was narrowed.

Using biochemical approaches, we studied this interaction by comparing the

functional and structural properties of these different enzymes and the 70
amino acids long-peptide by itself. Experiments reveal unexpected structural
properties of the N-terminal domain which is involved in tRNA binding.
[1]: Plltz J. et al. Science, 252, (1991) 1696.
[2]: RuffM. et al. Science, 252, (1991) 1682.
[3]: Sauter C. et al. to be published

(We/7.3/243)

Autoaminoacylation of E. coli valyl-tRNA synthetase

C.Hotmtondji, P.Dessen, C.Beauvallet*, J.C.Pemollet*, S.Blanquet
Lab.Biochimie,Ecole
Polytechnique,91Palaiseau,*Bioehirn~e
et Structure des Prot~ines, lNRA, 78Jouy-en-Josas, France.

Upon valyl-adenylate formation, valyl-tRNA synthetase (ValRS) from

coli undergoes a slow covalent modification, with isopeptide
bonds resulting from the reaction of the adenylate with lysyl residues on the
enzyme. Such a reaction is believed to occur at the binding site of the
adenylate, at least partly [1]. Matrix-assisted laser desorption-ionization mass
spectrometry designated lysines- 154, - 162, - 170, -533, -554, -593, -894, -930
and -940 ofE. coli ValRS as the target residues of L-valine.
Under identical experimental conditions, in the presence of the non
cognate isosteric substrate L-threonine instead of valine, a faint covalent
modification of ValRS can be revealed, corresponding to the threonylation of
lysines-162, -170, -178, -277, -291, -554, -580, -593, -861, -894 and -930.
Comparison of ValRS to the 3-D structure of T h e r m u s t h e r m o p h i l u s
IIeRS [2], allows to locate the residues specifically modified by threonine.
Two of them, Lys-291, and Lys-277 which is conserved in all ValRS
sequences but those of archaebacteria, may be predicted to belong to the CP1
domain. This domain was proposed to be involved in the edition of noncognate aminoacyl-adenylate by class Ia aminoacyl-tRNA synthetases [2].
Such a role of CP1 is discussed on the basis of sequence alignments.

Active site mapping of yeast aspartyl-tRNA synthetase

J. Gangloffa, L. Adora, J. Cavarelli b, D. Morasb and G. Eriani a

We present here the characterisation of a set of lethal mutants of yeast

aspartyl-tRNA synthetase isolated by a genetic selection. AspRS is one of the
ten class II aminoacyl-tRNA synthetase; its crystallographic structure has been
solved in the complexed form with the tRNA and the active site of the molecule
has been characterised by structural and functional approaches. Although this
enzyme is well known, it remains a model of study for this family of molecules
which regroups more than one catalytic challenge. These molecules catalyse the
same global reaction, with the same cofactor (ATP), but they have to
discriminate for their specific amino acid among twenty amino acids, and for
their specific tRNA isoacceptor(s) among more than several dozen of different
species present in the cell. In this study we have designed a genetic selection in
order to isolate mutations which inactivate the AspRS molecule. A coloured
genetic screen was used to select clones which cannot lose a rescuing vector
carrying the AspRS gene in a chromosomal context disrupted for this gene. This
approach looked efficient to map the active site residues since of the twenty
three different mutations isolated thirteen are in direct contact with the
substrates. Most of the mutations are located in a 15 A radius sphere around
the ATP molecule, where they affect the very conserved residues of the classdefining motifs. The results also showed the importance of the dimer interface
for the enzyme activity: a single mutation of the invariant proline residue of
motif 1 led to a structural defect inactivating the enzyme. From in vivo
complementation studies it appeared that the enzyme activity can be recovered
by reconstitution of an intact interface through the formation of heterodimers.
We also showed that a single mutation affecting an interaction with G34 of the
t R N A can inactivate the enzyme by inducing a relaxation of the tRNA
recognition specificity. Endly, several mutants were selected, which functional
importance couldn't be assessed from the structural data, demonstrating the
importance of this type of approach in the context of a structure-function
relationship study.

(We/7.3/244)

Wanted: tRNA partners

F. Martin, G. Eriani and J. Gangloff
IBMC - UPR du CNRS n 9002,
15 rue Ren~ Descartes, 67084 Strasbourg, France

Escherichia

[1]. Gillet et al.,.Protein Science, 6, (1997) 2426.

[2]. Nureki et al., Science, 280, (1998) 578.

For relatively small molecules, the biogenesis of functional mature tRNAs is

an amazingly complicated process. All tRNAs are transcribed in the nucleus
initially as precursors containing 5' leader and 3' trailer sequences that must
be removed by processing. Pre-tRNA also undergo a complex set of base
modifications that are carried out by a series of enzymes that recognise
specific features of tRNA structure. In addition, eukaryotic tRNAs have
CCA added to their 3' ends by a specialised nucleotidyl-transferase. A
subset of eukaryotic pre-tRNAs also contain intervening sequences, which
are removed by a dedicated set of tRNA splicing enzymes. Lastly tRNA
must be exported from the nucleus to the cytoplasm where some of them
undergo a set of additionnal modifications. The mature tRNAs are then
aminoacylated by their cognate aminoacyl-tRNA synthetase and bound by
translation factors to participate in the protein biosynthetic process. Each of
these biogenesis steps requires a bunch of proteins which interact with
several part of the tRNA molecule. Lots of these tRNA-binding proteins
remain unknown and we propose to characterise them by using the recently
developed yeast three hybrid system [1]. Two tRNAs have been used as a
bait: yeast tRNA Arg3and tRNA xyr2 to screen a yeast cDNA library.
[1] SenGupta et al., PNAS 93, (1996) 8496.

Abstracts FEB S' 99

(We/7.3/245)

s339

Glycyl-tRNA synthetase from Thermus thermophilus:

wide divergences with the other glycylation systems.

(We/7.3/246)

The dual identities of mammalian tRNA see for

SerRS and selenocysteine synthase
T. Mizutani

M.-H. Mazauric, G. Keith, R. Gieg~ and D. Kern

UPR 9002 CNRS/fl]MC, 15, Rue Ren~ Desca,fes, 67084 Strasbourg, France

Nagoya City Universit3, Nagoya, 467-8603 Jap~

The tRNA glycylation system belongs to the most complex aminoacylation

systems since neither the oligomeric structure of the enzymes nor the
discriminatory base N73 in tRNAs are phylogenetically conserved. To better
understand this structural diversity and its functional consequences we
investigated the glycylation system from extreme thermophile T. thermophilus and
analyzed its structural and functional interrelations with the systems of other
origins.
Alignment of the sequence of the dimeric thermophilic GIy-tRNA
synthetase derived from gene we cloned and sequenced with sequences of other
dimeric GIy-tRNA synthetases reveal the atypical character of motif 1 in all these
class II synthetases. Interestingly, the sequence of the prokaryotic thermophilic
enzyme resembles eukaryotic and archaebacterial GIy-tRNA synthetases, all
dimeric, and diverges drastically from the tetrameric enzymes from other
prokaryotes [ 1].
Cross aminoacylations with tRNAs and synthetases of different origins
show conservation of the recognition process in prokaryotes despite strong
structural variations of the synthetases. However, GIy-tRNA synthetase from T.
tRNA Gly whale
the charg'ng
1
thermophilus acylates eukaryotlc
ability of the E. coli
enzyme is restricted to prokaryotic tRNA Gfy. A similar behaviour is found in
eukaryotic systems where the restricted species specificity for tRNA glycylation of
mammalian GIy-tRNA synthetase contrasts with the relaxed one of the yeast
enzyme[2].
Comparison of the consensus sequences of the tRNAs charged by the
various Gly-tRNA synthetases reveal conservation of only G I - C 7 z from the
acceptor arm, Cj.~ and C~ from the anticodon and of the (G~o-Yz0-G4.~ triple
involved in tRNA folding. Conservation of these nucleotides is indicative of their
key role in glycylation and suggests that they were part of the ancestral set of
glycine determinants. Analysis of the contribution of the non conserved
discriminatory base (U in prokaryotes and A in eukaryotes) to glycylation
efficiency indicates non involvement in thermophilic identity whereas this position
determines glycine identity in E. coli and mammalians; nevertheless this nucleotide
is involved in specificity of tRNA charging in T. thermophilus since its
substitution by G73 strongly hinders thermophilic glycylation.
These features are presented in the context of the phylogenic connections
between prokaryotes, eukaryotes and archaebacteria, and of the particular place of
T. thermophilus in this phylogeny.
[1] Logan, D. T. et al. EMBO J., 14, (1995) 4156.
[2] Mazauric, M.-H. et al., Eur. J. Biochem., 251, (1998) 744.

(We/7.3/247)

Thermodynamics of aminoacyl-tRNA 3'-end analogues

B.Nawrot ~, P Acharyab, M. Sprinzl*, Ch.Thibaudeau b,

J.Chattopadhyaya b
Polish Academyof Sciences, 90-363 Ldd2, Poland. b Dept. Bioorg.
Chem., Biomed. Centre, 75123 Uppsala, Sweden, Biochem. Lab.,
Bayreuth University, 95440 Bayreuth, Germany
During in vivo protein biosynthesis many enzymes and factors has to discriminate
between flee and charged tRNA. The differences up to 5 orders of magnitude in
the equilibrium dissociation constants between tRNA and aminoacyl-tRNA and
elongation factor Tu (EF-Tu) prompted us to investigate the tRNA 3'-end
molecular features. It has been shown by us [1,2] and others [3] that anthranilic
acid charged tRNAs are able to form stable complexes with bacterial elongation
factor Tu and GTP, and the 2'- and 3'- O-anthraniloyladenosines ( l a and 2a) are
the minimal models mimicking aminoacyl-tRNA

o-c

ta: R=H(2'-ant-Ado)
ib: R=FO3H t2"-am-Aiv~)
North (N)-sugar

(C3"-endo-C2'-exo)

o.c

")a:R=H (3'-ant-Ado)
2b:R=PO3H ~3"-ant-.advlP)
South (S)-sugar
(C3 '-exo-C2"-endo)

By NMR studies we examined the thermodynamic parameters of l a and 2a, their

5'-phosphate counterparts l b and 2b, as well those of adenosine As evidenced
from the relative temperature dependency of N to S population, and from the
relative preference for S-type sugar conformation in 3'- and 2'-isomers of
anthraniloyl derivatives of adenosine and AMP, 3'-isomers have relatively more
stabilized S-type conformation (AG = -4.6 kJ/mol and AG = -3.9 kJ/mol for 3"ant-AMP and 3'-am-Ado, relatively) driving the aminoacyl residue in the
pseudoaxial orientation and aglycon moiety in the pseudoequatorial orientation
because of intrinsic gauche effect O4'-C4'-C3'-O3' These features play key role
in enzymatic recognition of 3'-isomers of aa-tRNA by elongation factor Tu.
[1] Limmer S. et al, Angew. Chemic Int. Ed. Engl., 1997, 36, 2485;
[2] Nawrot B. and Sprinzl M., Nucleosides Nucleotides 1998, 17, 815;
1-31 Servillo L. et aL Eur. ~ Biochem. 1993. 213. 583.

Selenium is an essential trace element and is found as selenocysteine in the

active sites of glutathione peroxidase, 5'Dt and thioredoxin reductase.

tRNA sec is first aminoacylated with serine by SerRS and further is
converted to selenocysteyl-tRNA by selenocysteine synthase. Mammalian
selenocysteine tRNA had dual identities with SerRS and selenocysteine

synthase. Key identity elements for selenocysteine synthase are the long 9
bp AA- and long 6 bp D-stem. Major serine tRNA was converted to a
mutant with a 9 bp AA-stem and 6 bp D-stem, instead of a 7 bp AA-stem

and 3 bp D-stem. This mutant was active for selenylation as well as

serylation. The relative kinetic parameter (Vmax/Km) of the mutant was
0.052 of the value (I .00) of wild-type Sec tRNA. This low activity may
come from some unknown and fine base specific for selenocysteine
synthase. For serylation, mutant having 12 bp and wild-type tRNA sec

having 13 bp of the total length of AA- + T-stems were active but the
mutants having 11 or 14 bp were inactive. This shows that SerRS
measures the distance between the discriminator base and long extra arm

for ~ecognition of tRNA ser.

T. Mizutani et al., Mol.Biol.Reports 25, 211-216 (1998).

(Wef/.3/248)

In vitro selection of active tRNA Asp variants missing major

identity elements in the anticodon loop
J. PiJtza, J. Wientgesb, C. Florentz a, A. Schwienhorst b, R. Gieg6 a
"UPR 9002/CNRS, 15 rue Rend Descartes, 67084 Strasbourg, France, blnst.
fur Mikrobtologie & Genetik. Griesebachstr. 8, 37077 GOttingen, Germany

The positive [1,2] and negative identity elements [3] of yeast tRNA ASp were
previously determined by steady-state kinetics for a series of unmodified
tRNA A~p variants. These studies were carried out by knowledge based site
specific mutagenesis. In order to screen for altemative anticodon loop
structures to be recognized by yeast AspRS, an in vitro approach based on the
selection of aminoacylated tRNAs [4] has been applied An initial library
covered a sequence space of 7 anticodon loop nucleotides generating
47 = 16.000 tRNA A~panticodon loop variants. Six rounds of selection created
pools with enhanced charging activity and sequence analysis of a total of 50
clones revealed molecular species containing predominantly the wt anticodon
sequence Steady-state kinetics of the aspartylation reaction were performed for
selected species after the 4th and the 6th round of selection. We also selected a
fully active tRNA Asp variant displaying a shifted anticodon G35U36C37.
Furthermore this variant does not present the conserved U34 nor a purine at
nucleotide 37. Mutations at these positions reduce the specificity constant k~,,/K~
for aminoacylation by 1-4 orders of magnitude, whereas mutations at the wt
anticodon G34U35C36 positions revealed losses by 2-3 orders of magnitude
[1]. We conclude that, compared to wt tRNA ASp, the changed anticodon loop
structures must be recognized in an alternative way.

[1]
[2]
[3]
[4]

Ptitz, J. et al., Science, 252, (1991) 1696.

Frugier, M. et al., Biochemistry, 33, (1994) 9912.
Pfitz, J. et al., Nature Struct. Biol., 1, (1994) 580.
POtz, J. etal., NAR, 25 (1997), 1862.

s340

(We/7.3/249)

Abstracts FEBS'99

S u l f o l o b u s solfataricus phenylalanyl-tRNAPhe synthetase

(We/7.3/250)

G. Raimo, B. Lombardo, V. Bocchini

Diparttmemo di Biochimica e Biotecnologie Mediche. Universitgt di
Napoh Federico II. I/ia S Pansini 5, 1-80131 Napoli, ITALIA

In current studies on the characterization of the macromolecutes involved in

the process of protein synthesis in extremophiles [1-3] we have focused our
attention on the phenylalanyl-tRNAP he synthetase (FRS) in S M f o l o b u s
solfataricus (Ss).
SsFRS has been purified from the SI00 fraction orS. solfalaricus cell
hornogenate with a final yield of 17%. Estimated by gel filtration at pH 7.8 the
Mr of the native protein was about 250 kDa. On SDS-PAGE SsFRS showed
two protein bands accounting one for 56 kDa and the other for 64 kDa. These
data indicated that in its native form SsFRS is a tetramer composed by two
molecules of each subunit, a feature common to FRS's isolated from other
organisms.
,%FRS is an ATP-dependent enzyme stimulated by spermine or spermidine.
It is specific for SstRNA being not able to amino acylate the tRNA from
Escherichia coli or S a c c h a r o m y c e s cerevisiae. In the range of 50-92C the
activity of SsFRS increased at increasing temperature, apparently without
reaching a maximum; the energy of activation of the amino acylation reaction
was 45 M/mot At 75C the kcat of SsFRS and the Krn for phenylalanine was 6.9
min -I and 1.5 [aM respectively.
The heat resistance of SsFRS tested at 87C (which is the optimal growth
temperature of S. solfataricus) showed a half life of 59 min. In addition,
fbllowing t0 min exposure in the 60-100C range the temperature for its halfinactivation was 88C, a temperature in the range of those determined for other
proteins isolated from S solfittaricus.
Researches on the interaction between subunits and their role in catalytic
and structural features of SsFRS will be performed.
This investigation was .supported by the E U Btotechnoloyw
6kmtract Nr. BIO4-CT97-2188 and by C.N.R. (Roma).

Programme.

[t] M. Masullo et al. Eur..Z Biochem., 199 (1991), 529

[2] G. Raimo et al. ,L Biol. Chem., 271) (1995), 21082
[3] G. Raimo et al. Biochim. Biophv,s. Acta, 1293 (1996), 106

(We17.3/251)

Structure of the free aspartyl-tRNA synthetase from yeast

and comparison with the enzyme associated to the tRNA
C. Sautcr". J C,p.'aJclh".D Moral"andR. Gicgd"
"UPR 90t)2/CNRS. 15 rm' Rc,~; I),sf e, t ~. 67t1S4 Sna~f, mr k, ~cdc~-f tam e
I'UPR 90(I.4/('NRS. ] ttl~' Lotlrt.tll klt.~. 874(t4 Illkt~ h ~edc~ Flare c

The aspartic acid system of yeast is one of the few systems where the
~tlUCture~ of the free tRNA and its co111131cx with the cognate ~;ynthela~c.
aspartyl-tRNA syntheta~e (AspRS). have been determined [I, 21. Their
comparison highlights the remarkable conformatlonal changes occtning to
the tRNA ~'e upon the complex formatinn [3]. In order to detect ~imilar
sn-uctural modifications affecting the enzyme, man', crystallization attempts
of fi'ec AspRS were condttcted during the hlst two decade,,. Howe,.cr. thls
protein proved to be vcr 3 difl'~cuh to crystalhzc m at form suilable fol
structtlre detcrminaIioll More reCelltly, cly~;tallogcllcM ~, ~,tudles illlpl'ovecl
the quality of free A,,pRS crystals and produced t,,vo crystal forms
diffracting X-rays to a resolutiml better than 3 A [41. The structure of the
free AspRS in the tetragonal space group (P41212) was ,,olved by molecular
replacement and refined to 2.95 ,~. Structural change,, between AspRS in its
flee state and its complexcd torm with the cognate IRNA Inillnly concern Ihc
catalync domain with two mobile Ioop~. also observed m procaryotic
AspRSs. that open and clo~e the acnve site of the enzyme. In addition, the
antlcodon hinditlg domain undergoe~ a ~hght rigid body mo~enlenl.
[t ] Mola~ D. et at.. NatLIte. 2g~. (I 9~0) 669
[2] Rulf M. et al.. Scmnce. 252. (1991) 1682.
[3] Rees B. et al.. BIochimm. 93. (1996) 624.
[4] Sauter C. et al.. Acta Cryst.. D55. 11999) [49.

Thermus thermophilus: a link in evolution of the

pathways of tRNA aspartylation and asparaginylation.
H. Roy, H. D. Becket and D. Kern
UPR 9002 CNRS/~MC, 15, Rue Rend Desccutes, 67084 Strasbourg, France

Thermus thermophilus possesses two aspartyl-tRNA synthetases

(AspRS) which differ functionally and structurally [1]. Both are c~2 dimers but
AspRS1 is significantly larger than AspRS2 (M, respectively 140 000 and 104
000); further AspRS1 aspartylates specifically tRNA a~p whereas AspRS2
aspartylates tRNA 7's" as efficiently as tRNA A~p.Aspartate mischarged on tRNA A~"
is then converted, in the presence of an amide group donnor and ATP, into
asparagine by a tRNA-dependent amidotransferase [2]. The tRNA-dependent
route of asparagine synthesis promoted by AspRS2 resembles that described for
Haloferax volcanii and may be the pathway of Asn-tRNA ~*" formation in the
archaebacteria lacking asparaginyl-tRNA synthetase [3].
To obtain an insigth into the origin of the two thermophilic AspRSs we
analyzed their structural interrelation. The two genes were cloned and sequenced
and the derived polypeptide chains compared. Their alignment with AspRSs
polypeptide chains of various origins reveals that AspRS 1 contains the peculiar
domains which characterize eubacterial AspRSs whereas AspRS2 is deprived
like archaebacterial AspRSs of these domains. This investigation establishes the
eubacterial and archaeabacterial nature of AspRS 1 and AspRS2 and demonstrates
the distinct evolutionary origin of the two thermophilic AspRSs. Taken together
with the functional analysis it establishes the peculiar structure-function
relationship distinguishing AspRSs of eubacterial and archaebacterial types :
eubacterial AspRSs aspartylate specifically tRNA a~p whereas archaebacterial
AspRSs by aspartylating tRNA As" as efficiently than tRNA ^s" are additionally
involved in an indirect pathway of tRNA asparaginylation. However in contrast
to archaebacteria deprived of asparaginyl-tRNA synthetase (AsnRS) T.
thermophilus contains an AsnRS able to charge tRNA AS" with free asparagine
despite of the unability of this organism, deprived of asparagine synthetase, to
convert free aspartate into asparagine.
Since Z thermophilus is the first organism characterized until now able
to ensure tRNA aminoacylation by the eubacterial and the archaebacterial routes
these findings shed a new light on the interrelation between the ancestral and the
modern pathways of tRNA aminoacylation. Simultaneous presence of AspRS 1,
AspRS2 and AsnRS confers to Z thermophilus the role of a link in evolution of
the pathways of tRNA aspartylation and asparaginylation.
[I] Becker H.D. et al Biochemistry 36, (1997), 8785.
[2] Becker H. D. and Kern, D., Prec. Natl. Acad. Sci. USA, 95, (1998) 12832,
[3] Curnow A. W.et al., Nature 382, (1996) 589-590.

(We/7.3/252)

An aminoacyl-tRNA synthetase-like protein required for

the catalysis of the first step in histidine biosynthesis
M. Sisslera, C. Delorme b, P. Renaultb, J. Bond c, C. Francklyn
aI~ept. ofBiochemist~. University of Vermont, Burlington, V'I] USA
-- G~ndtique Mtcrobienne, INRA-CRJ,, 78532 Jouv-en-Josas, France
: MMG, Vermont Cancel- Center. University of Vermont, USA

T h e his operons of gram negative and gram positive bacterial species

are regulated by diverse mechanisms. They all involve proteins which share
a domain related to the catalytic core of histidyl-tRNA synthetase (HisRS)
and involve binding interactions with uncharged tRNA. In gram positive
organisms such as Lactococcus lactis, a HisRS homologue (his Orf3) has
been found within the his operon [1]. Recently, Orf3 has been shown to be
required for histidine prototrophy, but its structure and precise function
remained uncharacterized. Orf3 shares significant sequence identity with the
E. coli HisRS catalytic core, but is missing several presumed catalytic
residues in motif 2 and motif 3. In addition, the C-terminal domain,
required in the functional enzyme for anticodon recognition, is absent. As
expected, Orf3 does not possess eminoacylation activity, yet binds cognate
and non-cognate tRNAs non-specifically. Paralogous HisRS have also been
detected in other genomes. Off3 and its homologue in B. subtilis are located
adjacent to the HisG gene, which encodes the first enzyme (ATPphosphoribosyl transferase) involved in the histidine biosynthetic pathway.
The length of the HisG polypepfides varies in relation to the presence or the
absence of an Orf3 homologne, such that organisms that posses one have a
short version of HisG (by -100 amino acids). This observation prompted
us to consider the involvement of Ort3 as a functional subunit of the HisG
protein. Several observations now provide direct evidence for this model.
First, an in frame deletion of HisG in E. coli (which does not possess Orf3
homologue) can be complemented by HisG from L. lactis only when Off3
is also provided. Second, HisG and Orf3 directly interact, as shown by coelution during affinity chromatography. Lastly, direct assay of the ATPphosphoribosyl transferase reaction shows that both Orf3 and HisG are
required for catalysis, and that neither subunit alone possesses activity.
Thus, we have identified a protein family based on the catalytic core
of the contemporary class II HisRS that serves as an essential subunit
responsible for catalyzing the first step in histidine biosynthesis. The
finding of a common protein domain linking amino acid biosynthesis and
protein synthesis suggests that the aminoacyl-tRNA synthetases arose early
in the transition from the RNA to the protein world, and implies and original
direct connection between the biosynthesis of amino acids and proteins.

[1] Delorme C. et aL, J. Bacteriol., 174, (1992) 6571.

Abstracts FEBS' 99

(We/7.31253)

taaRSAt, a database of tRNAs and aminoacyl-tRNA

synthetases from A r a b i d o p s i s t h a l i a n a

s341

(We/7.3/254)

I. SmallqK. Akama~,K Akashi".A.Chaplon~.N.Choisne'.A Dietrich'~.L.Drouard".

A-M.Duchene".A Giritch',D.Lancelin~.N Peelers~,G.Sumer~.H W]ntz"

A. Th6obald-Dietricha, G. Kotzorek b, R. Gieg6a

'Station de Gdngtique. INRA, Versailles, France: "Shimane Untversi O,

Japan; 'Genoscope - CNS. El'tT, France; ~TBMP, Strasboltrg. France

Transfer RNAs and aminoacyl-tRNA synthetases are central components in all

translation systems. Large-scale genome sequencing is starting to permit a
complete description of these components from several organisms. Prokaryotes
generally contain 30-60 tRNAs to read the 61 sense codons and 18-20
aminoacyl-tRNA synthetases (aaRSs) to aminoacylate these tRNAs specifically
with one of the 20 primary amino acids. Most eukaryotes contain added
complexity as they have two translation systems, mitochondrial and cytosolic.
Plants are more complicated still, containing a third translation system in their
plastids. This complexity makes the study of the first plant nuclear genome to be
extensively sequenced, that of Arabidopsis thaliana, particularly interesting. The
four labs hsted above are collaborating to prepare a database of all tRNA genes
(nuclear, mitochondrial and plastid) and all aaRS genes (all are nuclear) from
ArabMopsis. Currently the database (accessible via the world-wide web at
http://www.inra.fr/USER/PRODUCTIONS/BDD/TAARSAT/)
contains nearly
300 tRNA genes and 26 aaRS genes, out of the approximately 40% of thc
genome sequenced so far. The database not only includes raw sequence data, it
also contains exon and intron predictmns, predicted peptide sequences,
sequence alignments, phylogenetic trees, secondary and tertiary structure
predictions, and even data from GFP fusions showing subcellular localization
of the various aaRSs. The data provide a wealth of information on these
important molecules that is essential for understanding translation in plants (and
particularly the way in which the organellar translation systems function) and
useful for comparisons with other organisms.
Some of the more interesting features to come out of this study so far will
be highlighted on the poster.

Role of the ribose of residues UI! and G27 in yeast

tRNA A'p on its aspartylation capacity
OUPR 9002/CNP~, I5 rue Rend Descartes, 67084 Strasbourg cedex-France
~NAPS Gottingen GmbH, Rudolf-Wissell-Str.28.37079 Gottingen-Germany

In the frame of structure-function studies on aminoacylation systems, an

effect of the substitution of ribonucleotides by deoxyribonucleotides in
yeast tRNA Asp was shown to affect the in vitro kinetic parameters for
aspartylation [1]. While the complete substitution of C or A nucleotides in
dC or dA in the tRNA produces only low changes in the aminoacylation
efficiency (-2 to 3 fold), that of G or U residues by dG or dU affects
drastically the catalytic efficiency (>1000 fold). These functional effects can
be correlated to the 3D-structure of the complex between yeast aspartyltRNA synthetase and tRNA Asp [2] in which the ribose moieties from
nucleotides U ll and G27 make contacts by their 2'OH groups with Asp210
and Glu202, respectively, of the synthetase. It was hypothesized that the
absence of 2'OH groups at the ribose of UII and G27 is responsible for the
decreased activity of the tRNA as a result of the loss of two functional
contacts between the tRNA and the synthetase.
To verify the hypothesis, tRNA Asp molecules, with only ribose 11 or ribose
27 replaced by a deoxyribose, were synthesized (i) by chemical synthesis
for the 5' half containing the modification, (ii) by in vitro transcription for
the 3' half, and (iii) by enzymatic ligation of the two halves.
First results show that the tRNA A~p (G27->dG27) recovers activity
compared to the completely dG modified tRNA. In the case of the tRNA A~p
(UII->dUll) it was not yet possible to show significant aspartylation
activity of the variant. These data agree with an involvement of the ribose
moiety of U II in the aspartylation capacity of yeast tRNA A~p.
[1] Aphasizhev R. et al., RNA, 3, (1997) 893.
[2] Cavarelli J. et al., Nature, 362, (1993) 181.

(We/7.3/255) Crystal structures of two Phe-tRNA synthetase complexes

Vassylyev D.G) 2 Reshetnikova L?, Moor N. 3, Lavrik 0. 3
InternationalInstitutefor AdvancedResearch,Kyoto, Japan
-' R1KEN Harima Institute,Sayo, Japan
Instituteof BioorganicChemistry.Novosibirsk,Russia

The crystal structures of Phe-tRNA synthetase (PheRS) complexed

with phenylalanine (Phe) and phenylalaninyl-adenylate (PheOHAMP) have been determined at 2.7A. and 2.5A resolution,
respectively. Both, Phe and PheOH-AMP are engulfed in the
active site cleft of the catalytic a-subunit of PheRS. The
recognition of Phe by PheRS is achieved through a mixture of
multiple van der Waals interactions and hydrogen bonds. The side
chain of the Pbe substrate is sandwiched between the hydrophobic
side chains of Phec~258 and Phec~260 on one side and the main
chain atoms of the two adjacent [3-strands on the other. The side
chains of Valc~261 and AlacG 14 form the back wall of the amino
acid binding pocket. In addition, PheRS residues (TrpcO49,
Serot180, Hiso~178, Argot204, Gln~218. and Gluc~220) form a total
of seven hydrogen bonds with the main chain atoms of Phe. The
conformation of PheOH-AMP and the network of interactions of
its AMP moiety with PheRS are reminiscent of the other class II
synthetases. The structural similarity between PheRS and histidyltRNA synthetase extends to the amino acid binding site, which is
normally unique for each enzyme. The complex structures suggest
that the PheRS [~-subunit may affect the first step of the reaction
(formation of phenylalanyl-adenylate) through the metal-mediated
conserved ~[3-subunit interface. The modeling of tyrosine in the
active site of PheRS revealed no apparent close contacts between
tyrosine and the PheRS residues. This result implies that the
proofreading mechanism against activated tyrosine, rather than
direct recognition, may play the major role in the PheRS
specificity.

(WeF/.3/256)

Analysis of functional groups and binding modes involved

in RNA-protein interactions: tRNA-synthetase complexes
C.S. V6rtler ab, R. Gleg6 b and F. Eckstein a
a MPl fiir exp. Medizin, Herrmann-Reinstr.3, D-37075 Gottmgen
b UPR 9002 du CNRS, 15, rue Ren~~Descartes, F-67084 Strasbourg

In vitro evolutionary methods open the way to discovery of new molecular

RNA species fullfilling known or entirely novel functions. For the case of
tRNA this is examplified by the recent work of Pi~tz et al. [1]. A detailed
understanding of these novel structures requires their structural
characterization. One method to obtam such information is the footprinting
and nucleotide interference mapping technique using phosphorothioatemodified analogs [2-4]. With a set of four experiments, one for each kind of
nucleobase, data for the binding mode or the importance of a particular
functional group for the RNA function can be obtained. Results for the tRNA
recognition by aspartyl-tRNA-synthetases will be presented. Comparison of
the footprinting and interference data of tRNA with available crystal structure
models underline the precision and thus the prediction power of the method.
[ 1] see corresponding abstract of J. Patz presented at this conference
[2] V6rtler, C.S. et al. R N A 4, (1998), 1444
[3] Strobel, S.A. Biopolymers 48(1). (1998), 65
[4] V6rtler, C.S. et al. Meth. in Enzymol. in press

s342

(We/7.3/257)

Abstracts FEBS'99

Study on the interaction between arginyl-tRNA synthetase

incorporated with 19F-tryptophan and its substrates by NMR
E. D. Wang, Q. S. Zhang
Shanghai Institute of Biochemistry, Academia Simca
320 Yue Yang Road, Shanghai 200031, China

Arginyl-tRNA synthetase (ArgRS) from Escherichia coh contains five

tryptophan residues which are Trp t62, Trp 172, Trp 228, Trp 349 and Trp 446. 4fluorotryptophan-substituted ArgRS was biosynthetically prepared and purified
from a tryptophan auxotroph, which could overproduce this enzyme. A method
was developed to separate 4-fluorotryptophan from tryptophan and to
determine accurately their contents in the 4-fluorotryptophan-containing
proteins. Using this method, the purified 4-fluorotryptophan-substituted ArgRS
was confirmed that tryptophan residues were more than 95% replaced by 4fluorotryptophan Furthermore, the effect of the 4-fluorotryptophan
replacement on properties of the enzyme was examined. As compared with the
native enzyme, the fluorinated enzyme exhibited approximately 20% lower both
specific activity and first-order rate constant with only slightly higher Km values
In addition, thermal unfolding studies revealed that the 4-fluorotryptophan
replacement led to a less stable enzyme with a 2.8C lower Tin. CD studies,
however, did not reveal any differences in the secondary structures of the native
and fluorinated enzymes The substitution of 4-fluorotryptophan in ArgRS has
no substantial effect on the structure and function of the enzyme
The 19F nuclear magnetic resonance (NMR) spectroscopy of the
fluorinated enzyme showed a well-resolved spectrum with strong resonance
signals. Their chemical shifts were located at -2.64, 0.91, 1.29, 1 67 and 4.04
ppm The binding of ATP on the fluorinated ArgRS did not affect the five
resonance peaks. In the absence of ATP and tRNA A~g,the binding of arginine
on the ArgRS shifted the resonance peak from 1.29 to 1.67 ppm which
overlapped with the original resonance peak at 1.67ppm, however the other
four resonance peaks were not changed. No matter whether the other two
substrates exist, tRNAA% may bind on the ArgRS and cause the shifts of the
two resonance peaks of 4.04 and 1.29 ppm, the other three resonance peaks
were not affected The five mutants of ArgRS in which each tryptophan
residues was separately replaced by alanine were fluorinated, purified and
analyzed by NMR spectroscopy. All resonance peaks may be assigned from
NMR spectra of the five fluorinated mutantsof ArgRS The tryptophan residues
involved in the binding of the three substrates were determined

Abstracts FEBS'99

s343

12.2 Proton pumps, redox-coupled systems and ATPases

(We/12.21258) Cyanide-resistant, ATP Synthesis-sustained, and Uncoupling Proteinsustained Respirations During Post-Harvest Ripening of Tomato Fruit

(We/12.2/259)

AM. Almeida~, W. Jarmuszkiewiczz, H Khomsi~, P. Arruda4, AE.

VercesiI, and F.E. Slase3
/Dept Patologla Cllntca,Umverstdade Estadual de Camptnas. Brazil: 'Dept
Bwenergetlcs. Poznan Umverstty, Poland; 3Lab Bwenergencs, Umvers:ty of Lt~ge,
Belgrum: ~Centro de Biologta Molecular e Engenhwqa Gen~nca, Camplnas, Braztl

Tomato (Lycoperstcon esculentum) mitochondria contain both

alternative oxidase and uncoupling protein as energy dissipating systems
that can decrease the efficiency of oxidative phosphorylation. Uncoupling
protein-sustained respiration, cyanide-resistant respiration and ATP
synthesis-sustained respiration of isolated mitochondna, as well as the
immunologically detectable levels of uncoupling protein and alternattve
oxidase were followed during tomato frutt ripening from the mature green
stage to the red stage. The alternative oxidase protein level as well as
cyanide-resistant respiration of isolated mitochondria decreased with
ripening from the green to the red stage. The ATP-synthesis-sustamed
respiration followed the same behavior On the contrary, uncoupling
protein level and total uncoupling protein-activity sustained respvation of
isolated mitochondria decreased only from the yellow stage. An acute
inhibition of the cyanide-resistant respiration by linoleic acid (p_M range)
was observed. These results suggest that the two energy dissipating
systems could have different roles during ripening processes.

Interaction ATPsynthase-tentoxin : towards a structural

understanding of the inhibition and reactivation processes.

J Santolini .1, J -M. Gomis2, F. Haraux l, C., Sigalat r, F. Andr6 I

PCEll, Section de Bio~nerg~tique, 2 CEA, Service des l~4ol~cules Marquees,
D.B.C.~4. Centre d'Etudes de Saclqy, 91191 Gif-sur-Yvette cedex, France

Tentoxin (TTX) is a cyclic tetrapeptide which induces chlorosis of many

plants. Tentoxin binds specifically to the soluble part of the ehloroplastic ATPsynthase (CF1) The effect of the toxin on CF1 is concentration-dependent:
potent inhibitor at low concentration and stimulatory above 10 laM. We
demonstrated that inhibition and over-activation effects are correlated to the
sequential binding of two molecules oftentoxin on CF1 [1,2].
In order to identify the structural features responsible for each activity, we
synthesised numerous analogues. Their kinetic and thermodynamic binding
parameters have been determined for both sites (through activity profiles,
competition experiments and kinetic studies). Besides, their conformational
properties have been determined through 2D NMR experiments and molecular
dynamics simulations. The analysis of the binding characteristics of the
analogues revealed that the first TTX-binding site is a tight, hydrophobic site,
insensitive to the catalytic state of CFI, and stressed the importance of
particular moieties of the peptide in molecular recognition and inhibitory
ability [3]]. The comparison of the 3D-structures of TTX analogues to their
functional properties enlightened the correlation between the conformational
features of TTX and its activities. Besides, the reactivation process, which is
dependent on the catalytic state of CF~, seems to result from a subtle and cooperative interaction between both TTX binding sites [2, 3].
In order to provide our data with a structural model, an analogue of TTX,
bearing a radioactive photo-affinity reagent, was synthesised This derivative
proved to be a good photochemical reagent which specifically binds and
inhibits CFI-e. The subunit carrying the binding site has been determined, and
the identification of the peptides involved in this binding is in progress.
These results gave a new understanding of inhibition and reactivation of
CFj-e by TTX A more extensive study of the structure-activity relationship of
tentoxin will be a support for investigating the regulation of chloroplast
ATPsynthase and the plant-species specificity of TTX toxic effect.
References

i. Pinet et al. Biochemtstry,35, (1996), 12804

2 Santolini et al, J Biol. Chem. (19991. on press
t. Santolini et al. J. Biol Chem 273, (1998). 3343

(We/12.2/260)

Characterization of residues close to the NADP(H)-binding

site in nicotinamide nucleotide transhydrogenase in E.coli.

(We/12.2/261)

Structural studies of the Yeast ATP synthase e-subunit

C. Aznar, P. Picard, S. Geoffre, C. Manigand, G. Pr~cigoux

UBS, UmversltO Bordeaux 1, Avenue des Facult~s, 33405 Talence. France

M. Axelsson and J. Rydstrrm

Departmentof Biochemistryand Bioohystcs,GoteborgUniversuy,Sweden
Nicotinamide nucleotide transhydrogenase constitutes a proton pump, which
links the NAD(H) and NADP(H) pools in the cell by catalyzing a reversible
reduction of NADP + by NADH according to the reaction:
H+out+ NADH + NADP + =~ H+,, + NAD + + NADP
where "out" and "in" denote the cytosol and matrix, respectively, m
mitochondria and penplasmlc space and cytosol, respectively, in
bacteria. Even though not yet fully established, the physiological role is
probably to provide reducing equivalents in the form of NADPH for
detoxificatton and biosynthesis. Transhydrogenase from Escherichia
coil consists of an a subumt of about 54 kDa and a 13subunit of 48
kDa, and an active form of ~2132. Like all proton-pumping
transhydrogenases the E.coli enzyme is composed of three domains,
i.e., the hydrophilic domain I containing the NAD(H)-binding stte, the
hydrophobic domain II containing the membrane-spanning c~-hehces,
and the hydrophilic domain III containing the NADP(H)-binding site.
Ola Fjellstrrm et al. has predicted a model for parts of domain III [1]. To test
this model, a couple of mutants (13A348C, 13A390C, 13R425C, 13D450C and
13A451C) were introduced in the intact as well as in a functional cysteine free
transhydrogenase. These residues were mainly chosen to investigate the area
around the NADP(H)-binding site. In addition to kinetic characterizations,
effects of sulfhydryl specific labelling with N-etylmaleimide (NEM) and 2(4'-maleimidylanilino)-naphthalene-6-sulfonic acid (MIANS) were
examined. ~A390 is located in the heart of the binding site, 13R425 is also in
the binding site but the side-chain points away from it and 13A348 is in a loop
close to the site. The results from these mutants so far support the predicted
model. The rest of the mutants have at the moment not been evaluated.
1. FjellstrOm, O., et al. 1999. J. Biol. Chem. ( in press)

The ATP synthase is a protein complex and reversible

enzyme which catalyses the ATP synthesis from ADP and
inorganic phosphate by extracting energy from the flow of
protons across the membrane. This complex is three-part-formed.
The catalytic and hydrophilie sector F1, the hydrophobic FO
sector, embedded in the membrane, which permits a protonconducting pathway, and a stalk connecting both sectors [ 1].
In order to understand how this enzyme works, numerous
researches are lead in the world. Our laboratory is interested in
yeast mithochondrial S a c c h a r o m y e e s cerevisiae ATPase. The FI
sector contains five different subunits: ct, 13, ?, 6, ~ with a
stoichiometry of 3:3:1:1:1. We are more particularly working on
the e subunit, which is the smallest FI polypeptide (6612 Da, 61
residues). The primary, structure of this subunit was obtained by
direct sequencing [2,3], but its function in the assembly of the
ATPase protein complex remains to be established. Therefore, the
knowledge of its three dimensional structure will provide very
useful information.
For this purpose, we synthesized the e subunlt by solid
phase method (Boc chemistry), purified it by HPLC and checked
its purity by Mass Spectroscopy. Circular dichroism studies were
undertaken to point out its conformational behaviour. In
particular, e subunit shows a random coil conformation in
aqueous solution, but adopts a defined a-helical structure upon
addition of TFE (up to 50%). CD investigations were also used to
set the fight solvent conditions. NMR experiments are in progress.
[ 1] Weber J. et al., Biochim. Biophys. Acta, ! 319, (1997), 19.
[2] Arselin G. et al., J. Biol. Chem, 266, (1991), 723.
[3] Gurlin E. et al., J. Biol. Chem, 268, (1993), 161.

s344

(We/12.2/262)

Abstracts FEBS'99

A voltage dependent equilibrium under periodical

perturbation:application to partial reactions of ion pumps
Alexandru Babes and Klaus Fendler

(We/12.2/263)

Deparnnent of Medical Chemistry, Institute of Biomedicine, University

of Helsinki, P.O.Box 8, 0(3914 UniverMty of Helsinki, Finland

Max-Planck-lnstitut fuer Bipohysik. Kennedyallee 70. FrankJurt am Main.

Germany

Membrane fragments containing purified ion pumps (NaK-ATPase, HKATPase or Bacteriorhodopsin (BR)) were adsorbed on black lipid membranes
forming a compound membrane system. A lock-in amplifier was used to monitor
changes in the impedance of the system associated with activation of the ion
pumps. The ion pumps were activated either by flash photolysis of an inactive
precursor of ATP (caged ATP), in the case of NaK-ATPase and ~K-ATPase, or
by illumination with visible light in the case of BR. In all cases, activation of the
pumps leads to a reversible change in the impedance of the compound membrane
system.
We attribute the change in impedance associated with the activation of ion
pumps to the voltage dependent equilibrium between two states in the enzymatic
mechanism of ion translocation. In the case of the NaK-ATPase it has been shown
using whole-cell patch clamp recordings that following a voltage jump the system
relaxes exponentially and the rate constant of the relaxation process was assigned
to the reaction E 1P(3Na+)cz>E2P+3Na*. Instead of a voltage jump we have used
a periodical perturbation and we have analysed our signals in the frequency
domain. The frequency spectra of the impedance signal could be interpreted as
the sum of two charge moving reactions. We have determined a rate constant for
the slow charge moving reaction of 393 s t at pH 7.4 and 24C. Preliminary
experiments with the HK-ATPase indicate a different behaviour, namely no
electrogenic transition with a rate constant between 60 s -a and 1800 s-~. Rate
constants as slow as 42 s -~ have been reported for the E 1P--+E2P conformational
transition which are in agreement with our results so far.
In the case of BR the impedance signals displayed a biphasic behaviour.
The slow phase dissapears upon permeabilization of the membrane system by
addition of the ionophores monensin and 1799.We atribute the fast phase to a
voltage dependent equilibrium within the photocycle (possibly the MIc=,M2
transition) and the slow phase to charge accumulation between the menthrane
fragments and the BLM. The action spectra of the impedance signal follows the
absorption properties of BR. Experiments performed with the mutant D96N
indicate slower charge accumulation, which can be explained by a slowing down
of the photocycle.
(We/12.2/264)

Control of cell respiration by endogenous NO.

Bellelli A., Mastronicola D, Zona C., Le~daro E., Ippohtl R.,
Brunori M., Xu F, Blanck T J J. and Sarti P.
Dept Biochemical Sciences and CNR Center of Molecular Biology. Uni "La
Sapienza", Dept Neurosctence. Um "Tor l'ergata"'andS. Lucta. IRUCS. Roma
halv. H.S.S.-Cornell Um Med. Coll. N L. L~A.

In addition to its role as an intercellular physiological messenger, it has been

proposed and assessed in a wide variety of m vitro conditions that NO
controls mitochondrial function by inhibiting promptly (milliseconds) and
reversibly (tens of seconds) cytochrome oxidase, complex IV (COX), and on
a longer time scale (hours) other respiratory complexes If the hypothesis
holds also m vtvo, then major effects on 02 metabolism parameters, such as
the apparent Km of cytochrome oxidase for 02, and related changes of
mitochondrial energization state are expected. By means of single cell
fluorescence microscopy, we have investigated the eleetrodriven import o f
rhodamine 123 (RDt23) into mitochondria of intact cells. The mitochondrial
fluorescence pattern and intensity of neuroblastoma cells has been studied
following the addition to the culture medium of the NO donor NOR~
(Calbiochem); variation of the intracellular NO concentration was also
induced by stimulating or inhibiting NOS with NMDA or 7-nitroindazole
Results were consistent with complete inhibition of COX induced by NOR~,
followed by spontaneous recovery of activity with k' = 0 1-0 01 s-t The
inhibition of NOS in cultured cells lead to a significant (30-50%) increase in
the ability to import RD~2s, as expected on the assumption that a significant
fraction of COX is inhibited by endogenous NO under normal cell growing
conditions These observations have been extended to freshly prepared
(primary) cerebellar neurons in the absence and presence of specific NOS
stimuli.
Work partially financed by the NATO grant CRG971556 to P S and T J J B.
and the Progetto Neuroscienze 95/95 to A B

Proton transfer to the binuclear center of cytochrome c

oxidase
C. Backgren, M. Wikstr6m, and A.Puustinen

The cytochrome c oxidases catalyse the reduction of dioxygen to water at

the binuclear heme iron-copper (CUB) site in subunit I of these membrane
bound enzymes. This reaction is coupled to proton translocation across the
mitochondrial or bacterial membrane. The CUA and heme a prosthetic
groups are involved in the transfer of electrons from the electron donor,
cytochrome c, at the outside of the membrane, to the 02 reduction center.
The four protons required for the formation of H20 are taken up to the
binuclear center from the inside of the membrane. In addition, four protons
are pumped across the membrane per 02 reduced. The enzyme therefore
must contain conduction paths for proton uptake into the binuclear site, as
well as for translocation.
Proton transfer in proteins can occur through chains of hydrogen
bonds formed by amino acid residues together with water molecules. One
possible path for proton input in the cytochrome c oxidases is the so called
K-channel (for the highly conserved lysine residue, K354 in
P.denitrificans), which is made up of residues in helices VI and VIII of
subunit I. The K-channel starts from Ser 291, which is in contact with
external H20 moleculcs, and continues via Lys 354 and Thr 355 to Tyr
280, which may donate protons to oxygen at the binuclear center. These
amino acids could be connected to each other by hydrogen bonds directly,
or via solvent molecules. Of interest is also Gly 296, whose backbone
C=O is located 4 ~. away from the hydroxyl group of Ser 291. It has been
proposed that the K-channel is required for initial reduction of the
binuclear center in the first half of the O~_reduction cycle. It has also been
suggested that the lysine side chain is flexible and might move or shuttle
protons from the serine to the threonine.
We have mutated these residues to perturb the possible proton
conducting pathway, and to fix the position of Lys 354 in order to prevent
its movement. The effects of these mutations on the mechanism by which
the heine-copper oxidases translocate protons will be discussed.

(We/12.2/265)

Study of the roles of protonable groups involved in the

reactions catalysed by H-transhydrogenase.
T. Bizouarn, O. Fjellstr6m, X. Hu, J. Rydstr6m
Department of Btochemtstry and Biophysws, GOteborg Umversitv and
Chalmers Umverstty of Technology, GOteborg. Sweden

Transhydrogenase couples the stereospecific and reversible transfer

of hydride equivalents from NADH to NADP* to proton translocation
across the inner membrane m mitochondrm and cytoplasmic membrane
of bacteria. The enzyme is constituted in three domains, two hydropbihc
domains harboring each a binding site for the nucleotidc NAD(H) or
NADP(H) and an hydrophobic domain spanning the membrane. In order
to obtain information on the couphng mechanism between the transport
of protons and the chemical reaction, studies on the pH dependence of
the various transbydrogenat~on reactions catalysed by the intact enzyme
and the asolated nucleotide binding domains have been performed. The
effect of the substitution of the NAD(H) binding domain of the E coil
enzyme by the corresponding domain from R. rubrum on the pH
dependence of the reverse and cyclic as well as the effect of mutations of
the residue 13His91 will be described.
From these results, the existence of two groups, one possibly located
in the NAD(H) binding domain regulating the hydride transfer step [1 ]~
the other ~His91 located in the transmembraneous domain regulating the
binding and release of NADP(H) [2], has been proposed. This work was
supported by the Swedish Natural Science Research Council
Relevant references:
[1] O. Fjellstr6m et al., Biochemistry, 38,415-422.1999.
[2] X. Hu et al., Biochemistry, 38, 1652-1658, 1999.

Abstracts FEB S' 99

(We/12.2/266)

s345

Cyanide complex of the fully reduced cytochrome bd

V.B. Borisov', J.P. Osborneb, S.A. SiletsloyL ILB Gennisb,
A.A. Konstantinov ~

(We/12.2/267)

G Btasseur~,J-P dlRa$ob, P Tron',C ntuel',PP StommsklbaladD Lemesle-Meumer~

*Labor~otee de Biodne tJ e el Ingdmerte des Protimes, C,V~, 31 ch, J Aigmer, 13402
Marseille cedex 20, ~ ' ~ Y ~
bCentre de Gdndtlque~loldculatre, CVRS, 91198 Gtf-sur-Yvette Cede.~ FI~4,VCE

"A N.BelozerskN/nstitute o f Ph.vsico-('hemlcal Biology, A/Iosco~ State

(.mversl~', 119899 Aloscow. Russia,
bDepartment ofBtochemastt 3. Unlver.sTty ofllhnozs at Urbana, 1L, L ~ t

Cytochrome bd (cytbd) is one of the two membrane quinol oxidases of E.coh

respiratory chain. The enzyme consists of two subunits carrying three redox
centers: a low-spin b-558, a high-spin b-595 and a high-spin heine d. According
to Mitchell, et al. [1], cytbd differs from the heine/copper oxidases in that it
does not bind cyanide in the reduced state. In contrast, evidence for KCN
reaction with the fully reduced cytbd (red-cytbd) was reported in [2]. In this
work we show that KCN reacts with red-cytbd from E.coh under anaerobic
conditions briuging about absorption changes indicative of ligand binding to
heine d. The KCN-induced difference spectrum of cytbd shows in the Soret a
W-shaped trough with minima at 425 and 443 tun very similar to that observed
in case of CO and NO binding to heme d. The spectral changes in the visible are
however quite different from the effects of NO and CO and reveal a peak at
590-610 am and a minimum at about 633 am. KCN-induced absorption changes
titrate with apparent I% of 50-100 mM and are reversed upon dilution of the
ligand-pretreated enzyme. The kinetics of the spectral changes is biphasic. There
is an unresolved fast phase (15 - 20%) and a major slow transition (80 - 85%).
The contribution ratio of the two phases does not change significantly with
[KCN]. The rate of the slow phase depends linearly on [KCN] in the range 5 100 mM giving a second order rate constant (1%,) ofO.05 - 0.1 M-~ff~. When
extrapolated to [KCN] = 0, the rate dependence intersects the ordinate axis at
L,e = 0.003 s"1. This is close to 1%a-=0.0025 s"~ as calculated from the measured
values of 1% - 50 mM aud I%, : 0.05 Mls -~. Different spectra of KCN and CO
complexes of red-c.vtbd in the visible allow to look at the competition between
the two ligauds CO added to KCN complex apparently extrudes cyanide and
CO adduct of the enz3ane is formed with 80-90% yield. However. much more
CO is required to saturate formation of the CO complex in the presence of 100
mM KCN as compared with CO binding with red-cytbd. We conclude that the
two ligands compete with each other for binding with heme d. The reduced
cyanide compound of cytbd does not show photodissociation in the
microsecond scale.

(We/12,2/268)

Evolution and adaptation of bc complexes: the example

of the green thermophilie bacterium Chloroflexus
M. Brugna, W. Nitschke
BIP, I B . S . . ~ - C , V R . S , 31 che mm ,1. Atguier, 13402 A lars~ille cedex

20. FRANCE

M.tatio. P197T in the Rieske FeS protein

suppresses the decoupling effect due to the cyt. b mutation GI37E in
the yeast be t complex.

The mitochondrial bc~ complex catalyses electron transfer between

quinol and cytocbrome c coupled to a proton translocation across the inner
membrane
The cyt b respiratory deficient (Gly') mutant GI37E is impaired in
the establishment of a membrane potential albeit maintaining a good
electron transfer activity. The defect is due to a partial decoupling of the
bc~ complex (Bruel et al. 1995). This strain is resistant to the Qp site
inhibitors myxothiazol and stigmatellin. An extragenic suppressor was
obtained by chemical mutagenesis of a plasmid containing the gene
encoding for the Rieske protein. The selected suppressor mutation P 197T
is localized in the conserved Pro loop in the Rieske protein. The mutant
cytb:G137E + FeS:PI97T is a respiratory competent strain (Gly +) albeit
its electron transfer activities are not higher than the corresponding
respiratory deficient mutant cyt b:G137E (18% bc~ complex activity
versus 23% for b:G137E). Thus the suppressor mutation must regain a
better coupling between electron transfer and proton translocation at the
Qp site of the bcl complex. Replacement of an apolar proline by a polar
threonine may account for the better proton conduction at center P. To
study the effects of the single mutation, the strain bearing only the
suppression FeS:PI97T was constructed and behaves like the wild type
strain. Moreover, this strain is not resistant to inhibitors showing that the
inhibitor resistance phenotype is only due to mutations in cyt b position
137 and not to the FeS position 197.
In the bct complex three-dimensional structure, cyt b:G137 and
FeS:PI97 are distant by more than 20A. Assuming that the Rieske FeS
protein can adopt different conformation during the bc~ complex turnover, it is conceivable that the recovery of proton translocation in the
suppressor is hnked to the Rieske FeS protein motion between a position
close to cyt b and a position close to cyt ct.
Bruel, C , Manon, S., Guerm, M
Biomembr 27, 527-539

(We/12.2/269)

and Lernesle-Meunier, D

(1995) J. Bioenerg

Role of cooperative coupling (redox Bohr effects)

at heme a and Cue in protonmotive cytochrome c oxidase
N. Capitanio, G. Capitanio, E. De Nitto and S. Papa
Institute of Medical Biochemistryand Chemistry, Universityof Bari,
Bail, Italy

bc complexes play a central and essential role in photosynthetic

and respiratory electron transfer chains, both in eukaryotes and in
prokaryotes In general, bc complexes contain 3 proteins involved in redox
reactions: a monohemic cytochrome c, a dihemic cytochrome b and a
protein containing a [2Fe-2S]-cluster of high redox potential called the
Rieske protein. Recent studies, however, have shown a considerable
diversity of structural and functional properties l1 ] of bc complexes spread
over the entire phylogenetic tree of the Bacteria [2].
Since the green filamentous thermophilic bacterium Chloroflexus
aurantiacus is situated at a strategical position in the phylogenetic tree of
the Bacteria (close of the root of the tree), we have undertaken the study of
the bc complex of this organism.
We developped a purification protocol to obtain fractions enriched
in bc complex. Staining of SDS/PAGE with TMBZ allow us to estimate
the molecular weight of cytochrome b (27 kDa) Moreover, a cytochrome
c of 23 kDa, which is partially copurified with the b-type cytochrome, is a
good candidate for the "cytochrome c subunit" of the complex.
We have furthermore characterised the complex. The redox
potentials of the two heroes b were determined in membranes (-240 and 60mV for bE and bH respectively) and in isolated complex (-100 and
+25mV) and are characteristic of b-heroes from bc complexes. The
orientation of the g-tensor of the [2Fe-2S]-cluster of the Rieske protein has
been studied by EPR : gy is parallel to the membrane plane as is the case
for all the Rieske proteins studied so far By contrast, gx is oriented at
about 45 to the membrane plane, which is a novel feature for Rieske
proteins.

Oxido-reductions of metal centers in cytoehrome c oxidase are cooperatively

linked to pK shifts of acidic groups in the enzyme (redox-Bohr effects).
Analysis in our laboratory of the pH dependence of I~ transfer associated
with redox transitions of the metal centers in isolated cytoehrome c oxidase
from bovine heart mitochondria allowed us to characterize the Bohr effects in
this enzyme [1]. The experimental H+/COX linkage ratios measured, in the
pH range 6-9, in the reduced/oxidized/reduced transitions of the soluble
oxidase, could be best-fitted with a minimum of four protolytic groups, each
undergoing pK increase in the oxidized/reduced transition. The four pK shifts
were attributed to the individual metal centers from their correspondence
with those obtained by best-fit analysis of H+/COX ratios measured in the
CN-liganded oxidase (heme a3 blocked in the oxidized state) and in the COliganded oxidase (heme a~ and Cue blocked in the reduced state). The
assignment of pK shiRs of the four protolytic groups to individual metal
centers does not exclude the possibility that the pK of a single group can also
be influenced by cooperative interaction of different metals. The membrane
vectoriality of the redox Bohr effects was then analysed by measurement of
proton transfer associated with oxido-reduction of the metal centers in the
purified bovine heart cytoehrome c oxidase, in the unliganded and COliganded state, incorporated in liposomes [2]. The results showed that the
proton transfer resulting from the redox Bohr effects linked to heine a and
CuB in the bovine heart eytochrome c oxidase display membrane vectorial
asymmetry, i.e., protons are taken up from the inner N aqueous space, upon
reduction, and released in the external P space, upon oxidation of the metals.
A model for the involvement of the cooperative linkage at heme a and Cue in
the proton pump in cytochrome c oxidase is presented [3].

[1] Schoepp et al.,Adv. Inorg. Chem., 47 (1999) in press.

[2] Olsen et al., J. Bact., 176, (1994) 1.

ll] Capitanio, N. et al., Biochim. Biophys. Acta, 1318, (1997) 255.

[2] Papa, S. et al., Biochimie, 80, (1998) 821.
[3] Papa, S. et al., FEBS Lett., 438, (1998) 1.

s346

Abstracts FEBS'99

Crystal structure of light-regulated pea

fructose-l,6-bisphosphatase

(We/12.2/270)

(We/12.2/271)

Ammoniums are transported by the colonic H,K-ATPase.

M. Cougnon 1, F. Jaisser;~, A. Edelman 1,
T. Anagnostopoulos, l and G. Planelles I

M.Chiadmi a'b, A. Navazza c, J.P. Jacquot a, J. Cherfils a

a

I[NSER3][ U 467 and 2U 478. Par~s, France

LEBS, 91198 Gif, France. Crtstallograp?+e et RMN, 75006 Paris,

France, CLP, Chaten~-Malabry France, IBP 91405 Orsay, France

Fructose-l,6-bisphosphatase (FBPase) catalyzes the hydrolysis of

fructose-l,6-bisphosphate to fructose-6-phosphate with liberation of
inorganic phosphate. In plants, the cytosolic FBPase has the same
characteristics than its mammalian counterpart, its activation being also
AMP and fructose-2,6-bisphosphate dependent. Whereas cytosolic FBPase
in plants is involved in the synthesis of sucrose, the chloroplast isozyme is
necessary for the Calvin cycle of photosynthetic carbon dioxide fixation, it is
light activated and inactive in the dark. The activation reaction involves the
reduction of a critical disulfide bond in the enzyme, via a light activated
ferredoxin-thioredoxin system. This disulfide bridge is likely to belong to an
insert specific to chloroplast FBPase which is rich in cysteins. The aim of
this crystallographic study is to locate the disulfide bridge involved in the
FBPase regulation, to solve the structure of the insert, to determine the
quaternary form of the active reduced and the inactive oxidized enzyme.
The results achieved from the crystal structure of the recombinant pea
chloroplast FBPase will be presented.

Various K-ATPases, including a P-ATPase cloned from rat distal

colon [1], are expressed along the renal collecting duct. We have
previously shown [2, 3] that, when expressed in Xenopus laevis
oocytes, this colonic (c) P-ATPase encodes for a ouabain-sensitive
H,K-ATPase which may extrude Na + from the cell, thus presenting
common features with the Na,K-ATPase. Since this latter is known
to transport NH4 +, we investigated whether the cH,K-ATPase
shares this property. To this purpose, we measured the effect of
NH4CI on 86Rb uptake, on intracellular pH, pHi, and on intracellular
sodium activity, Na~, in X. oocytes expressing either a 13 subunit
alone (control oocytes) or the cH,K-ATPase.
The following results were obtained. 1) Adding I to l0 mM NH4CI
significantly decreased 86Rb uptake in a dose-dependent fashion in
cH,K-ATPase-expressing oocytes, but not in control oocytes. 2)
Adding 10 mM NH4CI to the bath induced a faster phi decrease in
cH,K-ATPase-expressing oocytes, (dpHi/dt = 0.1+ 0.01 UpH/min,
n=9) than in control oocytes (dpHi/dt = 0.06 + 0.01 UpH/min,
p<0.001); adding 2 mM ouabain reduced dpHi/dt to 0.05 + 0.01
UpH/min in cH,K-ATPase-expressing oocytes. 3) Exposing cH,KATPase-expressing oocytes to another amine (TMACI 10 mM) had
no effect on 86Rb uptake nor on dpH~/dt, although it depolarized the
oocyte membrane as did NHaCt, 10 mM. 4) In a K+-free and
NH4Cl-supplemented solution, Nai was lower in cH,K-ATPaseexpressing oocytes than in control oocytes. 5) cH,K-ATPaseexpressing oocytes induced extracellular acidification of K+-free
medium in the presence of NH4CI. 6) Kinetic analysis revealed
competitive inhibition of NH4 + at a K + site (apparent Kt 6.3 _+0.8
raM).
We conclude that, when expressed in X. oocytes, the colonic H,KATPase mediates NH4 influx. Due to the localization of this pump
in tubular segments involved in NH4 + excretion, NH4 + transport by
the cH,K-ATPase may be of physiological relevance.
[1] Crowson et al., J. Biol. Chem., 267, (1992) 13740.
[2] Cougnon et al., J. Biol. Chem., 271, (t996) 7277.
[3] Cougnon et at., Proc. Natl. Acad. Sci. USA, 95, (1998) 6516.
+

(We/12.2/272)

Sarco/Endoplasmie Reticnlum Ca2+ATPase 3 (SERCA3)

Multiple, diverse and species specific isoforms.
J. Enouf, V. Martin, R. Bredoux, R. Bobe and B. Papp
IZ 348 INSEPJvI. IFR 6, 8, Rue Guy Patm, 75475 Paris Cedex 10, France

Recent advances in understanding of Ca2+ signalhng pointed for distinct

roles of Sarco/Endoplasmic Ca2+ transport ATPase (SERCAs) isoforms.
Recently, we and others described two novel SERCA3 isoforms (b and c) in
addition to SERCA3a. Here we show this identification of SERCA3b and
SERCA3c m human platelets and report on a novel SERCA3 splice variant
in rat.
RT-PCR amplification of human platelet T-end SERCA3 RNA disclosed, in
addition to SERCA3a, new SERCA3 species. Based on data in the litterature
indicating two mouse SERCA3a and SERCA3b cDNAs and on the analysis
of the Y-end part of human SERCA3 gene, we identified a new 101 bp exon
(21), located in the intronic sequence linking the two last exons used in
SERCA3a (now termed 20 and 22). The partial or complete alternative
insertion of this exon gives rise to two human SERCA3b and SERCA3c
species respectively. In rat platelets, instead of SERCA3b and 3c, a new
variant was identified by 3'-end RACE RT-PCR and by analysis ofT-end rat
SERCA3 gene. In this form, part of the Y-end ofexon 21 is excluded and
the T-end is extended. It is due to the use of a second polyadenylation signal
located in the last intron and gives rise to a 4-kb mRNA species.
Taken together, these results suggest that the 3'-end of the SERCA3 gene is
organised as follows.

[laumanSERCA3

......... a ' " ~ . ,

sc sa Sb

122

pA ]
Ip_~

The existence of multiple SERCA 3 gene products may correspond to the

functional specialization of the calcium homeostasis of subcompartments of
the endoplasmic reticulum of non-muscle cells.

(We/12.2/273)

Over-expression of the calcium-transporting ATPase

SERCA1 in yeast.
F. Corre, T. Menguy, L. Bouneau, P.A. Pedersen, D.
Thynes, M. le Maire & P. Falson*.

The Ca~+-ATPase is a large membrane protein of 100 kDa

belonging to the family of P-types ATPases whose actively transport
cations across biological membranes. The gene encoding the Ca2"-ATPase
of rabbit sarcoplasmic reticulum, SERCA1, was previously cloned in a
yeast expression plasmid, pYeDPI/8-10 [1] in which it was under control
of the galactose-inducible gall0 promoter. Yeasts containing the plasmid
could grow in minimal medium to a maximal density of 4"109 yeasts/L
and produced after induction 0,9 mg of Ca2"-ATPase corresponding to 0.3
% of the proteins [2]. We have now improved the production of Ca~+ATPase by modifying several parameters.
We have used a new type of yeast expression plasmid,
essentially differing from the previous one by the fact that the transformed
yeasts can grow in rich medium [3]. After one galactose induction, the
yeasts reached a density of 1"10" yeasts/L and produced 10 mg of Ca 2+ATPase, corresponding to 0.6 % of the proteins. The number of galactose
inductions was optimized allowing a 1.7 fold increase of expression that
was also stable for several hours. The host yeast was genetically modified
by integration of the gene encoding the gal4 protein also under the control
of the gall0 promoter. This also gave a 1.7 fold increase in the expression.
These modifications allowed the production of 30-40 mg Ca2+-ATPase/L
but we found that when yeasts were cultivated at their optimal growth
temperature of 28C, only a small fraction of the expressed Ca2+-ATPase
with a mild detergent. Lowering the temperature to 18C reduced the
amount of expressed protein to 20 mg/L but increased to 2 mg Ca2+ATPase/L the amount of soluble and active material. This result will be
compared with the yield obtained for this protein with other expression
systems and, more generally, with the yields obtained for other eucaryotic
multi-span membranous proteins.
[1] Pompon, D. (1988). Eur. J. Biochem, 177, 285-293.
[2] Centeno, F. et al. (1994). FEBS let., 354, 117-122.
[3] Pompon, D. etal. (1996) Methods in Enzymol., 272, 51-64.
(*) pierre.falson@.cea.fr, CEA, CE Saclay, DSV/DBCM/SBPM, URA
CNRS 2096, Bt 528, 91191 Gif Sur Yvette Cedex France

Abstracts FEBS'99

(We/12.2/274)

REDOX DRIVEN CHANGES IN AXIAL COORDINATION

OF PHE82HIS ISO-1-CYTOCHROME C
B.A. Feinberg,
X. Liu, M.D. Ryan, A. Schejter, C. Zhang, E. Margoliash
Dept. Chem., University of Wisconsin-Milwaukee. MiL WI, USA 532l I

Heme proteins and enzymes play key roles in biological electron transfer
reactions, e.g., Williams et al. found that for cd~ nitrite reductase, which
catalyzes the reduction of nitrite to nitric oxide, the 6 ~" ligand interchanges
between histidine and methionine during its catalytic cycle. In our work,
we have examined the Phe82His/Cys102Ser variant of iso-1-cytochrome c.
For wild type iso-l-cytochrome c, the normal 5 ~hand 6'h ligands in both
redox states are histidine and methionine. Direct square-wave and cyclic
voltammetric electrochemical examination of the Phe82His/Cys102Ser
variant revealed the intricacies of redox driven changes in axial
coordination, concomitant with intramolecular rearrangement. Cyclic
voltammetry (CV) and square-wave voltammetry (SWV) are ideally suited
for such a redox study, since they provide a direct and quantitative
visualization of specific dynamic events. SWV showed that the primary
species in the reduced state is the Mets0-FeZ-Hisls coordination form, while
in oxidized state the Hiss2-Fe3+-Hists form predominates. Thus the addition
or removal of an electron to the appropriate form of this variant serves as a
switch to a new molecular form of the cytochrome. Using the 2X2
electrochemical mechanism, simulations were done for the CV experiments
at different scan rates. These, in turn, provided relative rate constants for
the intramolecular rearrangement/ligand exchange and the equilibrium
redox potentials of the participating coordination forms: E ' = 247 mV for
Mets0-Fe3+/Z+-Hisls couple, E ' =47 mV for His82-Fe3+/Z+-Hisls couple, and
E ' = 176 mV for the cross reaction couple: Hissz-Fe3-Hiszs + e
Mets0-Fe2+-His~s. The entropy of reaction, AS~,, was determined for the
net reduction/rearrangement: Hissz-Fe3+-His~s + e- "~ Mets0-Fe2+-Hisas
and compared to that for wild type cytochrome: Mets0-Fe3+-His~ + e- +
Mets0-Fe2+-His~8. For the Phe82His variant mixed redox couple, AS~o =
-80 J/mol.K compared to AS~, = -53 J/mole.K for the wild type cyt c
couple without rearrangement. Comparison of these entropies indicates that
the oxidized Hiss2-Fe3+-His~s form, is highly disordered, and this may
facilitate its rapid rearrangement to Metso-Fe2+-His~8 upon reduction.
[1] B.A. Feinberg, et al., Biochemistry 37 (1998) 13091.

(We/12.2/276)

CHARACTERISATION OF THE MOUSE GENE

CODING FOR A TRANSHYDROGENASE
E. Lagberg Arkblad, J.Rydstr6m,
Department of B~ochemistcT and Biophystcs, Chalmers Umverstty of
Technology, Goteborg University, Goteborg. Sweden

Nicotinamide nucleotide transhydrogenase (EC 1.6.1.1) is a membranebound proton pump which catalyses the reversible reduction of NADP + by
NADH linked to proton translocation across the membrane.The
physiological role of the enzyme still remains unclear. The chromosomal
localisation of the transhydrogenase gene has been determined by
fluorescence in situ hybridisation in mouse, rat and human using mouse and
human cDNA as probes [1]. The positions were mouse chromosome
(MMU) 13D2, rat chromosome (RNO) 2q 16 and human chromosome
(HSA) 5p 13.1, respectively, suggesting that the transhydrogenase gene is
included in a group of genes that exhibits conserved synteny between
MMU 13, RNO2 and HSA5 [2]. Cloning of the genomic gene and restriction
enzyme mapping and sequencing of the exon/intron borders show that the
gene is more than 100 kb large and consist of_>21 exons. The cloned part of
the gene cover the area from eight amino acids into the mature protein until
the 3' untranslated region.

Arkblad, L. E. et al. Biochlm. Biophys. Acta. 1273 (1996), 203.

Arkblad, L. E. et al. Mammalian genome 8 (1997), 703.

s347

(We/12.2/275)

Expression of biqtinylated sarcoplasmic reticulum

Ca'* ATPase in yeast.
C. Jaxel, J. M. Fuentes, P. Falson, T. Menguy and
M. le Maire.
DSV,DBCM,SBPM, CEA & CNRS URA 2096, 91191 Gif/Yvette. F r a n c e

Sarcoplasmic reticulum (SR) Ca 2+ ATPase from skeletal

muscle is a membrane protein that actively transports Ca 2 within the
sarcoplasmic reticulum as a result of ATP hydrolysis. In order to purify
this protein, we have used an approach based on the construction of
chimeras between the Ca 2 ATPase and a 100 amino acid residue
polypeptide containing the biotin accepter domain from the oxaloacetate
decarboxylase of KlebsieUae pneumoniae [ 1]. Chimeras were constructed
with a thrombin proteasc site between the Ca 2 ATPase and the biotin
accepter domain. Biotinylated Ca 2 ATPase may be then purified in one
step by affinity chromatography on monomeric avidin-Sepharese.
Conditions for expression of Ca 2+ ATPase in yeast have
been previously described [2, see also the abstract of Corre et al. in tilts
meeting]. The biotin aceeptor domain corresponding DNA was inserted
at the C-terminus of the Ca 2 ATPase by using the overlap-extension PCR
method. The obtained construction was verified by DNA sequencing. For
the Ca 2. ATPase - biotin accepter domain chimeras, the level of
expression is similar to that of the wild type protein expressed in
Saccharomyces cerevisiae. The Ca 2 ATPase is biotinylated in rive and is
present in the microsomal fraction. No other biotinylated protein from
yeast is present in a significant amount in this microsomal fraction.
Finally, the ATPase enzymatic activity of biotinylated Ca 2 ATPase is
similar to that of the wild type protein in identical conditions.
[1] Consler et al., Prec. Natl. Acad. Sci. USA, 90, (1993), 6934.
{2] Falson et al., J. Biol. Chem., 272, (1997), 17258.
E-mail: jaxel@dsvidf.cea.fr

(We/12.2/277)

Loop L6-7 is involved in a high affinity Ca2+-binding site

fonctionally essential for rabbit SERCA1 Ca2+-ATPase.
T. Menguy, F. Corre, P. Champeil, M. le Maire & P. Falson.
DSV, DBCM, SBPM, CEA & CNRS LIRA2096, 91191 Gif/Yvctte.

Limited proteolysis of the rabbit SERCA 1 Ca/-ATPase by proteinase K

generates a number of fragments which have been identified previously. A Cterminal peptide of 20 kDa, p21, starting at G808 after transmembrane
segment M6, i.e in the L6-7 loop, binds Ca 2+ as deduced from (i) its change in
migration rate when running in SDS-PAGE in the presence of Ca 2, (ii) its
labeling by 45Ca2 overlay experiments. In contrast, p19, a proteolysis
fragment identical to p21 but for 10 amino acids missing at the N-terminal
side, i.e starting at D818, does not bind Ca 2.
Two cluster mutants in the L6-7 loop, D813A-D818A and D813A-D815AD818A, have been expressed in yeast. Both mutated ATPases have less than
10 % Ca 2+ dependent-ATPase activity. They are also unable to become
phosphorylated from ATP up to 1 mM Ca 2. They are phosphorylated by

inorganic phosphate even in the presence of moderate concentrauons

of Ca 2+
(K0.5 in the millimolar range). The reduction in Ca z affinity for
phosphorylation from Pi is observed both at acidic and at neutral pH,
suggesting that these mutations interfere with binding of the first calcium to
bind as proposed for some of the intramembranous residues essential for
Ca2-binding.
The CaZ+-dependence of the pattern of Ca2+-ATPase proteolysis by proteinase
K was used to monitor the conformational changes induced by Ca 2+ binding
to the ATPase. In the wild-type ATPase, this change was observed at Ca 2+
concentrations in the micrornolar range, while it occurred only at Ca 2+
concentrations in the millimolar range with the cluster mutant D813AD818A.
This again suggests that the residues of the loop L6-7 control ATPase
activation by Ca 2 and that aspartic residues in the L6-7 loop interact with
Ca 2 during the initial steps of Ca z binding.
Falson et al, J. Biol. Chem., 272, (1997), 17258.
Menguy et al, J. Biol. Chem., 273, (1998), 20134.
E-mail : menguy@dsvidf.cea.fr

s348

(We/12.2/278)

Abstracts FEBS'99

FTIR study of the interactions of the heine propionates

with their surroundings in heme-copper oxidases
Anne Puustinen and M~rten Wikstr6m

(We/12.2/279)

Department of Medical Chemistry, Universtty of Helsinki, Finland

The heine-copper oxidases catalyse proton translocation that is linked to

the reduction of 02 to water in the binuclear heine iron-copper (Cua)
center of the enzyme. Properties of this binuclear active site may be
studied by examining the stable CO bound adducts of the enzyme by
Fourier transform infrared (FTIR)-difference spectroscopy. Some
structural characteristics of the heme pocket are known for the fully
reduced CO-bound heine-copper oxidases. Photolysis of CO bound to the
reduced heme iron leads to fast transfer of CO to the nearby Cua in the
bmuclear center. At low temperatures, CO remains bound to the copper,
and difference FTIR spectra of this photolysis event show a through near
1960 cm -1 due to the loss of CO from the heme and a peak around 2065
cm -~ for the CO stretch of CuB-CO. This low-temperature FFIR procedure
has been used to probe the effects of site-directed mutations on the
binuclear site. For example there is a carboxylic acid IR stretch, which we
were able to assign to the carboxylic group of Glu286 residue [1]. This
result suggest that there is connectivity between Glu286 and the binuclear
center.
The X-ray structures of cytochrome c oxidases show that the well
conserved Arg473 and Arg474 interact with the propionate groups of
haems a and a3 and several water molecules may connect this domain to
the outside of the membrane, perhaps providing an exit pathway for
pumped protons. Mutations of these arginines revealed that the Dpropionate of the oxygen-binding berne is involved proton translocation
[2]. Further analysis of these mutations is focused on the identification and
assignment of the IR modes of the heine propionates and arginine side
chains. Implications of these results for the mechanism of proton
translocation are discussed.

The Ca2+ binding site in cytochrome aa 3 from P. denitrificans

S. Riistama, L. Laakkonen, M. Wikstr6m, M. Verkhovsky and A.
Puustinen
Department o f Medical Chemist~, University of He/sinki, Finland

Cytochrome c oxidases of mitochondrial and bacterial origin catalyse the

reduction of 0 2 to water at the bimetallic haem iron (a3) - copper (CUB)
center in subunit I. This reaction is coupled to proton translocation across
the mitochondrial or bacterial membrane. Two other prosthetic groups, Cu A
and haem a, are needed for electron transfer from cytochrome c to 02.
A shift in the spectrum of haem a, induced by calcium or proton binding has
been attributed to interaction of Ca 2 or H + with the vicinity of the haem
propionates in mitochondrial cytochrome c oxidase, and proposed to be
associated with the exit path of proton translocation [1]. This is not observed
in highly homologous oxidases from bacteria or yeast. A new metal-binding
site was recently reported in the crystal structures of cytochrome c oxidase
from both Paracoccus denitrificans [2] and bovine heart mitochondria [3].
Site-directed mutagenesis of the cytochrome aa 3 of Paracoccus
denitrificans has been used to study the role of the new metal binding site in
the Ca2+-induced spectral shift seen in the mitochondrial enzyme. Results
indicate that the shift is due to binding of the cation to this site. Comparison
of the structures of this site in the two types of enzyme allow rationalization
of their different reactivity with cations.
[1] H. Saari et al., J. Bioenerg. Biomembr., 12, (1980), 325
[2] C. Ostermeier et al., Proc. Natl. Acad. Sci.USA, 94, (1997), 10547
[31 S. Yoshikawa et al., Science, 280, (1998), 1723

[1] Puustinen et al., Biochem., 36, (1997), 13195.

[2] Puustinen & Wiktr6m, Proc.Natl.Acad.Sci.USA, 96 (1999), 35.

(We/12.2/280)

ATP synthesis on disk membranes isolated from rod outer

segments of bovine retina.
I. M. Pepe ~, I. Panfoli2 , L. Notari 2 and Alessandro Morelli2.
lstituto dt Biofisica, Facolta ~di Medicina and 2 lstituto di Chimica
Biolojzica, Umversita' dt Genova, 16132 Genova, ltaly

Disk membranes of rod outer segment are known to store calcium at millimolar
levels making a steep gradient with the cytoplasm where Ca 2 has a
concentration of about .5 IxM. A Ca 2. -ATPase was first localized by
cytochemical studies on ROS disk membranes of the toad retina [1] and then
isolated on polyacrilamide gel from bovine ROS disks [2] and successively
characterized as a Sarco- or Endo-plasmic Reticulum Calcium ATPase
(SERCA) type with a 100 kDa phosphorylated intermediate [3]. This Ca 2+pump has a k m for Ca 2+ = I0 laM [2] and a negligible activity at Ca 2+
concentrations between . 1 ~tM and 1 p_M, which means that, at physiological
Ca2*concentrations, the pump does not work to uptake calcium ions into the
disks. Although the actual reversibility of an ion - ATPase has rarely been
demonstrated in eucariotic cells, our results suggest that the Ca ~+ -ATPase of
the disks is able to reverse its function by acting as a synthesizer of ATP at the
expense of the Ca 2+ gradient. In fact, Ficolt purified disks from rod outer
segment, in the presence of ADP and phosphate, released calcium and
synthesized ATP at the physiological range of Ca 2+ concentrations present in
rod cytoplasm. The ATP synthesis had a k~ for Ca 2. _= 10 laM and was inhibited
by vanadate, a strong inhibitor of phosphorylated ATPases. These results are
important in terms of the need of an immediate source of AtTP on the disk
membranes where the energy is requested to supply the rapid*reactions of the
photoreception processes.
1. Davis, W.L.et al. Anat.Rec. 221 (3), 1988, 761.
2. Panfoli, I. et al. J. Photochem. Photobiol. 24, 1994, 187.
3. Panfoli, I. et al. Biochem Biophys. Res. Commun. 204(2), 1994, 813.
This work was supported by the PRIN project "Bioenergetica e Trasporto di Membrana" of
the Italian MURST.

(We/12.2/281)

Structural studies of Bacteriorhodopsin: from the

Ground-State towards Intermediate States
A. Royant a, H. Belrh alia
European Synchrotron Radiation Facility, 38043 Grenoble, France

Bacteriorhodopsin (bR) is a light-driven proton pump found in the purple

membrane of Halobacterium salinarum which has become a model among
membrane proteins. The mechanism of the proton translocation process has
been investigated extensively over the last two decades. However, a detailed
atomic understanding of the entire process has not been reached yet.
Nevertheless, it is well established that the absorption of a photon by the
chromophore retinal, which is bound covalently through a Schiff base. causes
an isomerisation from a all-trans to a 13-cis conformation. Then a series of
intermediate states follows, showing both local and global structural changes of
the protein, resulting in the release of a proton to the extracellular side of the
membrane. In a subsequent step the intermittently deprotonated Schiff base
becomes reprotonated. The photocycle is finally completed by a backisomerisation of the retinal.
High-resolution time-resolved crystallography seems to be a promising method
to reveal, at once, numerous features of the structural changes occuring during
this photocycle, bR crystals grown from lipidic cubic phases have demonstrated
their diffraction power [1], [2], and they made the determination of the first Xray high-resolution structure of this membrane protein possible [2]. Moreover,
it has been shown that the protein in these crystals undergoes virtually the same
photocycle as in the purple membrane [3]. Therefore bR crystals in lipidic
cubic phases appear to be a suitable material to carry out crystallographic
experiments to elucidate bR intermediate state structures.
The high-resolution structure of the ground-state protein will be presented, as
well as the first attempts of intermediate state structures determination.
[1] Landau et al., Proc Natl Acad Sci U S A , 93(25), (1996) 14532
[2] Pebay-Peyroula et al., Science, 277, (.1997) 1676
[3] Heberle et al., 3 Mol Biol, 281(4), (1998) 587

Abstracts FEBS'99

(We/12.2/282)

Interaction ATPsynthase-tentoxin : towards a structural

understanding of the inhibition and reactivation processes.
J. Santolini*', J.-M. Gomis2, F. Haraux t, C., Sigalat', F. Andr6
I CEA, Section de Biodnergdtique, 2 CF~, Service des Moldcules Marqudes.
D B.C~L, Centre d'Etudes de Saclay, 91191 G~f-sur-Yvette cedex, France

Tentoxin (TTX) is a cyclic tetrapeptide which induces chlorosis of many

plants. Tentoxin binds specifically to the soluble part of the chloroplastic ATPsynthase (CF0. The effect of the toxin on CFI is concentration-dependent:
potent inhibitor at low concentration and stimulatory above 10 MM. We
demonstrated that inhibition and over-activation effects are correlated to the
sequential binding of two molecules oftentoxin on CFt [1,2].
In order to identify the structural features responsible for each activity, we
synthesised numerous analogues. Their kinetic and thermodynamic binding
parameters have been determined for both sites (through activity profiles,
competition experiments and kinetic studies). Besides, their conformational
properties have been determined through 2D NMR experiments and molecular
dynamics simulations. The analysis of the binding characteristics of the
analogues revealed that the first TTX-binding site is a tight, hydrophobic site,
insensitive to the catalytic state of CFI, and stressed the importance of
particular moieties of the peptide in molecular recognition and inhibitory
ability [3]]. The comparison of the 3D-structures of TTX analogues to their
functional properties enlightened the correlation between the conformational
features of TTX and its activities. Besides, the reactivation process, which is
dependent on the catalytic state of CF~, seems to result fi,om a subtle and cooperative interaction between both TTX binding sites [2, 3].
In order to provide our data with a structural model, an analogue of TTX,
bearing a radioactive photo-affinity reagent, was synthesised. This derivative
proved to be a good photochemical reagent which specifically binds and
inhibits CFve. The subunit carrying the binding site has been determined, and
the identification of the peptides involved in this binding is in progress.
These results gave a new understanding of inhibition and reactivation of
CF~-e by TTX. A more extensive study of the structure-activity relationship of
tentoxin will be a support for investigating the regulation of chloroplast
ATPsynthase and the plant-species specificity of TTX toxic effect.
References
t. Pinet et al., Biochemistry,35, (1996), 12804
2. Santolini et al. J. Biol. Chem. (19993, on press
3 Santolinl et al, J. Biol. Chem. 273, (t99g), 3343
(We/12.2/284)

ATPase activity of cardiac human ventricular myosin at

dilated cardiomyopathy
L.Sidorik, D.Ryabenko*, V.Bobik, T.Kovenya, G.Matsuka
Institute of Molecular Biology and Genetics NAS of Ukraine, Ukraine
Institute of Cardiology, Kyiv, Ukraine

The study of molecular mechanisms of myocardium contractile

apparatus function is very important for understanding of mechanisms
of cardiovascular diseases development and progression. Idiopathic dilated cardiomyopathy (IDCM) is a chronic heart muscle disease of unknown origin characterized by left ventricular or biventricular cavities
extension and impaired myocardial contractility. For this time pathogenesis of IDCM remains unclear. Over recent years an important pathogenic
role of immune mechanism at the certain part of patients with IDCM becomes more and more obvious. Data from experimental and clinical
studies showed that cardiac myosin is a major autoantigen at IDCM. The
results of immunological examination showed the significantly increased
immunoreactivity (ELISA method) of myosin isolated from left ventricular (LV) myocardium of IDCM patients (myo-D), in comparison
with myosin, purified from myocardium of LV of practically healthy person who died from casual trauma (myo-N). Results also showed that
both myo-D and myo-N preparations had identical electrophoretical mobility (10 % PAAG in presence of SDS) and isoelectric points. The study
of ATPase activity of myosin preparations revealed that ATPase activity
of myo-D in conditions of a high ionic force (in presence of Ca 2, Mg 2,
K ) is considerably reduced (practically in four times) in comparison with
myo-N. Almost similar differences were revealed in condition of low
ionic force. Myo-D ATPase activity was significantly lower (almost
twice) compared to myo-N.
The obtained data indicate the presence of structural modifications, of
human cardiac myosin molecule. It is possible to assume, that alterations,
probably affecting active center of myosin molecule, may not only lead to
changes of protein immuno- and antigenicity but probably cause the decrease of contractility of myofilaments well known at IDCM.

s349

(We/12.2/283)

Resolution of the membrane domain of bovine

Complex I into subcomplexes:
implications for the structural organisation of the
enzyme.
L. A. Sazanov and J. E. Walker
MRC Laboratory of Molecular Biologyi Hills Road. Cambridge CB2 2QH.
U.K.

NADH:ubiquinone oxidoreductase (Complex I) was purified from bovine

heart mitochondria and subsequently treated with the detergent N,Ndimethyldodecylamine N-oxide (LDAO). In previous work it bas been
established that under similarconditions Complex I dissociates into two
subcomplexes termed Ict and 113 [1]. These subcomplexes contain mostly
hydrophilic and hydrophobic subunits, respectively, but several subunits of
Complex I, particularly hydrophobie subunits encoded in the mitochondrial
genome, were not found in either subcomplex. We have shown that most of
these subunits (ND1, ND3, ND4L and KFYI, as well as ND2) elute separately
from Ict and 113during anion-exchange chromatography, in a fraction referred to
as I~/. Size-exclusion chromatography of the I~, fraction and subcomplex 113
indicates that: a) at least ND1 and ND2 (from Iy) form a subcomplex, and that b)
113 dissociates further into two smaller subcomplexes, one of which contains
ND4 and ND5. These results strongly suggest that the NDI and ND2 subunits
are likely to be present in different regions of the membrane domain than the
ND4 and ND5 subunits. The implications of our findings for the structural
organisation of Complex 1 in the context of the known low-resolution structures
will be discussed.
1. Finel, M., Skehel, J.M., Albracht, S.P.J., Feamley, I.M. and Walker, J.E.
(1992) Biochemistry, 31, 11425-11434.

s350

Abstracts FEBS'99

15.2 Plant organ development

(We/15.2/286)

Expression of 3-hydroxy-3-methyiglutaryI-CoA synthase in

(We/15.2/287)

Brassica j u n c e a
D.Alex and M-L.Chye
Department of Botany. The Universzty of Hong Kong, Hang Kong

lsoprenoids form a wide range of compounds produced by plants that are

essential for normal growth, development and defense. Mevalonate, which is
the precursor to cytosolic-derived isoprenoids in higher plants, is synthesized
from acetyl-CoA and acetoacetyl-CoA by the action of
HMG-CoA
synthase(HMGS) and HMG-CoA reductase(HMGR). Unlike HMGR, HMGS
has not been well-characterized at the molecular level in plants. As a first step
towards understanding its regulation and expression, we have isolated a
cDNA encoding HMGS from Brassica juncea, a vegetable crop in Asia. B.
juncea HMGS shows 94.6% amino acid identity to Arabidopsis HMGS and
48.5% to human HMGS, with the active site being highly-conserved.
Genomic Southern blot analysis indicates the presence of a multigene family
encoding HMGS in B. juncea. We investigated the temporal and spatial
expression of H M G S during plant development using northern blot analysis.
Our results show that H M G S is developmentally-regulated in flowers, with
high expression early in floral development. High H M G S expression is also
observed during germination and transcripts can be detected as early as 6 hrs
after imbibition. In germinating seedlings, H M G S expression ns downregulated by abscisic acid and osmotic stress, the effects of which arrested
seedling growth. Rapidly elongating etiolated seedlings show increascd levels
of H M G S expression. These results suggest that H M G S expression is
triggered during active cell division and growth so as to supply cells with the
necessary isoprenoid precursors for plant development.

(We/15.2/288)

AGOI : a gene involved in Arabidopsis

developmental signaling?
I. Camus ~, H. I. McKhann', C. Benning ~ and C. Bellini'.
: INRA, Versailles. France
"*Dept. of Btochemtstt T, Michigan State Universio.. USA

As part of our strategy to identify genetic loci controlling cell

elongation in Arabidopsis, we visually examined mutagenized populations for
individuals with short hypocotyls. An allelic series of the mutant argonaute
was isolated. The agol mutants have pleiotropic defects in the plant
architecture. The shoot apical meristem generates narrow and succulent rosette
leaves, unbranched inflorescence stems bearing filamentous structures instead
of cauline leaves and abnormal infertile flowers.
The phenotype and the physiology of the mutant have been investigated in
more detail. Cross sections of the different agol-3 mutant organs showed
abnormal tissue organization. In addition, light and scanning electron
microscopy revealed defects in cell differentiation and cell wall structure.
Mutants also demonstrated elongation defects. Thus, m dark conditions or in
response to gibberellic acid in the light, the mutant hypocotyl did not elongate
as did the wild type.
Two independent T-DNA insertions into the AGO1 locus led to the isolation
of genomic sequences and a complete cDNA encoding a putative 115 KDa
protein with sequence similarity to a novel gene family present in wide range
of organisms, including humans [1 ]. However, no specific function has been
assigned to these genes yet.
In an attempt to link the molecular data on the AGO1 locus and the phenotype
of agol mutants, we examined the presence of the AGO1 transcript and
protein in different wild-type organs and in wild-type seedlings of different
ages. Northern and Western blot analysis showed that the AGO1 transcript
and protein are present in all organs investigated and throughout the
development.
Given the plelotropic nature of the mutation, we propose that AGO1 encodes
a protein that may be involved in the plant's response to and/or integration of
specific developmental signals. Currently, efforts are underway to elucidate
the function of this gene.
[1] Bohmen et al., EMBO J., Vol. 17, (1998) pp. 170

Expression of cafp, a CDC48 homologue, during cell

division in bell pepper fruits and BY2 cells
S. Akrim, G. Houln6, M.L. Schantz, R. Schantz
IBMP/CNRS-Universit( Louis Pasteur, 12, rue du G(n(ral Zimmer,
67084 Strasbourg Cedex, France

Bell pepper fruit development can be divided into three phases. The first
one corresponds to an active program of cell divisions, it is followed by a
phase of cell expansion until the fruit reaches its final size, allowing the
third phase, the ripening, to take place. In order to elucidate the regulation
mechanisms involved in gene expression during the early stages of fruit
development, we have set up a strategy for the isolation of cDNAs
specifically expressed at this stage.We have isolated a eDNA encoding
CAFP (Capsicum annuum fusion protein), a protein from the AAA family
(ATPases associated with diverse cellular activities) homologous to the
yeast Cdc4gp. A structural characteristic is the presence of the AAA family
is the presence of a conserved sequence (AAA module) of 220-250 residues
that includes 3 signature sequences, Walker A and B domains and SRH
(Seconde region of homology) also present in RNA/DNA helicases.
CAFP encodes a protein of 805 amino-acids, a calculated MW of 89,5 KDa
and a pI of 5,01. The protein contains 2 AAA modules highly conserved, 2
nuclear targetting signals in the N-terminal region and several putative
phosphorylation sites over the entire sequence. Southern blot analysis
suggests that the corresponding gene is unique in the bell pepper genome.
This result was confirmed by a PCR approach on genomic DNA allowing
to establish the structure of the gene. Analysis by northern blot reveals a
transcient accumulation of transcripts during the early development and
during ripening. However, the highest protein level, as estimated by
western blot is mainly found during the early stages of the fruit. These
results suggest different mechanisms of gene expression depending on the
fruit development stage. To further characterize CAFP and to test its
potential involvement in cell division, we have extended our study to an
experimental system less complex than the fruit : the tobacco BY2 cells.
In a first attempt we have isolated the cDNA (n~p), counterpart of cafp. The
cDNAs from the 2 species share 73% homology, ntfp gene expression is
also regulated in BY2 cultured cells : transcripts and proteins are
preferentially accumulated during the exponential phase of growth.
Immunolocalization studies suggest that the majority of proteins is present
within the nucleus consistent with the presence of putative nuclear target
signals. In addition, in order to determine more accuratly and in a dynamic
way the cellular localization of NTFP, we have developped an other
approach : the protein fused to a cellular marker, the GFP. After transient or
stable transformation of BY2 the fusion protein is detected in the cells and
results of the expression at different development stages will be discussed.

(We/15.2/289)

The influence of Rhi~blum nodules setting on the intensity

of chlorophyll fluorescence and field bean crop yield.
I Czyczy|o-Mysza, F Dubert
The Franciszek G6rski Departmentof PlantPhysiology,PolishAcademy
of Sciences,Podha~na3, 30-239 Krak6w, Poland

Ecological and economical considerations caused that still increased

significance of researches about ecological conditions of nitrogen fixation
efficiency in the Rhizobium leguminosarum and field bean plants.
The aim of the study was to determine the effect of setting and growth
of the root nodules o f Rhizobium on the intensity of chlorophyll fluorescence
(FC) and the crop yield of the field bean plants in hydroponic cultures. The
effectiveness of the photosynthetic apparatus may depend on the nitrogen
content in the plants nutrition. For this reason it has been tested whether
different nitrogen doses in the nutrient and the resulting different development
of the Rhizobium system, as well as the bulk of the biologically fixed nitrogen
affect the dynamics of changes of the measured FC parameters.
The investigations were conducted on self-completing field bean
variety Tibo. The plants were grown for a period of 11 weeks in aired
hydroponic cultures with Hoagland's nutrient solution, modified with respect
to nitrogen which was equal to 0, 25, 50, 100 (control), 200% of this element
in typical Hoagland's nutrient. The hydroponic solutions were inoculated by
rhizobial strain "P". Chlorophyll fluorescence parameters (Fv/Fm, h/2, Fv, Fro,
Fo) from dark-adapted leaves was measured using a Plant Stress Meter
(BioMonitor prod Sweden).
The nitrogen free nutrient stimulated the setting and growth of root
nodules to the greatest extent. The largest nodules were observed on a nutrient
with the highest nitrogen concentration With increasing concentration of
nitrogen in the nutrient nodules number diminished. The highest mass of
nodules was observed on plant with the biggest crop yield.The values of the
parameter Fv/Fm indicating the potential quantum yield PSII did not exhibit
great variation, similar as the remaining values of the fluorescence parameters
induction. An analyses of changes in the Fv/Fm values as depending on the
development period of the plants shows that the beginning of vegetation and
during the pods setting they are the highest (average 0.815). The Fv/Fm ratio
is lowered during the plants flowering and at the end of vegetation (0.634).
Positive correlation between dry mass of nodules, the number of nodules and
Fv/Fm values occurred during plants flowering. This correlation depends on
nitrogen concentration in nutrient.

Abstracts FEBS'99

(We/15.2/290)

Effect of cadmium ions on the accumulation of zinc and

manganese in callus of Acer pseudoplatanus L
M.Filek, M.Pllipowicz
Polish Academy o f Sciences, The Franciszek G6rski Dept. o f Plant
Physiology, Podtu2na 3, 30-239 Krak6w, Poland

Cadmium ions are widespread in polluted environment and relatively easy

derived by plant cells. The aim of this study was to examine sensitivity of
two lines (white and green) of Acerpseudoplanus tissue culture, grown in
presence of various CdSO4 concentration in the medium: 0 (control), 10,
33, 66, 100, 215, 330 and 660 p,M. Observations were done after 14 days
culture. The rate of callus growth was influenced by Cd 2+ concentration. In
the presence of 215 .aM cadmium ions, ealli of white line showed severe
reduction in tissue growth, whereas green line reacted already at 100 p.M.
In examined concentration range, the ultrastructure disturbances were
observed in stressed cells (changes in cell shape, plasmolysis, loss of
chlorophyll in green line). The lines differed also in respect of Zn 2+ and
Mn 2" ions deriving ability. In the case of white line, concentration of both
ions was similar as compared with control, when green line caUi showed its
accumulation already at 100 p.M Cd 2~. This could be the result of different
membrane permeability of both Acer pseudoplatanus lines. Analyses of
membrane fipids proved higher unsaturation level of fatty acids (18:3/18:2)
in membrane of green line cells. Other possibly reason is an interaction
among Zn 2~, Mn 2+ and Cd 2+ cell accumulation. Both callus lines
accumulated cadmium ions during its growth but in the green line intensity
of this process was about 1.5 times higher in all examined concentrations.

s351

(We/15.2/291)

Arabidopsis Mutants of Early Chloroplast Development :

A Genetic Approach using a Plastid Ribosomal Protein.
J-L Gallois, G. Green, C. Bisanz, P. Carol, R. Mache
G~ndtique Mol~culaire des Plantes, Umv. J Fourier-CNRS
BP 53. 38047 Grenoble Cedex 9 France

The differentiation of chloroplasts in plant cells is essential for

photosynthesis and thus autophototrophic growth. Our understanding of
the genes leading an undifferentiated proplastid into a fonctionnal
chloroplast is still fragmented, especially in its early events. In this work,
we attend to develop a method allowing the selection of early chloroplast
differentiation mutants.
We have been interested in the plastid translationnal machinery. In
spinach, several nuclear genes encoding plastid ribosomal protein were
characterised. The promoter region of two of them (L21 et S1) share
common features, suggesting common regulation mechanisms. These genes
are expressed very early during seed germination in developping
photosynthetic tissues (cotyledons) and are not light-regulated. They are
useful markers to focus on the very early stages of chloroplast development.
Also, the Arabidopsis RPL21 gene was cloned and sequenced. Both the
promoter structure and its expression pattern are closely related to those of
the spinach counterpart.
The luciferase gene was fused to the spinach RPL21 promoter and
that construct was introduced by transformation into Arabidopsis. Primary
transformants harbouring one copy of the T-DNA insertion at homozygous
state were selected. To assay for luciferase activity, a low photon counting
CCD camera was used. One line was selected, showing an uniform luciferase
expression pattern in the siblings, consistent with endogenous RPL21
expression. The test was conducted over 2 000 seeds of the selected line.
20 000 seeds (M1) were chemically mutagenised by E.M.S
treatment. M1 Seeds were sowed by pool of 50 and the progeny (M2) was
harvested. A screening for luciferase activity using the CCD camera is in
progress on M2 plantlets. It consists mainly of screening luciferase
underexpression in albino mutants, but others screenings are envisaged (i.e.
overexpression in roots where the luciferase expression is under the
detection threshold). The first results of this screening are shown.
Keywords : plants, chloroplast, ribosomal protein

(We/15.2/292) The tobacco extensin Ext 1.2 gene family: cloning and
regulation of expression
P. Guzzardi and E. Jamet
Institut de Biologic Molgculaire des Plantes/CNRS- Universitd Louts Pasteur,
12, rue du Gndral Zimmer, 67084 Strasbourg Cedex, France

The plant cell wall is a complex structure composed of two main networks,
the cellulose/xyloglucan network and the structural proteins network,
embedded in a pectin matrix. Extensins are HRGPs (Hydroxyproline-Rich
GlycoProteins) and are major structural compounds of higher plant cell
wall. We are interested in the molecular events which take place after
Nicotianeae mesophyll protoplast isolation. Indeed, the cell wall
regeneration might be a main step in plant regeneration via protoplasts. An
extensin gene family named Ext 1.2 is transcribed immediatly after
protoplast isolation. As a first step towards the characterization of Ext 1.2
genes, a cDNA clone was isolated from a cDNA library made from 6h-old
mesophyll protoplasts of Nicotiana sylvestris (Plant Mol. Biol. (1995) 29:
279-292). The aim of the present study is to characterize the Ext 1.2 gene
family and to study its regulation, especially in response to wounding.
The Ext 1.2 gene family was characterized using different PCR techniques,
including direct and inverted PCR, directly applied on genomic DNA.
Three genes were found: Ext 1.2A and Ext 1.2B which were completely
sequenced, and Ext 1.2C which consisted in a 3' non-coding region devoid
of intron. This was consistent with results from Southern blot analysis and
from Northern blot experiments which showed the existence of two
mRNAs transcribed from Ext 1.2 genes which might originate from
Ext 1.2A and Ext 1.2B respectively: 1.2 kb mRNAs in protoplasts, in
wounded tissues and in roots, and 0.9 kb mRNAs in stems.
The expression pattern of Ext 1.2A was further studied in transgenic
tobacco plants containing chimeric fusions between fragments of the
Ext 1.2A promoter and the coding region of the 13-glucuronidase (GUS)
gene. Histochemical stainings showed that the Ext 1.2A/GUS gene fusion
was expressed in wounded tissues, in root meristems and unexpectedly in
cells located at the edge of leaves.
The analysis of the Ext 1.2A promoter in transgenic plants will identify
different regulatory elements. This study will also allow to test the
hypothesis of a role for Ext 1.2 in cell wall repair after wounding.

(We/15.2/293) Laccase(s) in tobacco : a gene searching a function

M-C. K efer-Meyer", V Gomorda, E Guiney b, A,
O'Connell b, D kienard", C Halpin ~ and L. Faye~
a lahoratotre de~ IhTnsport,~ bm'acelhdalre~, ("AIRS t~514 6037,
II't%~[P 23, lrntverstt( de Rotten. 76821 Mont Saint Aigmm
ccdex, I')'~lncc
~' Zene~z7 Phmt 5'~wnce, ,leah~tt',~ Htll Re~earch Statton,
Bracknell, Berk.~hlre, RGI2 6E)~ ( "K
c l)el~lrtme m f)f ]3lologlcal 5k'wme~, IOm'erstty o[ Dundee,
I~mtdee l)l)l 4HN, I 'h"

Laccase(s) and peroxldase(s) are both believed to catalyse the

polymerisahon of monohgnols, the last step in tignin
biosynthesis. In order to study the possible role of laccase(s) in
lignificatton, three laccase-encoding cDNA were cloned from a
tobacco stem cDNA hbrary Two clones correspond to partial
sequences lacking the region coding for the N-terminal side of the
laccase but the third one (pTL3) contains a full-length sequence
coding for a cationic laccase (pl 10) Expression pattern of this
laccase gene was studied both by reverse transcriptionpolymerase chain reaction (RT-PCR) and Northern-blot
experiments and the results of these analysis will be discussed
With the aim to obtain further reformation about tbts laccase and
to check if the pTL3 cDNA encodes a functional protein, overexpression of this laccase gene in tobacco BY2 cells was done.
The full-length laccase cDNA was also used to prepare plant
transformation vectors carry'ing different gene constructs in order
to alter the Icvels of expression of thts hlgh-pl laccase in
transgemc tobacco plants and to analyse the effects of the
modified expression on hgnin content and/or composition.

s352

Abstracts FEBS'99

(We/15.2/294)

Molecular characterization of aquaporins, sucrose and hexose

transporters in grape, Vitis vinlfera L
S.Picaud ~, A.Ageorge~b, L.Fillion ~, C.Rornieubet S.Delrot'.
Laboratory of Plant BiochemisfiTv and Physiology. ES'_4 C.VRS 6161.
t_~ntversityc?fPoitiers blnstttzades Produits de la ['Tgne I.Vt~. .~,Iontpellier.

In the grape berry, ripening (veraison) is marked by massive water

and carbohydrate import. Carbohydrates are mainly synthesized in mature
leaves during photosynthesis and translocated in the form of sucrose in the
phloem, Sucrose is then accumulated as hexoses into the vacuoles of berry
mesocarp cells. In spite of the economical importance of grape, events
triggering these processes and the pathways of assimilate transport are still
poorly known. Consequently, it is interesting to study proteins involved in
assimilates and water transport. We cloned five cDNAs homologous to
plasma membrane aquaporins Four of them encode very close amino acid
sequences, and the last one showed a distinct sequence. The transcript level
of the four cDNAs decrease along berry ripening. These mRNAs are
especially present in developing organs as berries or young leaves. The
characterization of the corresponding genomic clones is in progress. A
similar work is led on the fitth clone.
A eDNA encoding an hexose carrier (Vvhtl) and the corresponding
genomic clone are already identified. Northern-blot analysis indicates that
the Vvhtl transporter is mainly induced during ben 3, maturation : the first
expression peak appears at anthesis, the second one is detected about five
weeks after veraison. Some potential cis-regulator dements were found by
computer analysis in the promoter sequence of Vvhtl, including ABA,
ethylene and sucrose boxes. Functional studies of this promoter are in
progress (see Leterrier et al.). Moreover, comparison of the promoters of
Vvhtl and the grape alcohol dehydrogenase genes, led to the identification
of a 15 bp consensus sequence, suggesting a possible co-regulation of these
genes. A similar work is performed on genomic and eDNA clones encoding
sucrose transporter. Functional studies of these transporters and aquaporins
in yeasts or in Xenopus lem,is ooeytes and their immunolocalization in berry
will allow to understand mechanisms involved in berry ripening.

(We/15.2/295)

CHARACTERIZATION OF THE y-TUBULIN GENE

AND STUDIES OF ITS EXPRESSION IN Lupinus albus
N. Saibo, E. Bekman and C. Rodrigues-Pousada
lnstituto Gulbenkian de Ci~ncia, Ap14, 2781 Oeiras, Portugal

"/-tubulin is a protein associated with microtubule-organizing centers

(MTOCs), recognized as an universal mierotubule nucleator and
consequently involved in cytoskeleton reorganization. So far, very little
is known about the role played by such proteins in plants. In order to
contribute to the study of their role in lupin, we have started the
characterization of the y-tubulin gene family in this plant. We have
amplified a eDNA fragment, synthesized from RNA poly A+ extracted
from the flowers, with degenerate oligonucleotides corresponding to
highly conserved N-terminal regions among several plant 7-tubulins.
This fragment was used as a probe to confirm the presence of 7-tubulin
gene(s) in L.albus genome by Southern blot and to screen the L. albus
genomic library. So far, we have isolated two recombinant phages that
bear an identical sequence coding for a 7-tubulin. The gene La-TubG1
isolated from L.albus encodes a polypeptide of 472 aa sharing a
92 - 95% identity with other plants 7-tubulins. The expression pattern of
gene 7-tubulin in L. albus was studied by Northern blot and RT-PCR.
La-TubG1 is expressed in all plant organs here presented. Transcript
accumulation in germinated embryonic axis is much higher than in
soaking seeds as well as in young organs, e.g. leaves, and in organs with
a high rate of cell division. After 4h treatment with the auxin IAA, the
steady state level of 7-tubulin mRNAs are not changed when compared
to control plants. This result contrasts with the one obtained for
La-ACS1 gene which belongs to a multigene family of ACC synthase
(1), an enzyme involved in the ethylene biosynthesis. The results taken
together will be discussed.
References
~- Bekman, E, Satbo, N, Cataldo, A. Regalado. A P. Rlcardo. C P and Rodngues-Pousada, C. Differential
expression of four genes encoding ACC synthases in Lupmusalbus during getmmatlon, growth and In
response to IAA, LI+ and ~oundmg Submttted to PlantMol Biol
Supported by PRAXIS XXL BIO/1146/94 and by fellowships given to N S (BD/13760/97) and (EB
B D/2808/94)

(We/15.2/296)

Identification of a pollen-specific sucrose transporter-like protein

NtSUT3 from tobacco (Nicotiana tabacum)
S. Sakr t, L. Barker 2, L. BiJrkle2. C. Kiihn2, M. Regnacq ~, C. Gaillard t,
S. Delrot ~,W. Frommer z et R. Lemoine ~.
~Laboratoire de Biochimie et Physiologic Vdg(tales. ESA CNRS 6161,
Universit( de Poitiers. ZBotanicalInstitute, University of Tiibineen. Gertnanv
Photosynthetic sucrose plays a major role in plant productivity. Sucrose

is not only a source of reduced carbone for sink organs but is now considered
as a signal molecule able to regulate the expression of several genes (sugar
sensing).
By screening a tobacco (Nicotiana tabacum) pollen cDNA library with
the potato sucrose cartier as a probe, we have identified a new eDNA (NtSUT3)
that shows high homologies to other plant sucrose carriers. However NtSUT3
has several remarkable features :
i) It has a small open reading frame upstream of the start codon. This kind of
sequence is frequently found among genes regulating growth and development
in plants.
ii) Studies in Northern blots showed that NtSUT3 expression is restricted to the
very late stages of pollen maturation and to pollen tube growth.
In order to understand the exact role of NtSUT3, we have developed
new tools (yeast expressing NtSUT3, antisense transgenic tobacco plants,
antibodies specific to NtSUT3). Some of these results will be presented and
discussed in relation to pollen maturation and pollen tube growth.

(We/15.2/297)

Identification d'un transporteur de saceharose sp~ifique du grain

de pollen de tabac (N icotiana tabacum)
S. Sakr L, L. Barker 2, L. Btirkle", C. Kiihn 2, M. Regnacq ~, C.
Gaillard ~, S. Delrot ~, W. Frommer: et R. Lemoine *.
ILaboratoire de Biochimw et Physiologie V#g~tales, ESA CNRS 616L
Universit~ de Poitiers. :Botanical btstintte, University of T#bingen, Germany

Le saccharose issu de la photosynthdse joue un rdle important dans la

productivit6 de la majorit6 des vdg&aux supdrieurs. En effet, le saccharose
constitue non seulement une source de carbone pour les organes puits mais
6galement une moldcule signal rdgulant l'expression de certains g~nes (sugar
sensing).
Au moyen d'ADNc de transporteur de saccharose de pomme de terre
(StSUT1), le criblage hdtdrologue d'une banque d'ADNc de grains de pollen
de tabac (Nicotiana tabacum) a conduit h l'identification d'un ADNc
(NtSUT3) ayant des homologies avec les ADNc des autres transporteurs de
saccharose. Cependant, NtSUT3 prdsente deux particularitds :
i) sa sequence nucldotidique est pourvue d'un cadre de lecture ouvert en
amont du codon d'initiation. Un tel motif est frdquemment retrouv6 dans les
g~nes rdgulant les processus de croissance et de ddveloppement des vdgdtaux.
ii) I'analyse des rdsultats de Northern blots et d'hybridation in situ
rdalisds avee la sonde NtSUT3 souligne une expression spdcifique dans les
grains de pollen. Cette expression ddpend du stade de maturation et localisde
darts le tube pollinique.
Enfin, l'utilisation en cours d'autres outils d'dtude (levures, plantes
transgdniques antisens) nous permettra /l trds court terme de mieux
apprdhender d'une part la fonction exaete de la protdine et d'autre part
l'importance de NtSUt3 clans le processus de ddveloppement et de germination
des grains de pollen.

Abstracts FEBS'99

(We/15.2/297)

M e m b r a n e composition changes associated with induction

and differentiation of Brassica n a p u s cani
I. Zur, A. Skoczowski, E. Niemczyk, F. Dubert
Pohsh Academy of Sciences, The Franctszek Gdrskl Dept of Plato
Physiology. Podtu-*na 3. 30-239 Krakdw, Poland

Establishment of callus cultures with high regeneration ability is an important

task for plant tissue culture and biotechnology. But early identification of high
morphogenic potential is difficult because of the complexity of the regeneration
process.
In the experiment, we studied changes in the lipids content and composition,
during calli induction and shoot regeneration in Brassica napus cultures. Calli
were induced on 5-days-old hypocotyles and transferred to the induction and
then, regeneration stimulating media. During the culture, lipids were extracted
[1,2] and analyzed [3] with Hewlett Packard 5890 gas chromatograph. The
obtained data were expressed as parameters describing ratios of total
phospholipids to galactolipids content (PL/GL) and each galactolipid fraction
(MGDG/DGDG), its fatty acid composition and unsaturation.
The obtained results indicated that process of callus induction
(dedifferenfiation of an explant tissue) altered membrane fatty acid composition
and its unsaturation. Further changes took plume during initiation of callus
regeneration. This process was associated not only with changes in membrane
fatty acids composition but as well as with changes in ratios of each lipids
fraction (PL/GL, MGDG/DGDG).
These results suggest that membrane composition could serve a good marker of
callus regeneration ability.
[ 1] E.G. Bligh, W.J. Dyer, A rapid method of total lipid extraction and
purification, Canad. J. Biochem. and Physiol., 37 (1959) 91 1-917.
[2] A. Ryypp6, E.M. Vapaavuori, Rikala R., M.-L. Sutinen, Fatty acid
composition of microsomal phospholipids and H +- ATPase activity in
the roots of Scots pine seedlings grown at different root temperatures
during flushing, J.Exp. Bot. 45 (1994) 1533-1539.
[3] C.M. Rivera, D. Penner, Rapid changes in soybean root membrane lipids
with altered temperature, Phytochem 17 (1978) 1269-1272.

s353

s354

Abstracts FEBS'99

17 Extremophiles
(We/17/298)

Enzymes from psyehrophilie micro-organisms:

What de the 3D structures tell us?

( W e l l 7/299)

N. Aghajari, R. Haser
lnstitut de Biologic et Chirnie des Prot~lnes, Bto-Cristallographie,
UPR 412 CNRS, 7 Passage du Vercors, 69367 Lyon cedex 07, France

Enzymes from psychrophilic microorganisms display optimal

activity at low temperatures where the catalytic activity of the
homologous mesophiles is drastically reduced.
With the aim of understanding cold adaptation on a molecular level,
i.e. structural factors which can explain the adaptation to low
temperatures as well as the relation between structure and activity,
an or-amylase from the Antarctic psychrophilic bacterium
Alteromonas haloplanctis and an alkaline protease from the
psychrophilic Pseudomonas TACII 18 have been crystallized. The
three-dimensional structures of the two enzymes in their native
state as well as in complex with different type of inhibitors in the
case of the a-amylase have been established to high resolution using
X-ray crystallography methods [1.2,3]. In the case of the
psychrophilic Or-amylase the 3D-structures of ternary complexes of
the enzyme binding simultaneously two inhibitors in the active site
region, suggest that a transglycosylation reaction has taken place in
the crystals [3], For both enzymes determinants of psychrophily
and details of the catalytic reaction mechanism as indicated by the
crystal structures will be discussed.

The py;B and pyrI genes encoding the aspartate carbamoyltransferase

(ATCase) of S. acidocaldarius were cloned on a 6.9 kb long DNA fragment
from a genomic Pstl bank by complementation of an ATCase deficient E.
cell mutant. An additional 1.7 kb of flanking DNA were amplified by
inverse PCR. Determination of the nucleotide sequence of the entire 8.6 kh
EcoRI-Pstl region revealed the presence of several pyrimidine genes: pyrB
and l, encoding the catalytic and regulatory subunits of ATCase, p y r C
(dihydroorotase), p y r D (dihydroorotate dehydrogenase), p y r E (orotate
phosphonbosyltransferase) and pyrF (OMP decarboxylase). In addition we
found four other orf's, the function of which is not yet clearly established,
and the lrp gene, encoding a homologue of the E. cell global transcriptional
regulator Lrp [i]. The gene cluster shows the following organization: pyrFpyrE-pyrB-pyrl-orfl-pyrC-pyrD-orf2-orf3-orf4-1rp. The pyrimidine genes
are organized in a bipolar operon, of which the pyrE, F and pyrB to off4
wings are transcribed in opposite orientations, a previously undescribed
organization for pyrimidine genes. Startpoints of transcription could be
determined for pyrB, pyrC, pyrE and lrp. In all instances, the transcriptional
initiation point precedes the translational codon by a few nucleotides only,
and is associated with a typical archaeal promoter element (boxA).
Transcriptional stop signals of Type I [2] are present at the 3' side of the
orf4 and Irp genes. Functional analysis of the bipolar operon by RT-PCR
indicates that the argC promoter ~s an internal, secondary promoter and that
transcription intiated at the p y r B promoter can proceed to off4. p y r F is
cotranscribed with pyrE from a promoter located at the pyrE-pyrB boundary.
Primer extension experiments with total RNA extracted from S.
acidocaldarius cells grown in different media support the existence in the
archaeon of a pyrimidine-specific repression mechanism operating at the
transcriptional level.
[1] Charlier, D. et at., Gene, 201, (1997) 63
[2] Reiter, W.-D. et at., Nucl. Acids Res., (1988) 2445

References
[1]
[2]
[3]

The bipolar pyrimidine operon of the extremely

thermoacidophilic archaeon Sulfolobus acidocaldarius.

D. Charliera'b, L. Thla Toonga, V. Durbecqc, M. Rooversa, N. Glansdorfta'b

a Microbtology, VUB and VIB. blRMW, CMicrobiolog3. ULB.
Av E Go'son l, B-lOTOBrussels. Belgium.

Aghajari N e t al. Protein Sci., 7, (1998) 564.

Aghajari N e t al. Structure, 6, (1998) 1503.
Aghajari N e t al. In preparation.

(We/17/300)

The crystal structure of glyceraldehyde-3-phosphate

dehydrogenase from Methanothermus fervidus
C. Charron ab, B. Vitoux b and A. Aubry b.
alBMC - UPR CNRS 9002. Strmbourg, France.
bLCM3B - ESA CNRS 7036, Universitg Nan0 I, France

The hyperthermophihc archaeon Methanothermus fervidus lives

in extreme conditions and grows optimally at 84C. We determined the
crystal structure of the glyceraldehyde-3-phosphate dehydrogenase frmn
this archaeon in the presence of NADP +. We solved the structure by
molecular remplacement techniques, as implemented in the program
AMoRe [l]. The three-dimensional structure ofSulfolobus so~Ttaricus
glyceraldehyde-3-phosphate dehydrogenase [2] was used as a model.
The refinement was performed without NCS restraints using REFMAC
to 2.1,~ resolution. The root-mean-square deviation of C a positions of
glyceraldehyde-3-phosphate dehydrogenase from M. fervidus and S.
solfataricus is 1.59A in the monomer. The structure of the M. fervidus
enzyme was compared to enzymes from both Bacteria and Eucarya in
order to highlight structural determinants responsible for molecular
hyperthermostability. We also compared cofactor-binding sites from both
M. fervidus enzyme, which exhibit dual cofactor specificity with a
marked preference for NADP +, and Bacillus stearothermophtlus
enzyme [3], which is strictly NAD+-dependent. Molecular features
involved in cofactor binding as well as discriminators for NAD + and
NADP + recognition are discussed.
[i] Navazza, Acta Crystallogr., A50, (1994), 157.
[2] Fleming et aL, Acta. Crystallogr., D54, (1998), 671.
[3] Skarzynski etal., J. Mol. Biol., 193, (i987), 171.

(We/17/301)

Production of antifungal molecules by extremophile bacteria

M. Fontaine", J. Coulon", J. BoudranP, R. Bonaly a, G. Barbier c.
J. Dietrich, J.L Simona
UMR 7564-54000 Nancy ; b UPR 6811o54505 Vandoeuvre ;
c IFREMER-29280 Plouzang ; aRHODIA-79500 Melle

Thermophilic and hyperthermophilic bacteria and archaea were described

consequently to studies achieved in deep-sea hydrothermal environment.
These bacteria are adapted to extreme conditions : temperature (above
100C), extreme pH and salinity, high pressure, low oxygenation, and
lightning environments.
One of the numerous industrial applications of these bacteria is the
production of macromolecules with therapeutic properties such as
antitumorat agents and antimetabolites.These compounds could be
macrolides or peptides with rare aminoacids.
Their effects should be very important as they can be detected in vitro at
very low concentrations.
Furthermore, in these submarine vents, no eukaryotic micro-organisms
could be detected up to now. This could be due to the extreme conditions
but also to antifungal agents produced by these bacteria.
Deep-sea hydrothermal vents are colonised by heterotrophic microorganisms, supported by auxotrophic micro-organisms. Our work focused
on the valorisation of these deep-sea hydrothermal micro-organisms whose
collection results from cruises in Pacific and Atlantic oceans using the
French submarine "Nautile".
More than one hundred bacteria strains were grown according to their
isolation conditions (temperature 60-97C, pH 5.5-7.5). Culture supernatants were screened for a possible antifungal activity. This disc method
was applied to culture supernatants, butanolic extracts and aqueous residues
on yeast belonging to the genera Kluyveromyces, Saccharomyces and
Candida. Results revealed antifungal activities which were compared with a
"gold" antifungal agent : amphotericin B.
These preliminary results evidence that some bacteria and archaea
growing in extreme conditions may contain molecules having potential
antifungal activities.

Abstracts FEBS'99

(We/17/302)

s355

IIl'~ t ' M i ~ l l i , l l l ~, on t ' % i r i l t l q h l l a r , . - a n | ~ , l a s r iw~,tlt)<li,.)n

,,%i|h t%%lJ alk;ih)t()lt.r;trlr P,,endonlolh')S str:l,nw

(We/17/303)

I.-I). I)inu. ~,1. Ju,'cna,e, Gh. ('aml)eam,

i,t~',l ~

~ ;, h , ,i,p

, }~

:,~

~., ,

/~,b/Rl,~ / ,

Dept. of BioL, Middle East Technical University, 06531 Ankara, Turkey

~olnble stmroh (1%), in ~ Vra~B~c~ of_ ,~z ~-d Mq z icc~ ~ ~ str~aL~

L 10 a.l~='-~t.n-.-e_(0.5%). ~]_~ ',,e s':._dlc-<'/the "---ffszt of initial ~H u~ t'-~ ,~,.i~m

,~/aly-,_~5,aty,:ic~ 3 -'2m-cgs ~ 20 ~ : g!~xfre-{,~CHh ~ . : : (gH !0.0] :~_~ r"e~

uee: ~ a l l e-zF-e ~ - , . ~
stuS,,~,.
Al~e-awlas~. ~ : s~=_in L Z] ~ s act!~ Ln a ~
::
(6.C-12.0) ~ith ~ e q ~ i ~ m cf aXivity at 30"C, ~ 7.0 (%Z.=~ U~?.) ard
10.5 (A.E.=@.8 U/~l..) ~ t-hat ~ L e ~
d~e ~,ds~2n o[ t n
aLkalq~ilic s ~ _ i ~ ~ :'~zil!~ ~ . (2). ~ e .a~iru~ t~q~,~--_~_ ~3. ~:
L iO straLn. (.~.E.=6.8 U/~I.). ~

b~th , ~ , , ~

csl,.~.z,n icrs ~

a ~r~v

~-e sd=s~-a~ s~ectficati~ ef a ~ l a ~

_'~--,an
h~th s ~ - s a ~
~ e ~ f e ~ cf ~i~f~ra~_~ h~C:r~
a~ a~les~ r,-~,.i,~_zy,,~s also ~.~b~'nedu

(i) L~D, Ei,~a et ~i., ~--~.~/-~. -2u~--~u Let(., %DI. !,(/Sc~9),c. I~

(2) K. ~2~52~i., Eqz.~r~ ~ Al~i,q/nil~ in ML.~i~-bi~I~
~ - ~ d : ~ . L~Le~, W.r:..~z~, C.T. lq~Ily ~-~.,(L~?C), ~. 275

(We/171304)

Thermostable Protease Activities of Thermoacidophilic

Microorganisms Isolated from Hot-Springs in Turkey
B.Erdem, S.Kocablylk

Effects of Glycine Substitutions in Interface Helices on

Stability of Thermoplasma acidohilum Citrate Synthase
i. Erduran and S. Kocablylk
Middle East Technical University, Dept. ofbiol.,06531, Ankara, Turkey

Proteins of known 3D structure could be stabilised by decreasing their

entropy of unfolding [1]. In the protein's unfolded state, glycine residue is
devoid of 13-carbon and has greater configurational flexibility than the other
residues which have 13-earbon. That is why, substituting any other residue for
glycine should decrease the entropy of a protein's unfolded state and stabilise
the folded protein. In this study, by using site-directed mutagenesis, we have
replaced Gly residues in the interface ct - helices ( consisting of four pairs ot
antiparallel helices - FF', GG', LL' and MM' ) of Thermoplasma ( Tp. )
acidophilum citrate synthase. The exchange of Gly 209 by Ala increased the
half-life of the Tp. enzyme at 85C about 23-fold, as compared to wild-type
enzyme [2,3]. The told-points of irreversible inactivation were at 1.6M
GdmCl and 2M GdmCl for G209A and wild-type enzymes, respectively. On
the other hand, catalytic activity of G209A enzyme was 15-times higher
(Vm~x for OAA and AcCoA, 44 mmoles / ml / min ) than that of the
recombinant wild-type enzyme. This substitution should stabilise the ~-helix
(Helix M), due to replacement of Gly by a residue (Ala) with high helix
propensity. Also, increased hydrophobicity should provide more intimate
subunit interaction which would also improve enzyme's processivity, because
citrate synthase is only active as a dimer. Second mutation is achieved by
introduction of Val in place of Gly 197 of the Helix F. This substitution
could not be tolerated in the subunit interface of Tp. enzyme and significantly
reduced the enzyme's stability (Tm at 85C is 1.5 min, and mid-point of
GdmCl inactivation is at 0.1M ). In addition, Gly 197 to Val replacement
resulted in the loss of catalytic activity, about 7-fold, as compared to wildtype Tp. citrate synthase. These results indicated that for Gly substitution to
stabilise the folded protein selection of the new residues should be critical.
References:
[1] Metthews et al. Proc. Natl. Acad. Sci. USA 84 (1987), 6663.
[2] Erduran, ] and Kocablylk, S. Biochem. Biophys. Res. Commun. 249
(1998), 566.
[3] Erduran, J and Kocablylk,S. J Prot.Chem. 17(1998), 531.

Thermophilic bacteria and archaea have been investigated for clues to

evolutionary processes as well as to highlight the mechanisms responsible
for the increased levels of biomoleeular thermostabilization. Many of the
enzymes isolated from these organisms are heat-stable and currently
become a focal target for their applications in commercial fields. Of the
industrial enzymes, pro(eases represent one of the largest groups, and
account for 60% of the total world wide sale of enzymes. They are not only
responsible for the complex processes involved in the normal physiology of
the cell, but also have successfull applications in food, detergent and leather
industries. Thermophilic and hyperthermophilic microorganisms are ideal
and invaluable sources for proteases.
In this study, we report the isolation and characterization of thermostable
microorganisms from dry hot-soils obtained from "burning cracks" and
water obtained from solfataric hot-springs in Turkey. The microbial flora of
the samples were enriched aerobically in Darland's medium [1] with pH
values between 1 and 3, at 60C. A total of 20 thermoacidophilic
microorganisms were isolated. Most of the isolates were Gram negative
long or short rods with varying organizations. Only two isolates were small
cocci with irregular shapes. Isolates were identified according to their 16S
rDNA sequences by PCR.
Intracellular neutral a n d / o r alkali proteolytic activities of the isolates were
determined spectrophotometrically using azocasein as the substrate [2].
Protease activities of the cell extracts varied between 0.5 and 4 Units which
were
analyzed by zymography using gelatin co-polimerized with
acrylamide gel. Cloning of a thermostable alkali protease gene from one of
the isolates is still in progress.
References:
[1] Darland,G et al., Science, 170(1970):1416,
[2] Morikawa,M et al., Appl. Environ. Microbiol. 60(1994):4559.

(We/17/305)

A COLD-ADAPTED CELLULASE
G, GARSOUX~, J. LAMOTTE*, M, CZJEZK#, E BARRAS, C. GERDA'
~"Laboratotre de Bloehtmleand CIP.' Instffut de Chtmt B~. Universitd de Lt~ge.
Sart-Tllman, 4000Liege. Belg~que,E-math ~tenevieve,~lar.~oux~ul~ a~he;
#AFMBand LCB CNRS,31~Chemlnl Atguler, 13402Marsellles Cedex 20, b?anc

The enzymatic hydrolysis of cellulose, a huge and renewable carbon

source, can generate numerous valuable p r oducts [1 Cellulases are usually
classified into endoglucanases, exoglucanases (or cellobiohydrolases) and
J3-glucosidases, and are thought to act synergistically in cellulose hydrolysis
Psychrophilic microorganisms - living in cold environmentsproduce enzymes which display, when compared to their mesophilic
counterparts, a higher catalytic efficiency over a temperature range of roughly
0-30C and a higher thermosensitivity P)Pl
The strain Alteromonas haloplanctts -isolated from Antarctic sea
water- secretes a cellulase : the endoglucanase G (EGG) The enzyme has
been purified and the sequencing of the 24 N-terminal amino-acids indicates
that the endoglucanase Z (EGZ) from E r w m i a chrysanthemi is a mesophilic
counterpart In order to compare the two enzymes, the mesophilic cellulase
has also been purified (using notably a chromatography on a cotton column)
As expected, the EGG shows an apparent optimum temperature shifted
towards lower temperatures, a higher specific activity at temperatures below
30C and a higher thermolability than EGZ
The gene has been cloned in E.coli and the nucleotide as well as the
amino-acid sequences have been compared to those of EGZ The recombinant
EGG displays an isology of 66% and 57% for the catalytic- and cellulosebinding- domains respectively but presents a large insertion (75 AA) in the
linking region
A 3D structure model has been built; this model and the analysis of
the amino acid sequence have highlighted some typical structural features
which might be involved in cold adaptation : fewer salt bridges, extended
surface loops, fewer proline residues, more glycine residues . leading to a
putative increase in flexibility
IHBayer, E A. et al Trends Biotechnol., 12. (1994), n 9. 379-386.
[2]Russell NJ. Adv Biochem.Eng. Btotechnol., 61. (1998). 1-21
PlGerday, C et al Blochim Bloph)s Acta. 1342. (1997). 119-131.

s356

(We/17/306)

Abstracts FEBS'99

Cloning and expression of the PCNA from two

hyperthermophilic archaea.
G. Henneke, J.P. Raffin, O. Lecompte, J Dietrich
Laboratoire de Biotechnologie des ~ticroorgantsmes Hydrothermaux,
Ifremer, DRV/VP, BP 70, 29280 Plouzan~, France

DNA replication is a highly organised process which implicates a whole set

of proteins which confer speed and high processivity to the system. This
high proeessivity is given by a clamp which forms a ring-like structure
around the DNA and which must be loaded on the DNA by a clamp-loader
in an ATP-dependent process. Recent genomic sequencing on archaen [1, 2]
have identified two genes as putative homologs of replication factor C (RFC) and one gene of putative homolog of proliferating cell nuclear antigen
(PCNA). These genes show sequence similarity with respect to the large
RF-C and p40 subunits of RF-C, and to the PCNA of eukaryotes.
We cloned and expressed the PCNA from two hyperthermophilic archaea,
Thermococcus fumicolans and Pyrococcus abyssi, which optimal growth
temperatures were of respectively 85 and 95C. In spite of a high sequence
similarity between the two PCNA genes, the Thermococcus PCNA could be
overexpressed when using a modified pET expression vector in
BL21(DE3)pLysS strain., while the Pyrococcus gene was very toxic to E.
coll. Therefore, the Pyrococcus PCNA was expressed by using the phage
CE6 induction system in BL21 strains.
Also the DNA polymerasa form I from the two arehaea strains were cloned
and expressed and, when tested with their respective PCNAs, their specific
activity and turnover rate were increased (about 10 times increase of dNTP
incorporation into primed L DNA, at 70C).
[1] Bult, CJ et at., Science, 273, (1996) 1058.
[2] Klenk, H P. et at., Nature, 390, (1997) 364.

(We/17/308)

1713-hydroxysteroid dehydrogenase in extremophilic

and mesophilic yeasts
T. Lani~nik Ri~ner, M. ~akelj-Mavri~
Institute of Biochemistry, Medical Faculty, Ljubljana, Slovema

1713-hydroxysteroid dehydrogenases (171]-HSDs) are enzymes responsible for

reversible interconversions of 17-hydroxy and 17-keto steroids. They are
widespread in mammals and to date eight types have been characterized.
In microorganisms 171]-HSDs have been found in bacteria, filamentous fungi
and yeasts. In spite of the presence of 1713-HSD in all representatives of
different fungal classes the enzymes were purified only from fungi
Cylindrocarpon radicicola and Cochliobolus lunatus, while complete
characterization and sequencing of 17!3-HSD was done only for the enzyme
from Cochliobolus lunatus. On yeast 1713-HSDs on the contrary there are
only scattered data. To obtain more detailed information about yeast 1713HSDs, three mesophilic yeasts; Candida tropicalis,
Cryptococcus
tsukubaensis and Saccharomyces cerevistae and three extremophilic halophilic
black yeasts; Hortaea werneckii, Trimmatostroma sp. and Phaeotheca
triangularis were tested for the presence of 1713-HSD 1713-HSD activity was
detected in all cases favoring mostly reduction of androstenedione and
estrone, similar to Cochliobolus lunatus 1713-HSD (1713-HSDcl). In
extremophilic yeasts, isolated from salterns, the effect of NaCI in growth
media on enzyme activity was observed. In Hortaea werneckii presence of
higher concentration of NaCl increased the enzyme activity 9 times, while the
effect of NaCI in other two extremophiles was not so significant. To
determine possible relationship between yeast 17!3-HSDs and 1713-HSDcl on a
protein and mRNA level Western and Northern blot analyses were performed.
The enzymes from extremophiles: Phaeotheca triangularis and
Trimmatostroma sp were found to be similar to 1713-HSDcl on a protein as
well as mRNA level, while that was not the case for enzymes of extremophile
Hortaea werneclai and all mesophiles The physiological role of 17!3-HSDcl
related 1713-HSDs from extremophiles: Phaeotheca triangularis and
Triramatostroma sp., as well as from other investigated yeasts is however still
under investigation.

(Wed17/307)

Bacillus thermoantarcticus producing enzymes for

bioconversion of xylao
L Lama B Nicolaus V Calandrelli E Esposito A Gambacorta
I('~IIB, ('.\'R l )a Tomno 6. 80072 .trco Fehce. .Vapoh. Ita(i

Hemicellulose is a naturally abundant resource and accounts for 20 to 30%

dry weight of agricultural residues Xylan, a heteropolymer of ([3-t,4) linked
xylose, is its major component. This abundance indicates that xylanolytic
enzymes can play an important role in bioconversion, in the preparation of
cellulose pulps, in fibre liberation technology, etc, Generally, the processes
are run at high temperatures and at extreme pH values Therefore, enzymes
tolerating these parameters are needed in order to make enzymatic processes
technically and economically more feasible.
Various microorganisms are able to grow on xylan, as sole carbon source
Exoenzymes degrade the polymer to D-xylose, which is transported into the
cell, isomerized from a D-xylose isomerase to D-xylulose and
phosphorylated to xylulose 5-phosphate which enters either the pentose
phosphate pathway or the phosphoketolase pathway
D-xylose isomerase has the largest market in the food industry because its
application in the production of high-fructose corn syrup. This enzyme is
also of industrial interest for the fermentation of hemicellulose to ethanol
with yeasts.
In view of this, a thermophilic bacterium, "Bacillus thermoantarctlcus",
isolated from geothermal soil in Antarctica was screened for its capacity to
produce thermostable enzymatic activities
This microorganism produces an extracellular xylanase and [3-xylosidase,
and an intraceUular xylose/glucose isomerase
In this communication we report the isolation and characterization of these
enzymes.
This work was financially supported by PNRA

(We/17/309)

Potential production of lactose-free milk by using an

immobilized thermostable recombinant 13-glycosidase
F. La Cara, E. lonata, F. Febbraio, R. Nucei, M. Rossi
Institute of Protein Btochemistry and Enz~nologv CNR, 80072Arco
Felice (NA), ltaly

The use of lactase ([3-D-galaetoside hydrolase, EC3.2.1.23) to prevent lactose

crystallization in dairy food, responsible for defects such as sandy texture or
deposit formation, is widely reported [1] Moreover the enzymic hydrolysis
&lactose in milk can avoid consequences from lactose intolerance in people
who are deficient in lactase. Fungal, yeast and bacterial sources of lactase
have been used to carry on the lactose hydrolysis and different support
materials have been tested for enzymes'immobilization. Thermostable
enzymes, that are able of continuous operation at high temperature in
immobilized enzyme systems, are preferable in order to prevent bacterial
contamination during the enzymic hydrolysis processes.
For this purpose we cloned and expressed in some yeasts (S. cerevisiae and
P. pastoris) a 13-glycosidase activity specific for 13-gluco-13-galactosides
isolated and purified from the archaebaeterium S. solfataricus [2,3] to
produce a large amount of enzyme We immobilized the ~-glycosidase on
several polysaceharidas supports to build up a bioreaetor suitable for a pilot
production of lactose-free milk. The yield of enzyme immobilization was
evaluated as 80%. The comparison of the free !3-glycosidase to the bound
enzyme showed a similar pH optimum (pH=7.0), the same thermophicility
above 90 C, improved thermostability and slight competitive inhibition by
glucose. The kinetic constants values for or-lactose and on ONPGal used as
substrates for free and immobilized enzymes were comparables Interestingly
the immobilized enzyme showed appreciable activity also at room
temperature (20% of that maximal at 90 C) differently from the free enzyme
(3% of that maximal at 90 C). The bioreactor activity did not significantly
change for at least one month at operative temperature of 60 C
The foregoing results have allowed us to obtain an hydrolysis of 80% of the
milk lactose content using the bioreactor in a single step approach, so that we
propose this recombinam immobilized !3-glycosidase for the
biotransformation of lactose in milk and dairy products.
[1] A. Dalhqvist et at., Biotechnol Bioeng, 15, (1973) 395.
[2] F.M. Pisani et al., Eur. J. Biochem., 187, (1990) 321
[3] R Nucciet al., Biotechool. Appl. Biochem, 17 (1993) 239.

Abstracts FEBS'99

(We/17/310)

T. ferroaxidans c y t o c h r o m e
purification and molecular characteristics.
P. Tron, D. Benan'och, C. Michel audD. Lemesle-Meunier.

s357

oxidase:

Laboratoir de BIod~rgdtlqlt tt lng~nler~ dej Pr~t~lne& CNRS, 31 ch J. Alguler, 13402

MarJelll ccdGx 20. FRANCE,

1: ferrooxidans is the main bacterium used in microbial leaching, which is an

industrial process currently used to extract metals from low grade ores, This
chemolithotroph organism is able 1o derive the energy required for its growth and cell
maintenance from the oxidation of reduced sulfur compounds or from the oxidation of
Fez' under acidic conditions, using O2 as the oxidant, the electron transfer chain which
mediates the oxidation of Fez' and the reduction of Oz to H2O, is the sole process
which release energy in the form of protonmofive force established through the
terminal oxidase. As cytochrome c oxidase of Zferroaridans shows the a absoq3tion
peak at 597 rim, it has been classified in cytochrome a v However, Kai et al showed
that the cytochrome c oxidase purified from 1: ferrooxidans strain F1 is similar to
cytochrome ass but exhibits some properties which differ from those of others a% type
cytochrome c oxidases so far studied.
In the present stady we described some properties of cytochrome oxidase purified to
homogeneity from a new strain of T.ferrooxidatls
The protocol is based on the use of the detergent dodecylmahoside and comprises
only three steps: snlubilisetion under acidic conditions, sucrose gradient sedimentation
and CM Toyopeari chromatography.
The difference spectra between reduced and oxidized cytochrome oxidas showed
absorption peaks at 439 and 449 tun in the Soret region and at 597 nm in the a region
The EPR spectra shows signals characteristics of heine as and CuA.
The subunit composition of the oxidas was analyzed by SDS PAGE decttophoresis
Staining with Comassie blue revealed three bands with apparent molecular weight of
approximately 60, 22 and 19 kDa. Nolle band was revealed by TMBZ staining. The N
terminal amino acid sequence of the three bands blotted on membrane were
determined,

The purified complex is active in transferring electrons from cytocbrome c to oxygen;

the optimal pH of the enzymatic oxidation of ferrocytochrome c was 3.5. This result
suggests that the O2 reduction by the bacterium may occur at the periplasmic side of
the plasma membrane.
The results are discussed with regards to the characteristics ofa t or aas type
eytoohrome oxidases.

(We/17/312)

Hydrolytic efficiency of a crude xylanase and a purified xylanase from

an SPS-0 Bacillus isolated from a hot spring in Portugal.

M. Bataillon ~, A-P. Nones Cardinali 2, E. LeginI and F. Duehiron k

] Universit6 de Reims-Champagne-Ardenne, France. 2 lnstituto Nacional
de lnvestiga~,~oAgraria, Portugal.
Large amounts of wheat bran are produced annually from milling process in many
countries of the world. A major share of these cereal by-products is the use in animal feed,
but in recent years, decreasing of cereal prices has stimulated research on the valorisation of
these products.
Industrial wheat bran is then one of the most representative available hemicellulosic
rich products. The main hemicellulosic component is the arabinoxylan, which consists of two
pentose sugars: arabinose and xylose (1). Arabinoxylan is easily degraded by chemical means
(2), however enzymatic degradation has recently received must attention, in particular with
thermostable enzymes, which are valuable candidates to replace chemical technologies.
The advantages of thermostable enzymes in industrial processes include decreased risk
of contamination, increased solubility of the substrate, increased diffusion rates and higher
stability of enzymes against denaturing agents.
In this work we discuss the influence of some parameters (delignification, powdered
wheat bran, enzyme concentration, substrate/xylan from birchwond and oat spelt and synergy
with others activities) on the efficiency of the hydrolytic capacity of a crude xylanase
preparation produced by a thermophilic Bacillus SPS-0 (3). This strain was isolated from a
hot spring in the northern Portugal.
In order to select an enzyme further used as a reference, the xylanases from four
commercial strains (NOVO SP-860, NOVO SP-473, NOVO SP-905, GENECOR Multifect)
were tested for their ability to hydrolyse destarched wheat bran. Xylanase from GENECOR
Multifect showed higher levels of productivity and was then used as a reference in the
hydrolysis experiments. The hydrolysis was evaluated in tenns of xylose and arabinose
production measured by High Performance Liquid Chromatography.
We finally compared the hydrolytic capacity of the purified xylanase (4) and the crude
enzyme preparation from SPS-0 against the xylanase from GENECOR Multifect. Results
clearly showed higher yields of xylose and arabinose when the purified enzyme was used.
This may suggest that some inhibitors are present in the crude xylanase preparation of
Bacillus SPS-O.
(1) Schmorak, J., Bishop, C.T., Adams, G.A., (1957) Can. J. Chem., 35, 108-114.
(2) Quiabi, A., Rigal, L.. Gaset, A. (1994) Industrial Crops and Products, 3, 95-102.
(3) M. Bamiltun, A-P. Nunes Cardinali and F. Duchiron. (1998) Bioteehnology Letters, 20,
I 1,10671071.
(4) M. Bataillon, A-P. Nones Cardinali and F. Duchirun. Purification and characterization
of a thermostable xylanase from Bacillus sp. strain SPS-0. Preparing for publication

(We/17/311)

Isolation and preliminary characterisation of an intracellular

aminopeptidase
from
the
hyperthermophilic
Thermococcus

hydrothermalis
L. PERROT, E. LEGIN and F. DUCHIRON
Universit~ de Reims-Champagne-Ardenne, France
Microorganisms growing in extreme ecosystems contain considerable
amounts o f enzyme potentially beneficial for the development o f bioeatalysis
and catalysis (1). Hydrolysis o f peptide bonds is a prerequisite for utilisation
o f proteins as sources o f amino acids for bacterial growth and represents an
essential mechanism o f modification o f proteins at the posttranscriptional
level. However, few reports about hyperthermophilic archaeal proteinases
have been published to date (2,3). The study o f archaeal aminopeptidases
could allow a better understanding o f the catalysis ofthermostable proteins.
The marine anaerobic hyperthermophilic Thermococcus hydrothermalis
producing an aminopeptidase (EC 3.4.11.-) was isolated from deep-sea
hydrothermal vent located on the East Pacific Rise. The purification steps o f
this enzyme included ammonium sulphate precipitation, Q Sepharose High
Performance chromatography and Sephacryl S-200 H R chromatography.
The enzyme was more active against H-Leu-para-hitroanifide (H-Leu-p-NA)
than against H-AIa-p-NA. The partially purified aminopeptidase displayed
optimum activity around pH 7.0 and the temperature optimum o f the
enzyme was 85C. This aminopeptidase showed a remarkable stabilit)
against heat and organic solvents.
(1) Borman S. (1991) Chem. Eng. News, 4, 31-33.
(2) C o w a n D.A., Smolenski, K.A., Daniel R.M. and Morgan H.W. (1987)
Biochem. J., 247, 121-133.
(3) Hanner M., Redl B. and St6fller G. (1990) Biochem. Biophys. Acts.
1077, 148-153.

(We/17/313) T h e

[LDH-like] Malate dehydrogenase

from
Methanococcus Jannaschii.
D. Madern and G. Zaccai.
lnstitut de Biologie Structurale, LBM, 41 avenue des Martryrs
38027 Grenoble cedexl, France.

L-Malate (MalDH) and L-Lactate dehydrogenase (L-LDH) are

members of an homologous class of 2-ketoacid NAD-dependent
dehydrogenases. They have been, purified and characterized from a
wide variety of prokaryotes and eukaryotes. Although,there are
similarities in their three dimensional structure and they present some
sequence identity, MalDH and LDH use specific substrats.The
sequence, structure analysis and folding pathways of various MalDH
have shown that they have diverged into three distinct groups. One
of them displays stronger similarities with L-LDH than with other
MalDH suggesting the existence of a so called L-LDH-Iike MalDH
group, compared to dimeric mitochondrial and cytoplasmic MalDH
[ 1 , 2 , 3]. In order to refine our knolwedge of this group enzymes,
we are characterising the properties of the recombinant LDH-like
MalDH from the hyperthermophilic archaeon Methanococcus
Jannaschii. A PCR DNA fragment was generated and introduced in
the expression plasmid pT7-7. The protein expressed in E. Coll. was
purified to homogeneity. A gel filtration experiment using Sephacryl
$200 calibrated with proteins of well defined Stokes radius showed
that the protein is a tetramer. The enzyme displays a high kinetic
thermal stability determined by residual activity measurements that
were in agreement with the high chemical stability determined by
changes in Far-UV circular dichroism spectra. The enzymatic activity
was typical of a hyperthermophilic protein with a continuous
activation up to 90C. A striking is that Mj. LDH-like MalDH uses
NADPH rather than N A D H for oxaloacetate reduction. Moreover,
the NADPH enzymatic activity was dependent on the presence of
divalent cations.
(1) Cendrin et al. (1993. Biochemistry. 32, 4308-4313.
(2) Richard et al. (1999 Submited to Stucture, folding and design.
(3) Madem et al. (1999) Submited to Stucture, folding and design.

s358

(We/17/314)

Abstracts FEBS'99

Antarctic Moraxella TAC II 25 elongation factor Tu

M. Masullo, B. de Paola, O. Fattoruso, P. Arcari, V. Bocchini
Dipartimento di Biochimica e Biotecwologie Meeh'che Universit~tdi
NapoR Federico 11, Via Sergio Pansim 5, 1-80131 NapolL Italy.

Structural studies on ubiquitous macromolecules are a convenient approach

for the understanding of the structure-function relationship in proteins. This
category includes the elongation factors Tu and lc~ which are the carriers of
the aa-tRNA onto eubacterial and archaeal or eucaryal ribosomes respectively.
In this communication the isolation and the characterisation of the elongation
factor "fu from the Antarctic Moraxella TAC II 25 (MoEF-Tu) is reported.
The Antarctic bacterial strain Moraxella TAC 1I 25 was grown on LB medium
at 4C. Followed by its ability to form a binary complex with [3H]QDP at
15C, MoEF-Tu was purified to homogeneity by chromatographic techniques.
A Moraxella TAC II 25 genomic library was constructed and screened using
as probe a degenerated oligonucleotide encoding the amino acid sequence
KNMITGA which is conserved in the N-terminal region of all EF-Ict and EFTu known so far (1). The screening allowed the isolation of a recombinant
clone which contained the gene encoding MoEF-Tu which most likely is part
of a str-operon like structure as it occurs in E. coll. Experiments are in
progress to clarify whether in Moraxella there is more than one copy of the
EF-Tu gene.
MoEF-Tu is a monomeric protein with a Mr of about 47500 with the Nterminal residue blocked. It binds GDP or GTP at 1 to 1 molar ratio with an
affinity at 15C for the former that is about 10-fold higher than that for the
latter. In addition, MoEF-Tu elicits a GTPasc activity stimulated by the
antibiotic kirromycin thus resembling the behaviour of E. coil EF-Tu (2).
Both nucleotide binding and GTPase activities were used as tools to evaluate
the heat stability and the thermophilicity of the enzyme. A comparison with
homologous proteins isolated from mesophilic and thermophilic sources will
be made.
1) Baldaufet al. Prot. Natl. Acted. Sci. USA, 93, (1996), 7749
2) Fasano O. et ah J. Biol. Chem. 257 (1982), 3145

(We/17/315)

Haloalkaliphili archaeon Natrialba magadii contains a family of

genes encoded flagellins.
L MesheheryakovaI. I. Serg~ova I. A. Serga.novI A. Metlina2,
V. Ksenzenkot, O. Fedorovt
tlnstttute of Protein Research. &4S, Pushehino, Russta. 2Belozerlo"
Institute of Physico-Chemical Biology. MSU. Moscow, Russta

Flagella of Archaea are distinct from bacterial ones in a

number of interesting features. Archaeal flagella are two times thicker
than bacterial ones and they are composed of multiple flagellins.
Archaeal flagellins undergo posttranslational modification, for
example glycosylation. Flagellin genes have been sequenced only
from two branches of Archaea. Haloalkaliphilic archaea, which live
under extreme conditions of high salt concentrations (4M NaCl) and
pH values near 9.5 are intriguing and poorly understood biological
objects. Up to now virtually nothing was known about the genetic
organisation and structure of flagellins from these organisms.
Our current work is devoted to the cloning and sequencing of
the genes encoding flagellins ofNatrialba magadii. To clone the genes
we have used several different strategies. Successful identification of
the fragment containing one entire gene (flaB2) and two fragments of
the other genes (flaBl, flaB3) has been achieved by screening of
genomic library with the flagellin gene from halophilic archaeon
Halobacterium salinarum. Following cloning of one more DNA
fragment and inverse PCR technique allowed us to complete
sequencing of the locus (flaB1 - flaB3) and to identify another
flagellin gene (flaB4). The four genes are arranged in one tandem
repeat. The N-termini of Natrialba magadii flagellins show a high
degree of homology among themselves and with other known archaeal
flagellins.
This work was supported by grant from the Russian Foundation
for Basic Research (.g_o98-04-48318).

The Moraxe[Ia strain was supplied by Pro[? ( ~ Gerday (Liege, Belgium).

Work supported 3)" MURST (PRIN 97) and (',,VR (Rome), (1taD,)

(We/17/316)

Extremophiles and Polysaecharides

B Nicolaus L Lama M C Manca A Panico A Gambacorta
1CAHB C . V R l'ta Tomno 6, 80072Arco Fehce I\a~, ftal~

(We/17/317)

Regulation of steroi biosynthesis in halophilic yeasts

isolated from salterns
U. Petrovi~ a, N. Gunde-Cimerman b, A. Plemenita~ a
Institute of Biochemistry, Medical Fac , University of Ljubljana, Slovema
National lnstttute of Chemistry, Ljubljana, Slovenia

Extremophiles, with their uniquely stable cellular constituents, will play an

increasingly important role in the biotechnological industry of the future. It
is widely accepted that extremophiles offer important insights into the
biology and evolution of many organisms, and they will provide a valuable
resource for exploitation in novel biotechnological processes
There exists relatively little information concerning polysaccharide
production by extremophiles and until now it was not clear whether these
organisms were likely to prove a useful source of polymers. However, the
number of extremophiles is rapidly increasing, making available new
resources for the exploitation of molecules with interesting properties.
Via controlled fermentation processes several bacterial polysaccharides can
now be "tailor made" produced starting from common cheep sugars
In this communication we report the production and the characterization of
extracellular polysaccharides produced by Haloarcula s p, Bacillus
thermoantarcttcus and new thermophilic isolate
B. thermoantareticus produces two different polysaccharides named EPS 1
and EPS 2, both containing sulfate groups. EPS 1 was a
heteropolysaccharide of which the repeating unit was constituted by four
different ct-D-mannoses and three different [3-D-glucoses. EPS 2 was a
mannan with a molecular weigth = 300,000 Daltons, and four different s - D marmoses were found as the repeating unit
Studies of the chemical structure of these molecules, substituent
identification, and improving them by using fermentation technology will be
reported.
This work was financially supported by PNRA and Regione Campania legge
n41

Halophilic yeasts are a novel class of extremophilic organisms. Representatives

of halophilic black yeasts, Hortaea werneckii, Phaeotheca triangularis and
Trimmatostroma sp. were recently isolated from saltems as their natural
habitat [1]. Halophilic microorganisms adapt to adverse environmental
conditions also through the alterations in membrane properties [2]. Sterols are
essential part of membranes in eucaryots. We studied the regulation of 3hydroxy-3-methylglutaryl coenzyme A reductase (HMG R) as a key regulatory
enzyme in the sterol biosynthesis. The activities of HMG R were measured in
halophilic yeasts / t werneckii, P. triangularis and Trimmatostroma sp. and
compared to that of mesophilic yeasts Saccharomyces cerevisiae and
Schizosaccharomyces pombe. The correlation between the abiotic factors of the
habitats from which yeasts had been isolated and optimal conditions for the in
vitro activity of HMG R was fotmd. Differences in basal activities of HMG R
as well as in sterol mediated regulation of HMG R between extremophilic and
mesophilic yeasts [3] were also found. These results suggest that halophilic
yeasts regulate sterol biosynthesis through the activity of HMG R in a different
way than mesophiles which might be a consequence of a different sterol
composition and ecology of the halophilic yeasts. We propose a new definition
for eucaryotic extremophiles.
[I] Gunde-Cimerman N e t al., Environmental biotechnology (Eds. Vemchtert
H, Verstraete W), (1997), 189.
[2] Driessen AJM et al., FEMS Microbiology Reviews, 18, (1996), 139.
[3] Hampton R et al., TIBS, 21, (1996), 140.

Abstracts FEBS'99

(We/17/318)

Adaptations of halophilic black yeasts to osmotic stress

a N. Gunde - Cimerman. ~'P. Zalar, b A. I~lemenita~

s359

(WeJ17/319)

a Nattonal lnstttute o! ('hemrstJ3'. Llubllana, Slovema

b lnstttnte o/Btochemtstt T. Medtcal Fa~ , U ~verstty o[ Llub/lanu Slovem,

Thermostable Protease and Esterase Enzymes

Important As Biocatalysts
H.S. Toogood ~, E. Hollingsworth ~, J.A. Linlechild ~, R.
Brown 2 & S. Taylor2 ~University of Exeter, Exeter, U.K. ~Chirotech
Technologies, Cambridge,U.K.

Hypersaline waters of salterns, with salinities ranging from 3 - 32% of NaC

provide very special living conditions for halotolerant and halophilic
organisms. Because of extreme salinities, low water activity and high U \
irradiation this habitat is often temed an extreme environment, so fa
considered to be inhabited only by certain genera of archaebacteria, eubacteri~
and algae. Recently we found out that certain genera of fungi are als~
inhabitants of such waters. The mycobiota of hypersaline waters is mainl,.
dominated with dematiceous meristematic polymorphic fungi. Among isolate~
black yeasts from hypersaline waters in the Mediterranean area in Slovenia am
Spain, isolated by us, are various ascomycetes belonging to the genen
Hortaea, Trimmatostroma, Phaeotheca, Cladosporium and Aureobasidium
They are extremely tolerant against high salinities (up to 32%), desiccation am
osmotic stress. Their temperature tolerance increases with increasin l
dehydration of the fungal thallus [1]. As response to high salinitie
multilayered cell walls are developed, and changes in membrane sterols occur
Considering the results of this study, a possible new definition of the tern
halophile is discussed, with regard to fungi present in the saltems as thei
natural environment.

Several industrially useful enzymes have been isolated from

hyperthermophilic archaeal species. These include a cysteine protease and
aminoacylase from Thermococcus litoralis, a deep sea isolate. The enzymes
have been cloned and overexpressed. The protease is part of a family of
pyroglutaryl carboxypeptidases and the structure of this enzyme has recently
been determined [1-2]. Site-directed mutagenesis experiments have been car
out with this enzyme to understand its thermostability (75C optimal
temperature) and to change the active site cysteine to a serine residue. Other
genes present on the 4kb clone insert are a permease and another protein of
unknown function also found in other archaeal genome sequences. In
addition, other Thermococcus species and other archaea are being used to
produce genomic DNA expression libraries which are being screened for
activity towards a variety of ester substrates.

[ 1] K. Sterflinger, Antonie van Leeuwenhoek, 74, (1999) 271.

1. Singleton et al., Structure, 7, (1999) 237

2. Singleton et aL, Extremophiles (1999) in preparation

(We/17/320)

Sterol composition of halophilic yeasts from salterns

M.Turk,a J.O.Grimalt,b N.Gunde-Cimerman,c A.Plemenita~ a

A major limitation in the use of enzymes in chemical synthesis is that

they tend to be relatively unstable under the conditions of the
biotransformation. Thermostable enzymes may be more suitable for this
purpose because higher temperatures can increase the solubility and diffusion
of reactants, and decrease the viscosity of the solution. They also tend to be
inherently more stable to organic solvents.

(We/17/321)

Biosynthesis and properties of an invertase from

psychrophilic yeast Leucosporidium antarcticum
M. Turkiewicz, M. Pazgier, S. Bielecki

o lnstttute of Btochemistry, Medical faculty. University ofLjubljana. Slovenia

b Department of Envtronmental Chemistry (CID-CSIC), Barcelona, Spare
' National Institute ofChemtstry, Ljubljana, Sloventa

Variety of microorganisms are capable of living in hypersaline

environments. Among them are also halophilic black yeasts which were
recently isolated from solar salterns as their natural habitat [1]. In the
adaptation to high salinity the compositional changes of the membranes may
play an important role [2]. Since sterols are an important part of the
membranes, we studied the sterol composition and portion in the
membranes of halophilie black yeasts Hortaea werneckii, Phaeotheca
triangularis,
Trimmatostroma sp. and halotolerant black yeast
A ureobasidium pullulans in comparison to mesophilic yeast Saccharomyces
cerevisiae. Differences in sterol composition between halophilic and
mesophilic yeasts were found. Ergosterol is not the major sterol component
in the cells of halophilic yeasts. Decrease in ergosterol content and increase
in 4ct,24-dimethylcholest-7-en-313-ol together with other sterols correlate
with the ability of microorganisms to adapt to high salt concentrations.
These observations allow us to speculate that membrane sterol profile is a
consequence of adaptation of extremophilic microorganisms to hypersaline
environments and it could serve as one of the markers for halophilic yeasts.
[1] Gunde-Cimerman N e t
al., Environmental biotechnology (Eds.
Verachtert h, Verstraete W), (1997), 189.
[2] Tunblad-Johansson I et al., Biochim. Biophys. Acta, 921, (1987), 116.

Institute of Techmeal Biochemtstry, Techmcal Umverst~,

4/10 Stefanowsktego Str, L6dY., Poland

Enzymes

of

extremophilic

microorganisms,

existing

under

unusual

conditions, are potentially useful. Recently more attention has been paid to
enzymes from microorganisms originating from permanently cold, polar
marine environment, which exhibit high activities and enhanced catalytic
efficiency at low temperatures [1]. We report on dynamics of biosynthesis
and some properties of an invertase produced by psychrophilie, endemic yeast
strain (optimal and maximal growth temperature, 15 and 22C, respectively)
of Leucosporidium antarcticum, isolated from Admiralty Bay waters.
Maximal enzyme biosynthesis occurs at 6C and is 50% higher than at the
growth optimal temperature. 90% of the invertase activity is bound to cells,
and only 10% is excreted to a growth medium. The enzyme bound to cells
can not be salted out and is a counterpart of the external invertase from
mesophilic S. cerevisiae [2]. The enzyme from L antareticum was purified
using DEAE-cellulose and ConA-Sepharose chromatography. Invertase is
stabilized and activated by Mg 2+ and Mn z ions and most active at 25-30C
(at 0C it exhibits more than 30% of maximal activity) and pH 4.5. It is
extremely thermolabile since it retains total activity up to 12C for 30
minutes. The enzyme prefers saccharose to raffinose as a substrate. Further
studies on invertase are in progress.
I.

Feller G. et aL, F E M S Mierobiol Rev., 18 (1996), 189-202

Goldstem A. and Lampen J.O., Meth Enzymol., 42 (1975), 505-511

s360

Abstracts FEBS'99

3.3 Programmed cell death

(We/3.3/322) Monomeric Bax is inactive in channel formation in liposomes
and in triggering cytochrome c release from mitochondria.
Bruno Antonsson. Sylvie Montessu]t, Robert Eskes, and Jean-Claude
Martinou. Serono Pharmaceutical Research Institute, Plan-les Ouates,
Geneva, Switzerland

Bax is a Bcl-2 family protein with pro-apoptotic activity that

can form channels in lipid membranes. The protein has been shown
to trigger cytochrome c release from mitochondria both in vitro and
in vivo. Recombinant human Bax isolated in the presence of
detergent was after purification found to be present as a Box
oligomer with an apparent molecular weight of approximately
160,000 Da on gel filtration. When Bax was isolated in the absence
of detergent the purified protein was monomeric with a apparent
molecular weight of 22,000 Da. Oligomeric Bax showed dimers
resistant to SDS on SDS-PAGE, no dimers were detected in
monomeric Bax. The Bax oligomer formed channels in liposomes
and triggered cytochrome c release from isolated mitochondria,
whereas the monomeric Bax was inactive in both respects.
Incubation of the monomeric Bax with 2 % octyl glucoside induced
oligomer formation and the protein subsequently displayed channel
forming activity in liposomes and it triggered cytochrome c release
from mitochondria. Triton X-100, NP-40 and n-deoxylmaltosid also
activated monomeric Bax, whereas CHAPS had no activating
effect. In cytosolic extract from mouse liver Bax migrated at a
molecular weight of 24,000 Da on gel filtration, after incubation of
the extract with 2 % octyI glucoside Bax migrated at approximately
140,000 Da.

(We/3.3/323)

Dexamethasone protects hepatocytes from

apoptosis
B. Bailly-Maitre, G. de Sousa, and R. Rahmani
Laboratoire de Pharmacotoxicologie Cellulaire et
Mol~culatre, Centre INRA, 06606 Antibes, France.

Hepatocytes in primary-culture dedifferenciate quickly and die

spontaneously by apoptosis. However, some types of cytochromes
P450 (CYPs) inducers were shown to exert a protective action, in
delaying these phenomena. During the last several years, a family of
aspartate-substrate cystein proteases - the caspases - have been
identified as the central executioner of apoptosis. The ca::oase 3 is
considered as essential for the execution of cell death, throu.~h the
cleavage of numerous nuclear (TIAR, PARP...) and cytoplasmic
substrates (1)
The aim of our work was to determine the intracellular factors
involved in this reduction of cell death. We studied the effects of 3
classical CYP
inducers:
dexamethasone, DEX
(CYP3A),
phenobarbital (CYP2B and CYP3A) and 3-methylcholanthrene
(CYP1A). Three days after seeding, human and rat hepatocytes
primary-cultures exhibit the typical features of apoptotic eelS, (DNA
fragmentation, membrane blebbing). We confirmed that DEX ~25gM)
increases the hepatocyte's survival, over 9 days of culture (MTY and
RN viability tests) and induces CYP3A expression (Norther- and
Western-blots, enzyme activity) (2). This effect is associate(, with an
absence of DNA fragmentation in treated cells (DNA-fragmentation
assay, TUNEL-effect). DEX also significantly reduces the activity of
caspase 3 (60%) and the expression (Western-blot) of its endogenous
substrate TIAR. Finally, the cellular apoptosis susceptibility protein
(CAS), which appears to play an important role in tumor necrosis
factor-mediated cell death and cell proliferation, is also inhibited,
On the whole, although DEX is a well-known inducer of apoptosis in
lymphoid cells (3), our data clearly demonstrate that this
glucocorticoid has anti-apoptotic effects in human and rat hepatocytes.
References:
1. D., Nicholsonet N., Thornberry, TIBS, 22, (1997) 299.
2. P., Maurel et al., in Cytochromes P450 Metabolic and Toxicological Aspects, C.,
Ioannides, Ed. (1996) 241.
3. M., Yamamotoet al., Hepatology, (1998) 959.

(We/3.3/324)

Proteome of human endothelial cells during apoptosis

B. Baudin a'b, A. Mailloux ~, A. Bruneel ~, B. B6n6teau-Burnat ~
"Biochimle ,4, H6pital Saint-Antoine, AP-HP, 75012 Paris
bBiochzmie G6n&ale et Glycobiologie, Facult6 de Pharmacie, Paris V,
75006 Paris, France

(We/3.31325)The serum responsive factor, a lie)' regulator of programmed

cell death in T lymphocytes. BertoIotto C. Ricci J. E. Luciano F
and Auberger P. (',IF 96-05. .qc'ttvalto;7 ties cell;;Ic~
hdmatopoidtiques, ,q v de valonthro.~ e, 061 O- .\l( 'E cede.v 2

We previously showed that a number of anticancer drugs induce apoptosis of

human endothelial cells cultured from umbilical vein (HUVEC), eather drugs
with both in vttro and in vtvo endothelial toxicities such as during cancer
chemotherapy

(bleomycin,

adriamycin...)

or

drugs

without

reported

endothelial toxicity (etoposide, paclitaxel...). We are now studying the

proteome of HUVEC during apoptosis in comparison to non-treated cells.
Using specific ELISAs and western-blots we have shown that HUVEC
undergo apoptosis, as characterized by DNA fragmentation, with Bcl-2 (an
anti-apoptotic protein) down-regulation but without Bcl-xl (also antiapoptotic) nor p53 (pro-apoptotic) expression. The proteome of apoptotic
HUVEC is completed by the determination of other pro-apoptotic proteins
(such as Bax and Fas), also caspases (such as ICE-1 and cpp-32) and the heatshock protein Hsp70. Moreover, by the establishement of their twodimensional electrophoretic pattern, we could characterize most of the proteins
implicated in the apoptotic process, and eventually specific endothelial
regulation factors. For example, we have demonstrated that the protein-kinase
C pathway is involved since HUVEC apoptosis was induced by staurosporine
whereas phorbol-myristate acetate overexpressed Bcl-2 protein. Furthermore,
a heat-shock at 42C also triggered HUVEC apoptosis with Bcl-2 downregulation; previous treatment of the cells with etoposide did not prevent this
effect. N-acetylcysteine, a thiol reducing agent, did not protect HUVEC
toward etoposide induced apoptosis.

Apoptosls or programmed cell death is a physiological process

that plays a crihcal role in the regulahon of inflammation and
immune response. Activation of death receptors such as Fas by its
ligand Fas-L or DR4 and DR5 by TRAIL, induces an apeptotic
program in Jurkat T ceils. We report here the identification of the
transcription factor SRF as a substrate for CPP32-1ike proteases
during Fas- and TRAIL-mediated apoptosis m Jurkat T cells.
Inhibition of CPP32-1ike proteases by Ac-Asp-Glu-VaI-Aspaldehyde (Ac-DEVD-CHO) prevents CPP32 and SRF cleavage,
indicating that CPP32-1ike proteases are responsible for the
cleavage of SRF. Further, PMA and anti-CD3 monoclonat
antibody, which are described to activate p42/44 MAP kinase
pathway in Jurkat T cells, inhibit the cleavage of SRF induced by
TRAIL or by an anti-Fas monoclonal antibody (CH11. Transfection
experiments with a luciferase vector containing the c-fos promoter
also show that CH11 and TRAIL inhibit the transcriptional activity
of the c-fos promoter and that pretreatment with PMA or anti-CD3
monoclonal antibody reverses the effects of the apoptotic stimuli.
Moreover, the effects of CH11 and TRAIL on c-fos activity were
completely blocked by Ac-DEVD-CHO. These results point out a
connection between survival and death cellular pathways in T
tymphocytes.

Abstracts FEBS'99

(We/3.3/326)

s361

Restoration of TNFcc-lnduced Ceramide Generation and

Apoptosisa in KGIa Cells
by the P-gp
Blocker
PSC833. .
a
a
I~
C.Bezombes, N.Maestre, G.Laurent, T.Levade . A.Bctta~eb.
J.P.Jaffrezou a

(We/3.3/327)

"INS~RM E 9910, lnstitut Claudius R~gaud. 31502 Touloll~e bl'an~~'

INSERM U466, CHU Rangued, 31403 Toulouse. Frum'~*

Tumor necrosis factor (TNFcQ is a cytokine having anfitumor

activity against several cellular models. TNFct-induced apoptosis seems
to be mediated by a signaling pathway termed "sphingomyelin-cera~lfide"
pathway, which consists in the hydrolysis of sphingomyelin and the
production of its breakdown product ceramide. Our study s h o ~ that
human myeloid leukemia KGIa cells, which are inherentl3 re~istam to
TNFot and which do not produce ceramide upon cytokine stimulatiotl.
can be sensitized by the use of the P-glycoprotein inhibitor. PS('833.
Co-incubation with 1 pM of this cyclosporin derivative restored lhe
apoptotic potential of 10 ng/ml TNFc~. This effect was associated ~ ith
the restoration of ceramide generation (215 %) and activation of neutral
but not acid sphingomyelinase activity (43 %).

Furthermore. \~c

demonstrate that treatment of KGla cells with 1 ~tM PSC833 led t~ a

three told increase in inner plasma membrane sphingom3eh~l cotltcm.
These results support the hypothesis whereby resistance to I\Vct

Regulation of the cyclin D1/RB signaling pathway during

neuronal apoptosis.
A.L. Boutillier, E. Trinh, P. Kienlen-C. and J.P. Loeffler.
UMR 7519 CNRS, ULP, 21 rue R.Descartes, 67084 Strasbourg. France

Apoptosis or programmed cell death (PCD) is a major mechanism for

eliminating excess cells during the development and the maturation of the
Nervous System. In primary cultures of cerebellar granule neurons,
depolarization and subsequent calcium entry exert essential neuroprotective
effects but the ultimate effector by which calcium blocks apoptosis is not
known. We show that inhibition of calcium entry into cerebellar neurons by
switching from high to low extracellular K concentrations (30 to 5 mM)
induces apoptosis and a rapid accumulation of cyclin DI, a key protein in
the GI/S transition of the cell cycle. Calcium does not regulate cyclin DI at
the transcriptional level but rather controls its rate of degradation. Using
highly specific proteasome antagonists: carbobenzoxyl-leucinyl-leucinylnorvalinal-H (MG 115) and clasto-lactacystin 13-1actone, we show the
degradation ofcyclin D1 by the ubiquitin-proteasome pathway is functional
only at neuroprotective concentrations of K + (30 raM). This suggests that
calciana controls neuronal death by regulating the proteasome-mediated
degradation activity of rapidly turning-over proteins (constitutively
expressed genes or preexisting pools ofmRNA).
Further, increased CD1 levels in pro-apoptotic conditions results in the
activation of its associated kinase, cdk 4. Studies are now directed on the
regulation of the retinoblastoma protein family, a central CDl-assoeiated
protein involved in proliferation and that has been also implicated in
apoptosis. We show that the RB protein, that is highly expressed in
cerebellar neurons, is cleaved during the onset of apoptosis. The molecular
mechanisms involved in this regulation are under investigation.

mediated apoptosis of certain leukemic cells is linked to the d isposabilit3

of the sphingomyelin signaling pool. These data suggest a role Ibr ['glycoprotein in sphingomyelin transverse plasma membrane as 3 nnncu'~

(We/3.3/328)

Caspase-8 activation is an essential event in Paclitaxelinduced apoptosis

D.Braguer, A.Gonqalves, G.Carles, C.Briand
UPRESA CNRS 6032 Facult6Pharmacie 27 bd J.Monhn 13005 Marseine

Amimicrotubule agents, especially taxoids such as paclitaxel (Taxol) and

docetaxel (Taxotere ) and depolymerizing agents such as vinorelbine
(Navelbine), have proven to be highly effective in the treatment of several
malignancies and represent a growing and promising class of cytotoxic
compounds. At cellular level, paclitaxel binds to the 13-tubulin subunits in
microtubules, thus promoting polymerisation of tubulin and disrupting
microtubule dynamics, leading to a sustained mitotic arrest and ultimately to
apoptotic cell death.
Antimicrotubule agents induce apoptosis in the human colon cancer cell line
HT29-D4. As shown by cell cycle analysis and Annexin V ligation-based
methods, such an apoptotic process required mitotic block in the
proliferating HT29-D4 cells. Moreover, G2/M arrest and subsequent
apoptosis were dose dependent (both observed with 100 nM paclitaxel or
docetaxel and 10 nM vinorelbine) and were associated with Bcl-XL
phosphorylation and procaspase-3 cleavage, as revealed by western blot
analysis. By using a colorimetric-based detection assay on living cells, we
showed for the first time that caspase-8 (FLICE/MACH) -a well-known
central component of the CD95/Fas ligation induced apoptotic pathway- was
significantly activated during paclitaxel-induced exposure, concomitantly to
occurrence of the apoptotic process. Such activation was observed only with
concentrations causing mitotic block and Bcl-XL pbosphorylation, suggesting
that these events could be linked. Similar results were also obtained with
depolymerizing agents. Consistent with recent data concerning cytokines and
other anticancer agents with different mechanisms of action, caspase-8 could
be a common mediator for various apoptotic stimuli. However, whether
caspase-8 activation induced by antimicrotubule agents is causally related to
mitotic events or results from an autocrine/paracrine CD95/CD95-L
interaction (both expressed by HT29-D4 cells) remains to be elucidated.

(We/3.3/329)

Molecular and cellular mechanisms of erythrocyte cell

death. An apoptotic phenomenon.
D Bratosin~, J. Estaquierb. F. Petitb, J-P. Tissjerk, 1. Trandabum d,
J-J. Huar(, J-C. Ameisenh & J. Montreuil~.

" hl.slttulul de Btochtmte, P 0 Box 2-2, R0-7~200 Bucure.~tz 2. ~ LVSERM (]]P 9707. 170
Boulevard Ney, b~75877 Parts Cedex 18: ' Statton de I'INRA, 269 Rue Jules Guev.le, b2
59650 l'tlleneuve d'Ascq, a ht~titutul de Btologw. P.O. Box 56-53, R0-79651 Bucurestt; ~
('entre Regtonal de ]?ansfuston Sangttme, B P 2018, 1"~59012 Ldle Cedex; t (buversJtd de.~
Sctence.~ et Tt'~qmol~L~le~ de Ltlle. Laboratotre de ('hsmie B~ologJque, F-59655 l)lleneuve
d 'A.wq t "edex.

Human red blood cells (RBC) survive in the circulatory system for about 120 days
and, every day. 360 billions of RBC are phagocytized. This phenomenon raises the
following question. "By what specific membrane signals do the macrophages
distinguish between the senescent RBC and others?>>. Even if it is generally
asserted that erythrocy~es cannot undergo apoptosis because they lack nucleus and
mitochondria, we have hypothetized that erythrophagocytosis by macrophages
could be regarded as an apoptotic mechanism, on the basis of a series of wellknown characteristics of senescent RBC which are characteristics of apoptosis :
progressive decrease of szze due to a progressive release of vesicles from RBC
membrane, budding of RBC leading to the echinocyte form and then to the spheroechmocyte form with filipodes due to cytoskeleton alteration, desialylation.
exposure ofphosphatidylserine (PS) in the outer leaflet of cell membrane, increase
of protease actlwty
We have undertaken experiments using erythrocytes submitted to conditions of
"accelerated senescence" by incubation at 37C for a few days. The results we
obtained could be summarized as follows: cystem-protease mbibitors inhibit t) the
PS exposure, tO the m vitro capture and phagocytosis of RBC, m ) the m vtvo
clearance of RBC in mice, tv) the RBC hemolysis. They maintain the heated RBC
in their natural discoid form. Cystein-proteases have been evidenced which are
inhibited by cystein-protease inhibitors Identical results were obtained using
nucleated RBC of R a n a incubated at 30C. In fact, cystein-protease inhibitors
block the fragmentation of nucleus, the exposure of PS, the fixanon of propldium
iodide (PI) and the protease activity.
Taking all together, these results strongly support the hypothesis that phagocytosis
of senescent RBC occurs at the end of an apoptotlc phenomenon.

s362

Abstracts FEBS'99

(We/3.3/330)

Anti- and Pro-apoptotic members of Bcl-2 family

control the conductance induced by Adenine
Nucleotide Translocator in planar lipid bilayer.
C. Brenner ~'b, H. Cadiou c, N. Zamzami b, I. Marzo b,
A.S.Belzacq ", H. Duclohier c and G. Kroemer b

(We/3.3/331)

aSchool of Biomedical Sciences. University of St. Andrews. Fife, UK

blnstitute of Gene Biology, Russian Academy of Sciences, Moscow. Russia

a." UPRESA 6022 CNRS-UTC, Compikgne; b: UPR 420 CNRS, Villejuif" c"
UMR 6522 CNRS-Univ. Rouen (IFRMP 23), Mont-Saint-Atgnan.

Apoptosis or programmed cell death can be divided in three phases : initiation,

effection, and degradation. Mitochondrial permeability transition (MPT)
constitutes the critical event that initiates the common phase of apoptosis, i.e.
the effector phase which includes the dissipation of the mitochondrial inner
membrane potential and the release of cytochrome c. MPT is an highly
regulated process, that is notably under the control of members of the Bcl-2
protein family and of the caspases activation cascades [1]. Recently, Kroemer
and co-workers showed that the pro-apoptotic Bax protein binds to the
permeability transition pore complex (PTPC), the proteaceous channel
involved in the regulation of MPT, and that Bax cooperates with the adenine
nucleotide translocator (ANT or ATP/ADP translocator) in the mitochondrial
control of apoptosis [2]. In an attempt to elucidate the molecular mechanisms
of cooperation between the anti-apoptotic protein Bcl-2 and the pro-apototic
Bax with ANT, we investigated the channel activities induced by ANT, and of
complexes of Bax/ANT and Bcl-2/ANT reconstituted into planar lipid bilayers.
Conductances were measured at both the macroscopic and single-channel
levels, and ion specificities were determined (see [3] for methods). In
summary, we found that : (i) atractyloside, both an inhibitor of the ATP/ADP
translocator function of ANT and an inducer of MPT, is able to convert ANT
into a channel; (ii) Bax converts atractyloside-induced ANT openings into
larger and more frequent openings of mild cationic specificity; (iii) Bcl-2 and
ATP inhibits this channel conversion by atractyloside and Bax; and finally (iv)
mutants of Bax and Bcl-2 which are inactive in apoptosis also fail to display
such channel modulation. In conclusion, Bcl-2, Bax and ANT cooperate in the
control of the permeability of the inner mitochondrial membrane in apoptosis
through drastic modifications of channel activity.
[I] Kroemer G., Nature Med., 3 (1997) 614.
[2] Marzo I., et al., J. Exp.Med., 187 (1998) 1261.
[3] Duclohier H.et al. (1997) Chemtracts - Biochem. and Molec. Biol. 10:189206.

(We/3.3/332)

Investigation of the role of mitochondria

in the apoptotic process caused by myocardial ischemia
A. Budriunaite, V. Borutaite, R. Morkuniene
Laboratory of B~ochemtstry. lnstztute for Biomedtcal Research.
Kaunas Medwal Umversity, Etveniu 4. LT-3007 Kaunax, Ltthuania

There is evidence that certain proteins of mitochondrial intermembrane

space (cytochrome c, AIF) when released to cytoplasm activate apoptosis
specific proteases - caspases, particularly, caspase-3 winch is responsible
for nuclear fragmentation [1]. Our previous investigations have shown that
myocardial ischemia causes the release of cytochrome c from mitochondria.
Thus, it might be possible that a part of cells injured by myocardial ischemia
are eliminated by apoptosis. In this study we aimed to determine whether
isehemia itself induces epoptosis in myocardium and if so, whether caspase3, activity of which is considered to be induced in eytoehrome c-dependent
way, is activated during ischemia. Apoptotic cells in myocardial tissue
sections were determined using immunohistochemieal staining method.
Activity of caspase-3 was measured by assaying cleavage of fluorogenic
easpase-3 substrate (DEVD-amc) in the cytosolic fractions of hearts
subjected to various time of ischemia. In the control tissue the number of
apoptotic cells/microscopic field was 0.45_+0.28. The number of apoptotic
cells significantly and progressively increased by 3, 8, 18 and 44 times after
20, 30, 45 and 60 min of ischemis accordingly. The activity of easpase-3
also was higher after longer periods of ischemia: in the control cytosolic
fraction it was 0.052:~0.012 nmol of substrate/min/mg protein, and after 15,
30 and 60 rain ofischemia it increased to 0.060_+0.012, 0.077_+0.009 and
0.148-+0.016 umol/min/mg, accordingly.
The data indicate that ischemia itself induces apoptosis in
cardiomyocytes and the process involves activation of easpase-3. As
ischemia-affected heart mitochondria release cytochrome c that is known to
stimulate caspase-3 activity it is likely that mitochondria are involved in the
development of apoptosis during myocardial ischemia.
[1] J. Cai et al., BBA 1366 (1998) 139.

Organisation, evolution and function of genes coding for

proteins of the d4 family
V. Buchman a.b, I. Mertsalovb, D. Kulikovab,
J. Adu,, L. Koroehkin b

The first member of the d4 family, neuro-d4, was cloned as a neurospecific,

developmentally regulated gene [ 1]. More recently we and another group
identified two other genes, requiem/ubi-d4 and err-d4, that encode proteins
closely related to neuro-d4 [2,3]. A hallmark of the family is the d4-domain,
a double-paired finger motif that consists of two tandemly arranged PHD
finger domains. A single K ~ p p e l - t y p e zinc finger was found in the Nterminal part of the most d4 proteins, but because of differential splicing,
some d4 proteins lack this finger along with a nuclear localisation signal and
a stretch of negatively charged amino acids. It was suggested that d4
proteins comprise a family of neurospecific nuclear factors, although some
of these proteins could have cytoplasmic function(s), gequiem/ubi-d4 is
involved in regulation of programmed cell death in the immune system
because in the IL-3 dependent myeloid cell line, expression of this gene is
necessary for apoptosis [2]. Using microinjections of neuro-d4 expression
plasmids into nuclei of cultured sensory neurons we demonstrated that
overexpression of neuro-d4 rescues these neurons from the death after
withdrawal of NGF. In the presence of growth factors increased level of
neuro-d4 has a stimulating effect on neurite outgrowth. These results and
the pattern of expression of neuro-d4 are consistent with the idea that this
protein plays an important role in embryonic and postnatal development of
the nervous systems. As the first step toward generation of mice with
targeted mutations of d4 genes we have cloned and elucidated genomie
organisation of three mouse d4 genes, d4 genes were also found in
invertebrates (D. melanogaster and C. elegans) but not in genomes of
prokaryotes and yeast. High degree of sequence conservation in evolution,
particularly in the region of d4-domain, once again suggests that these
proteins are involved in important biological functions in multicellular
organisms.
[1] Buchman et al., Nucleic Acids Res., 20, (1992) 5579
[2] Gabig et al., J. Biol. Chem., 269, (1994) 29515
[3] Chestkov et al., Genomics, 36, (1996) 174

(We/3.3/333)

The
death
effector
domain-containing
phosphoprotein enriched in astrocytes PEA-15, is
involved in apoptosis regulation.
B. Canton, M. Fauquet, J Glowinski & H. Chneiweiss
I N S E R M U114/' Collkge de France, Paris, France.

PEA-15 (Phosphoprotein Enriched in Astrocytes, Mr=l 5,000)

is an acidic serine-phosphorylated protein highly expressed in the
central nervous system where it might play a protective role against
cytokine-induced apoptosis. PEA-15 was characterized on the basis
of its high level of expression and degree of phosphorylation in
astrocytes. This protein is phosphorylated on two different seryl
residues, Serl04 being identified as a site of regulation for protein
kinase C (PKC) whereas Serl 16 is regulated by Calcium/calmodulin
kinase lI (CaMKII).
The N-teminus of PEA-15 has a close homology with the
death effector domain (DED) characterized in FADD. the adaptor
molecule apical to the caspase-dependent apoptosis cascade triggered
by FasL and TNF alpha. DED domains mediate the binding of
FADD to caspase-8. We therefore investigated PEA-15 ability to
interact with FADD and/or caspase-8 using in vitro binding assays.
35S-radiolabeled FADD, caspase-8 or PEA-15 were precipitated
with PEA-15 or FADD glutathione S-transferase (GST) fusion
proteins immobilized on glutathione-Sepharose beads. FADD and
caspase-8 associated specifically with GST-PEA-15, although the
interactions of FADD and caspase-8 with GST-PEA-15 were weaker
than those observed with GST-FADD. In addition, GST-DEDPEAl5 (containing the AA 1-79 from PEA-15) was also able to
precipitate FADD and caspase-8 but to a lower extend than the
whole PEA- 15 protein. These results suggested that PEA-I 5 interacts
with DED domain-containing molecules. To further investigate PEA15 function in apoptosis, a GFP tagged molecule was constructed
and stably transfected in NIH-3T3 cells. Clones were selected at high
concentrations of G418. Preliminary results indicate that PEA-I 5
expression protects the cells from TNF-induced apoptosis. Thus
PEA-15 may act as an inhibitor of DED-mediated caspase-dependent
apoptosis.

Abstracts FEBS'99

(We/3.3/334)

Aspirin induces cell death and caspase-dependent

phosphatidylserine externalization in HT-29 cells.
1~ Castafio, M. Dalmau, M Barragan, R. Bartrons and J Gil

s363

(We/3.31335)

tJlltl~l| de Bioquimlca Deparlamcnl de Cirncies Fisiolbgiqucs II

tJlll~CrSItilldC B;ircelon;i Ctllllpus dc Bellvltg 08907 L'Hospitalet S~Illl

r pidemiological studies, clinical observations and animal studies

dcm~nstrate that nonsteroidal anti-inflammatory drags (NSAIDs) can
!,~'~ ent colorectal cancer Apoptosis plays an important role in the control
~,~ colon epithelial cell number NSA1Ds have been shown to induce
,~!,~,ptosis of different cell types, including human colorectal tumor cell
Into, Whether or not aspirin induces apoptosis is still controversial
Ihe induction of cell death by aspirin has been analysed in HT-29
c~lon carcinoma cells Aspirin induced two hallmarks of apoptosis'
Pta'lcal chromatin condensation and increase in phosphatidylserine
c\!crnalization ttowever, aspirin did not induce neither oligonucleosomal
I)~gmentation of DNA, decrease in DNA content nor nuclear
I'~,~:-'rnentation The effect oF aspirin on Annexin V binding was inhibited
b~ 1lie caspasc inhibitor Z-VAD-fmk. indicating the involvement of
<,H~ascs in the apoptotic action of aspirin t Iowever, aspirin did not induce
!,~,le~l?/sis of PARP, suggesting that aspirin does not increase nuclear
, a,pase 3-like activity in HT-29 ceils, This fact could be related with the
.~!\pmal'" fealures of aspirin-reduced apopmsis in HT-29 cells

REACTIVE OXYGEN INTEILMEDIATES REGULATE

CELLULAR RESPONSE TO APOPTOTIC STD, IULI
M-V. Cl6ment a and S. Pervaiz h
~'Optcoh)g3. ' Research Institute, NU~[I. and ~Department of PItvsto/og 3,
A~ttonal Umverstty of Singapore, Singapore 117601

We have previously shown that superoxide anion is a

natural inhibitor of Fas-mediated cell death [1]. We have
nov, extended these studies to the dismutation product of
superoxide anion, hydrogen peroxide (H_,Oz), which is a
known mediator of apoptotic ceil death. The precise
mechanism by which H_,O_, triggers apoptosis is currently
not well understood. Here we show, that cells exposed to
apoptotic concentrations of H,O: (<0 5raM) display a
significant decrease in their intracellular levels of
superoxide anion, whtch is associated with a reduction of
the intracellular milieu, as measured by an increase in
GSFL~GSSG ratio and a decrease in intraceltular pH. Our
hypothesis that a decrease in intracellular superoxide anion
concentration facihtates apoptosis is supported by the
observation that H,Oz-mediated apoptosis could be retarded
in cells in which the intracellular superoxLdc level ts
maintained at or above the cellular baseline b.~ inhibition of
the major superoxide scavenger enzyme CuJZn SOD.
Taken together, our observations indicate that a decrease in
superoxide anion concentration, reduction and acidification
of the intracelinlar milieu constitute a signal for HzOzmediated apoptosis, thereby inducing a reducti~e as
opposed to an oxidative stress.
[ 1] Cldment. A4- !~ amt Stamenkovw, 1. Superoxtde anzon l.~
a natural mhtb~tor o f Fa.~-me~hated cell death. ~'3[BO J.
15(2):216-225. I996.

(We/3.3/336)

Quaternary ammonium-induced
apoptosis in conjunctival cells in vitro
C. Debbasch~, P. Rat a, J.-M. Warne:, C. Baudouin b

(We/3.3/337)

*Uniti de Pharmaco-Toxicologie Cellulatre. C.H.N.O. des XV-XX,

75571 Parts cedex 12, bService d'Ophtalraologle, Hdpital AmbroisePard, AP-Hp, 92104 Boulogne cedex, France

The aim of this study was to evaluate the effects of different

quaternary ammoniums currently used in ophthalmic preparations on
a continuous human conjunctival cell line (Wong Kilbourne derived
human conjunctiva)
Cytotoxicity tests were assessed according to ECVAM
recommendations using microplate cold light cytofluorimetry [1]
Membrane integrity (neutral red test), reactive oxygen species (ROS)
production (dichlorofluoresceine diacetate and hydroethidine tests)
and DNA condensation (Hoechst 33342 test) were evaluated on
living cells treated with different concentrations of benzalkonium
chloride, benzododecinium bromide and cetrimide (0.00001 to
0 01%) alter 15 minutes of treatment
All the molecules tested showed the same in vitro cytotoxicity a
decrease in membrane integrity at 0.005% and 0.01% (p<0.001) was
observed after a short time (15 minutes), using the neutral red test.
Moreover, there was a free radical production associated with a
chromatine condensation due to an apoptotic phenomenon. Such cell
conjunctival damage observed in vitro for low concentrations of
quaternary ammoniums can explain some of the ocular surface
disorders caused by long-term use of preserved eye-drops.
[1] ' P. Rat e t a l Meth. Enzymol., 252, (1995), 331.

Ag-Tetrahydroeannabinol induces apoptosis in prostatic

PC-3 cells via a receptor-independent mechanism.
I. Diaz-Laviada and L. guiz
Departamento de Bioquimica y Biologia Molecular. Facultad de
Medicina. Universidad de Alcald. 28871 Madrid, Spain.

The biologically active compound of marihuana, A9tetrahydrocannabinol (THC), promotes specific effects in humans. Two Gprotein-coupled receptors have been identified as cannabinoid receptors and
are refered as central CB 1 and peripheral CB2 receptor (reviewed in [ 1]), CB 1
receptor is principally expressed in central nervous system although it has also
been detected in other tissues like testis [2] and spleen cells [3]. Cannabinoids
exert a wide spectrum of effects such as alteration in cognition and analgesia.
In addition to psychotropic effects, cannabinoids also have inhibitory effects
on immune function.
We have studied the effects of THC in the human prostatic cell line PC3 which is androgen refractory. First of all, we assessed whether PC-3 line
expressed CB1 receptor. Western blotting results revealed two bands which
correspond to different glycosilation states of the receptor. Addition of THC
to cultured cells caused cellular death detected by depression o fmitochondrial
oxidative metabolism. To investigate whether the cellular death was apoptotic
or necrotic we determined anexin V binding by fluorescence microscopy and
nuclear fragmentation by 4',6-Diamidino-2-phenylindole (DAPI) binding to
DNA. Results showed that THC induced apoptosis in a dose-dependent
manner that was tmximal at 5 gM and 6 days of treatment. Apoptosis induced
by THC was not counteracted either by pretreatment with pertussis toxin nor
by the antagonist AM 251. Moreover, the potent cannabinoid receptor agortist
WIN 55212-2 bad no effect on PC-3 cellular death. Results thus show that
although cannabinoid receptor is present in prostatic PC-3 ceils, THC-induced
cellular death is not mediated by the caunabinoid receptor.
1.
2..
3.

FELDERC. C. and GLASS M. (1998) Cannabinoid receptors and their

andogenonsagenists.Annu. Rev. PharmacoL Toxico138:179-200
G[~RARDC.M. et al. (1991) Molecularcloning of a human cannabinoidreceptor
which is also expressedin testis Biochem. 279: 129-134.
KAMINSKI
N.E. et al. (1992) Identificationofa functionallyrelevanteannabinoid
receptor on mouse spleen cells that is involved in cannabinoid-mediatedimmune
modulation. Mol. Pharmaco126: 532-538.

s364

Abstracts FEBS'99

(We/3.3/338)

Cicloheximide preventS I o v a s t a t i n induced apoptosis in rat brain neuroblats.

N.Garcfa ~, AM. Alvamz~, MI TorC, A. Montes ~, MJ LorenzC.
aBiochemistry and ~Physiology Department, Alcal~ University, Madrid,
Spain. ~-'low Cytometry Center, Complutense University, Madrid, Spain.

Lovastatin is a competitive inhibitor of 3-hydroxy-3methylglutaryl coenzyme A reductase, a key enzyme in

cholesterol synthesis. We have previously showed that
lovastatin is a potent inducer of apoptosis in spontaneously
immortalized rat brain neuroblast (P cells). Many studies have
shown that inhibitors of macromolecular synthesis abolish
neuronal death induced by a broad range of stimuli. The aim
of the present work was to investigate the effect of
cicloheximide, a protein synthesis inhibitor, on the lovastatininduced apoptosis.
P cells were grown in Ham's F-12 supplemented with
10% fetal bovine serum. Twenty-four hours later, the medium
was changed to one containing lovastatin (10p.M) in the
presence or absence of different concentrations
of
cicloheximide (0.001-0.5 ~tM) and the incubation was continued
for a further 24 h. The following aspects were examined in all
the experiments: cell viability by the M'IT assay, cell
morphology by confocal microscopy and DNA fragmentation
by agarose gel electrophoresis . The exposure of cells to
lovastatin (101.tM) for 24h produced a decrease in the number
of viable cells, chromatin condensation, fragmentation of
nuclei, and DNA fragmentation. These apoptotic features were
prevented by cicloheximide in a dose-dependent fashion.
Ciclohexiraide alone produced no effect on celular growth.
These findings suggest that apoptosis
eficited by
lovastatin in rat brain neuroblasts requires functional protein
synthesis. Further work will be necessary for the identification
ofprotein/s implicated in the apoptosis mechanism activated by
lovastatin in these cells.

(We/3.3/340)

TOBACCO, GENOME AND SQUAMOUS CELL CARCINOMA

OF THE HEAD AND NECK
D GIMONET*, F L1AUTAUD-ROGER,K MOUTEL
Centre R6gionalde lutte contre le cancer BP 171, 51056 REIMS CEDEX

The patients with an environmental cancer have a genetic fragility (1).

Furthermore, an apoptosis deficiency is involved in carcinogenesis. This
study aimed to look for thts fragility and alteration of apoptotic functions on
the circulating lymphocytes (Ly). We have taken three groups of 15 donors '
healthy no smoker controls (NSC), healthy smoker controls (SC) and patients
with squamous cell earcmoma of the head and neck (P).
For each patient, age, alcohol consumption and number of "year-packets"
were registered. The circulating Ly are cultivated m vitro, sttmulated by PHA
and treated by bleomycine. The percentage of necrotic Ly was assumed by
Trypan blue, that of apoptotic Ly by Giemsa staining and flow cytometry as
well as the chromatin texture by cell image analysis after Feulgen staining.
The latest parameter that expresses the genome fragility, distinguishes the P
from the SC . 91,4 u.a. _+ 10,5 (SD) vs 111,5 + 12,8 (t: 4,7; ddl: 28,
p < 0,001). This result is independent of age, tobacco and alcohol
consumption The percentage of apoptosis is different between NSC and SC :
1,15 (arcsin~/AP) _+0,36 (SD) vs 1,76 _+0,46 (t : 3,6 ; ddl : 28 ; p < 0,01 )
The chromatin texture, which can detect a simple reciprocal translocation
(2), appears to be a cancer marker of squamous cell carcinoma of the head
and neck.
[1] Cloos J e t al. Cancer Lett; 74, (1993), 161-5
[2] Liautaud-Roger F et al. Analyt Cell Pathol : 4, (1992), 421-8

(We/3.3/339) P11KP IS k STRONG APOPTOSIS INDUCERIN HELA CELL LINE

E Giannoni., P. Cirri, P. Paoli, T. Fiaschi, G Camici, G
Manao, G Raugei and G. Ramponi.
Dlpartmtento dt Sctenze Btochtmiche, Universita degli Studt dt P)renze,
wale Atorgagm 50. 50134 Firenze, ]taha

p l l KP is a protein composed of 98 amino acids that is widely

distributed in many mammalian tissues where it is present in two
isoforms Until now, p l l KP was classified as an acylphosphatase since
it is able to hydrolyse tn vitro several physiological and synthetic
acylphosphates Very little is known about its physiological function.
An involvement of the protein in cellular calcium and sodium
homeostasis was hypotized since p l l KP can hydrolyse lit vttro the
phosphoenzyme intermediate of different membrane pumps. In
addition, some evidences suggested that p l t Kv is involved in cellular
differentiation process. In fact, increase of both isoforms can be
observed in the K562 erythroid cell line after induction of
differentiation.
In order to study the m vivo behaviour o f p l 1Ke, HeLa cell line were
transiently transfeeted using an eukaryotic vector expressing the
fusion protein pl IKP-EGFP which allows to follow the expression of
the protein by cytofluorimetric analysis. A growth curve of the
transfected cells performed using Trypan Blue, showed a high level of
death-rate in cells expressing the fusion protein
p l l r:p-EGFP wi th
respect to control cells expressing EGFP alone. The analysis of the
pl tKP-EGFP level revealed that cells transfected with pl Ir'V-EGFP
showed a lower expression with respect to GFP transfected cells,
demonstrating that a very low level o f p l IKP-EGFP is allowed for cell
survival. Surprisingly, the transfection of a mutated form of pl ]KP
with no acylphosphatase activity shows the same behaviour as wild
type enzyme with respect to cell survival. Hence, the physiologic role
of pl 1Kris not linked to its acylphosphatase activity The same data
were obtained in mouse fibroblast NIH3T3 cells, demonstrating that
the induction of cell death due to overexpression of p l l r:P is not
restricted to a single cell line
Cytofluorimetric analysis of DNA fragmentation of plI~P-EGFP
transfected cells evidenced a pattern typical of apoptosis These
results, even supported by time lapse analysis, suggested that pl I r;P is
a very powerful inducer of apoptosis in HeLa and N1H3T3 cell lines.

(We/3.3/341)

Interaction of MPP with the plasma membrane of rat

cerebeHar granule cells
Gonzalez-Polo RA, Mofa A, Centeno F, Soler G and Fuentes JM
D.Bwqufmaca y Biol. Mol. y Gen. Fac. de Veterinaria. Cdceres. Espmla.

MPP + is a neurotoxin used as a tool for studing various

neurodegenerative injuries like Parkinson disease, it is an inhibitor of the
mitochondrial respiratory complex I, the way MPP penetrates into the cell
and its putative interaction with membranous components are still a matter
of debate. We investigated this problem using primary cultures of rat
cerehellar granule cells and determined apoptosis cell death by M T r
reduction and DNA fragmentation. It has been hypothetized that MPP
modulates the aperture of the calcium channel associated with the Nmethyl-D-aspartate receptor (NMDA) resulting in an increase intracellular
calcium concentration which is believed to be responsible of various
necrotic and apoptotic processes [1]. However our findings indicate that in
the presence of a specific inhibitor (MK-801) of this calcium channel,
apoptosis cell death induced by MPP + is unaffected suggesting that MPP is
not modulating the Ca z~ entrance via the NMDA receptor. Moreover, we
investigate whether the cationic aminoacids and the dopamine transporters
play a role in the entrance of MPP + into the cells. Since MPP was found to
be a good substrate for the dopamine transporter in dopammergic cells [2].
Our results indicate that in the presence of MPP + cell death was
significantly reduced (35%) when incubated with either lysine, arginine or
histidine. Similar effects were observed (death reduction by 40%) when the
cells in the presence of MPP + were incubated with a specific inhibitor
(GBR-12909) of the dopaminergic transporter. Moreover the addition of a
cationic aminoacid and GBR-12909 results in additive protection effects
against apoptotic cell death. Taken together our experimental data suggest
that both transporters may facilitate the entrance of MPP +into the cells.
[1] Camins et ak, J.Neural.Transm., 104 (1997), 569.
[2] Schinelli et al., J.Neurochem., 50 (1988), 1900
This work was supported by the Grant of the spantsh CICYT PB 95-1227.
E-mall :jfuentes@unex.es

Abstracts FEBS'99

(We/3.3/342)

Role of FA proteins as a regulator of

executioners of apoptosis
C. Guillouf, Moustacchi E., Rosselli F.
lnstitut Curie, UMR-218 CNRS, 26 rue d'ulm, 75005 Paris, Franc

Fanconi anemia (FA) is a human genetic disease featuring cancer

predisposition, genetic instability and DNA damage hypersensitivity.
Although abnormalities in DNA repair and cell cycle checkpoint have been
proposed as the underlying defect in this syndrome, these hypothesis did
not provide fully explanations of the complex phenotype. Although not
exclusive of such possibilities, alterations in the control of apoptosis might
account for the pleiotropic phenotype of this syndrome. Indeed, we and
others have previously reported a deregulation of the apoptotic response to
DNA damaging agents and also to others apoptotic stimuli non-related to
DNA damage, suggesting that the products of the FANCC protein
contributes to the regulation of apoptosis. To better define the role of the
FANCC protein in the apoptotic pathway, we have extended our work to
the analysis of different molecular parameters modified during the execution
of the apoptotic program. Our results clearly show that apoptosis is not
responsible for the hypersensitivity of FA to cross-linking agents, a
characteristic of these cells. Moreover, it is shown that the FANCC protein
acts at a step of the apoptotic program preceding the activation of the
caspase 3 et after the modification of the mitochondrial transmembrane
potential. This has been described as a switch where cells can be pushed
towards either apoptosis or necrosis. Altogether, our data indicate that the
FANCC protein acts as a regulator of the commun execution phase of
apoptosis.

(We/3.3/344)

RANTES regulates Fas-L expression and killing by

HIV-specific cytotoxic T cells.
F.Hadida, V.Vieillard a, UMollet, I.Clark-Lewisb,
M.Baggiolini c , PDebr6 UMI~CNRS 7627Pitid-Salpbtridre.Paris

s365

(We/23.3/343) Activation of MAP-kinases by urea-induced cell death

through small G-protein Rho family in mIMCD3
Guivarc'h D., Badier C., Ripoche P. and Rousselet G.
Dept. Biol. Cell. MoI./SBCe,CEA-Saclay, 91191 Gxf/Yvette~France.

mlMCD3 (renal inner medullary collecting duct) cells adapt to

hyperosmotic medium and serve as an useful tissue culture model of
cellular responses to hyperosmolarity. NaCI and urea represent the two
principal components responsible of the high medullary osmolarity. In
a first time, we have examined the role of urea in cell survival. We
showed that the increment of osmolarity up to 400mOsm/kg H20 urea
(U400) induced either necrosis or apoptosis depending of the starting
levels of cultured cells osmolarity, isotonic medium (ISO) or
200mOsm/kg H20 urea (U200) respectively.
Mitogen-activated protein (MAP) kinases are known to be
activated quickly in response to various extracellular signals. Three
types of MAPK were described: the ERK (extracellular signal
regulated kinase) which participates in cell growth and differentiation
process, the SAPK/JNK (c-jun N-terminal kinase) and p38, more
implicated in growth arrest and apoptosis events. We have investigated
their role in urea-induced cell death. We showed that U400 induces a
rapid and transient activation of ERK and SAPK/JNK in cells cultured
in ISO condition, whereas it induced a rapid and sustained activation of
ERK and SAPK/JNK and a transient inhibition of p38 in cells cultured
in U200
Recent studies have shown that the small GTP-binding protein
of the Rho family are an integral part of the pathway linking cell
surface receptors to JNK activation. In our studies, we have analyzed
their possible role in the activation of ERK and JNK in different
conditions where urea induced cell death.We have transiently
transfected dominant negative mutant of each small G-protein Rho in
mlMCD3 in ISO or U200 condition. Activation of ERK and JNK m
U400 was blocked by Racl and cdc42 mutants but not by RhoA.
These results shown the principal implication of small G-proteins in
the activation of JNK and ERK where urea induced cell death.

(We/3.3/345)

Role of proteases in P.gingivalis induced T-cell apoptosis

J.I.Harris and J J TaylorDepartment of Oral Biology, Universtty of Newcastle upon Tyne,
Framhngton Place, Newcastle upon Tyne, NE2 4BW, United Kingdom

aUMR 146 CNRS, L Curie, Orsay DB.R.C., Vancouver "

I. Kocher, Bern

Based on the previous observation that RANTES mediates the cytotoxic

activity of human HIV-specific CDg + T cells via the chemokine receptor
CCR3 [1], we have studied the effect of this chemokine on different
effector CD8 + T cells requiring Fas/Fas-L or pefforin-dependent
cytolytic pathways. In CTLs derived from PBMCs of HIV infected
patients, both the spontaneous and the RANTES-induced cytotoxicity
were inhibited by anti-Fas-L neutralizing antibodies. By contrast,
allogeneic CTLs or NK cells killing through a perforin-based
cytotoxicity, were not affected by RANTES and anti-Fas-L monoclonal
antibody. Accordingly, RANTES significantly enhanced the expression
of Fas-L in a concentration and time-dependent manner in HIV-specific
CTLs while a neutralizing anti-RANTES antibody led to a marked
decrease in Fas-L expression. Finally, cell surface expression of Fas-L
protein in HIV-specific CTLs was also up-regulated by Eotaxin, a
selective ligand for CCR3. The present observations show that the action
of RANTES via CCR3 is necessary to regulate Fas-L expression on HIVspecific CD8+ T cells that kill through the Fas/Fas-L pathway.
[1] Hadida et at. J.Exp.Med. 188 (1998) p.609.

We have previously shown that the periodontal bacterium Porphyromonas

gingqvalis promotes apoptosis in human T-cells; this may be a
pathogenieally important mechanism in periodontal disease. Recently we
have found that the activity of caspase-3, a cysteine protease involved in
apoptosis, was increased during apoptosis induced by P.gingivalis. The aim
of the present study was to determine which bacterial products are
mediators of P.gingTvalis induced apoptosis in this system. To determine
whether the products involved in apoptosis are cell bound or secreted
extracellularly we incubated P.g~ngivalis whole cultures, supematant and
washed bacterial cells with J16S T-cells for 24 hours. The activity of
caspase-3 was measured in a fluorometfic assay using a fluorescently
labeled substrate (DEVD-AFC); incubation of J16S T-cells with an
antibody to the Fas receptor was used as a positive control. We found that
after 24 hours incubation there was an increase in caspase-3 activity with
P.gingtvalis whole cultures and supernatants (p < 0 05) compared to
control cells, incubated with bacterial growth medium, but no increase was
observed with washed bacterial cells. We assayed protease activity using a
BODIPY fluorescently labled casein substrate and found that protease
activity resided in both the P.gingivalis supernatant and washed bacterial
cells but activity was 170% higher in washed bacterial cells. To further
identify the role of proteases from P.gingivalis in the induction of apoptosis
two peptidyl-chloromethylketones (Phe-Phe-Arg-chloromethylketone and
Phe-Lys-2,4,6-trimethylbenzoyloxymcthylketone) which inhibit P.gingivalis
R- and K-gingipains were used. These inhibitors reduced protease activity
in P.gmgivalis cultures by 95% and did not themselves induce apoptosis in
JI6S T-cells. However, introducing the inhibitors into the co-culture
experiments with P.gingtvalis did not cause a decrease in caspase-3 activity
compared to P.gingivalis alone, with levels remaining significantly higher
than controls. These findings suggest that the mediators of apoptosis from
P.gingivalis are secreted into extracellular medium and that proteases from
P.gingivalis do not play a role in the activation of caspase-3 activity in this
system.

s366

(We/3.3/346)

Abstracts FEBS'99

Characterization of the cell death process in SH-SY5Y

human neuroblastoma cells induced by bilirubin
L. S. Hang, 1. Aa. Torgner and A C. Ostvold,

(We13.31347)

'~Leidetl/Amsterdam Center for Drug Research. Leiden Univer.~in'. Leiden.

the Netherlands'

Neurochemical Laboratory, University of Oslo, Norway

The induction of apoptosis in the human neuroblastoma ceil line SH-SY5Y

has been extensively studied, and staurosporine is a well-known inductor of
apoptosis in these ceils. Increased serum concentrations of bilirubin
(hyperbilirubinaemia) are known to induce selective damage to striatal
neurons in neonates. The mechanism for this neuronal damage is unknown.
We have treated SH-SY5Y cultures ~vith bilirubin and staurosporine to
compare these reagents and to examine the cell death process they induce, in
an effort to elucidate whether bilirubin induces death by apoptosis in these
cells. We used antibodies to poly-(ADP-ribose)-polymerase (PARP) to detect
apoptosis by Western immunoblotting, and we detected condensed chromatin
(apoptotic bodies) by light microscopy in cell nuclei stained with aceto-orcein.
Bilirubin-treatment induced cleavage of PARP, indicating apoptotic cell death.
However, chromatin staining showed cytoplasmic irregularities in cells treated
with bilirubin, while typical apoptotic bodies were seen in cells treated with
staurosporine
Comparison of immunoreactivity for neuronal proteins revealed that the level
of some proteins may be regulated by the death processes. The level of IP3
receptor is reduced and spectrin is cleaved into breakdown products by
staurosporine treatment, whereas other proteins remain intact. Spectrin
breakdown products are generated after apoptotic activation of calpain or
caspases. The IP3 receptor is a substrate for calpain [1], and decreased levels
of IP3 receptor are found in several conditions where apoptosis is suggested to
be involved, ~
Alzheimer's disease [2]. The ER-proteins SERCA2 and
calreticulin, the mitochondrial protein phosphate-activated glutaminase (PAG)
and the synaptic vesicle protein synaptophysin were also studied in relation to
the cell death induced. The cells treated with bilirubin and staurosporine and
the reactions of the different parameters were compared using time- and doseresponse studies.
1. A. Magnussonet al, FEBSLett. 323, (1993) 229.
2 L. S Haugel al. Neurodegeneratmn. 5 (1996) 169.

(We/3.3/348)

A protective effect of NF-~B against

apoptosis in Ewing tumor cells.
D. Javelaud, J.Wietzerhin and F. Besan~on.

In in vitro studies aiming at identifying the molecular mechanism leading to

apoptosls, it is of vital importance to have a reliable assay that gives a correct
assessment of the extent of apoptosis at a given moment. We compared
phosphatidyl serine (PS) exposure, cell cycle analysis, caspase activity and
caspase-substrate cleavage to detect apoptosis in rat mammary
adenocarcinoma cells (MTLn3). Three anticancer drugs were used:
doxorubicin, etoposide and cisplatin. First we determined apoptosis by PS
exposure using Annexin V/propidium iodide (AV/PI) staining and flow
cytometry. Doxorubicin (17 pM) caused an increase in percentage of
AV+/PI- cells, with a peak after 16 hrs (33%). This was followed by a
decrease to 28% and 23% after 24 and 48 hrs, respectively, and by a
concomitant increase in percentage of AV+/PI+ cells, indicating necrosis.
With cell cycle analysis, the percentage of cells with subGl/G0 DNAcontent, was also increased after 16 hrs (39%) , but in contrast to AV+/PIanalysis, it further increased to 46% and 67% after 24 and 48 hrs,
respectively. Doxorublcin caused a strong p53 upregulation already alter 8
hrs, which occurred prior to an increase of caspase activity (69, 324, 836 and
654 pmol DEVDase/min/mg after 8, 16, 24 and 48 hrs, respectively).
Cleavage of PARP- and PKC-delta, two typical substrates for caspase-3,
closely followed the increase in caspase activity. The sustained increase of
subG1/G0 cells and caspase activation, but not of AV+/PI- cells, suggests
that analysis of AV+/PI cells does not fully reflect the actual percentage of
apoptosts in time. To investigate this in more detail, we analyzed the effect of
zVADfmk, a general caspase inhibitor, on the above parameters, zVADfmk
(100 ~aM) was slightly effective in inhibiting the formation of AV+/PI- cells
(from 42% to 25%), however, it reduced the number of cells with subGl/G0
DNA-content almost to control levels (from 32% to 10%). This effect of
zVADfmk was associated with inhibitxon of PARP and PKC-delta cleavage.
Similar data were obtained with etoposide and cisplalin. Altogether, the data
indicate that cell cycle analysis ldennfies apoptotic cells more accurately than
PS exposure due to its cumulative character. Combination of cell cycle
analysis with caspase activity assays assures that an increase in the
subGI/GO population is directly related to activation of the apoptotic
machinery.

(We13.3/349)

INSERM u365 In~tltut ('uric. 26 rue d'L'hn. ":5005 Paris. France

The Ewing's sarcoma familx, of tumors are characterized by a translocation

that joins the EWS gene located on chronmsome 22 to the DNA binding
domain of fi~e different members of the ETS family', namely FLI-1, ERG.
ETV1. E I A F and FEV. These tmnors are thought to have a neural
histogenesis based on evidence of neuroectodermal differentiation.
Several studies have revealed a role of NF-~cB in the resistance of different
cell types to apoptotic stimuli although the role played by NF-~B in the
survival of cells of neural origin is controversial. In this study, we
investigated whether NF-KB exerts a protective effect against apoptosis in
Ewing sarcoma (ES) cells. We generated stable transfornlants expressing an
undegradable super-repressor Ibm1 of the NF-~cB inhibitor bcBc~ in an ESderived cell line, E\VV. In these transformants, basal NF-~cB activity Was
reduced and was barely enhanced by TNFc~ which is a rapid and strong
inducer of NF-v,B in EW7 cells. TNFc~. was much more effective at inducing
apoptosis in the bcBc~ super-repressor-expressing cells than in the controls.
In contrast, expression of the super-repressor I~Bo~ did not elthance the
apoptotic response to the cancer chemotherapeutic drugs doxorubicin and
etoposide, which were inefficient inducers of NF-~B in EW7 cells. The antiinflammatory drug sodium salicylate which has been shown to inhibit NF~:B
activation by preventing the degradation of IlcB, also enhanced killing of
EW7 by TNFc~.. These observations sustain the hypothesis that the
resistance of EW7 cells to TNFc~.-induced apoptosis is due to strong and
rapid activation of NF-~B by the cytokine.
The basal levels of NF-~zB activity in two primary and three metastatic ESderived cell lines were also compared. A higher activity was present in the
metastatic cells which correlated with a reduced sensitivity to doxombicin
and etoposide as compared to the primmy ES-derived cells.
Altogether, our data converge to indicate that NF-~eB is protective against
apoptotic killing in Ewing tumor cells. Furthermore they suggest that this
transcription factor CoLdd be a target for therapeutic intervention in this type
of tumor.

Kinetics of anticancer drug-induced apoptosis in mammary

adenocarcinoma cells
M. Huigsloota, R.B. Tijdens a, G.J. Mulder a, B. van de Water a

Mitoehondrial Kvl.3 is crucial for ceramide

triggered apoptosis.
A. Jekle, I. Szabo, L Kun, C. Mueller, F. Lang, E.
Gulbins Dept of Ph3,stolo~'. Umverstty of Tuebmgen, 72076
Tuebmgen. German3

Induction of apoptosis by Fas/CD95 receptor crosslinking or ceramide

treatment results in phosphorylation and inactivation of the voltage
gated K* channel Kvl.3. Using a genetic model, we show that Kvl.3
plays a crucial role in the regulation of caspase 3 activation.
cytochrome c release, depolarization of the mitochondrial membrane
potential and DNA-fragmentation. but not in phophatidylserine
translocation during ceramide triggered apoptosis.
Immunostaining experiments demonstrate that the Kvl.3 channel is
not only localized in the cytoplasmic membrane, but also in the
mitochondria. Further. patch clamp experiments on isolated
mitochondria revealed currents typical for Kvl.3, that could be
blocked by the highly specific inhibitor margatoxin.
Only mitochondria isolated from Kvl.3 expressing, but not from
Kvl.3 deficient ceils show depolarization of the mitochondnal
membrane potential and release of cytochrome c after incubation with
cytosol prepared from ceramide stimulated cells. Similarly, treatment
of isolated. Kvl.3 expressing mitochondria with margatoxin induces
release of cytochrome c and alterations of the mitochondrial
membrane potential.
These
(1)
(2)
(3)
(4)

data suggest that

Kvl.3 is essential for ceramide triggered apoptosis
Kv1.3 is in part localized in the mitochondrial membrane
mitochondrially localized Kvl.3 is regulated by ceramide
inactivation of mitochondrially localized Kv1.3 results in
cytochrome c release and depolarization of the mitochondrial
membrane potential

(supported by DFG and IKFZ)

Abstracts FEBS'99

(We/3.3/350) Supression of Apoptosis Induced by Anchorage and

Serum Removal by p42/p44 MAPkinase

s367

(We/3.3/351)

M. Le Gall, J.C. Chambard, D. Grail, J. Pouyssrgnr and

E. Van Obberghen-Schilling

K. Lemke, M. Hyzy, J. Konopa and A. Skladanowski

Department of Pharmaceutical Technology and Biochemistry,
Technical University of Gdansk, Gdansk, Poland

C~ntre de Biochmde. CNRS UMR 6543. Ntce. France

In addition to growth factors, most non-transformed cells require

anchorage to extracellular matrix components in order to survive and
proliferate. Suppression of anchorage, like growth factor removal, induces
not only cell cycle arrest but also cell death. In the present study we have
characterized this phenomenon using a non-transformed lung fibroblast line,
CCL39, highly growth factor and anchorage-dependent. We analyzed the
intracellular signaling systems that protect cells from death, particularly the
p42/p44 MAP kinase and P[3K/Akt pathways.
Following anchorage and serum removal, apoptosis can be detected by
Annexin-V labeling, TUNEL analysis and caspase-dependent cleavage of
PARP (Poly-ADP Ribose Polymerase). In order to dissect the PI3-K and
MAP kinase signaling pathways, we used CCL39 cells expressing an
estrogen-inducible activated-Raf-1 construct (~.Raf-I:ER). In these cells,
activation of P13K and MAP kinase by insulin and estradiol respectively, is
mutually exclusive. Raf-1 activation by estradiol supresses both Almexin-V
labeling and caspase activation and this effect was found to be mediated by
the RatTMEK/MAP kinase module. In constrast, stimulation of the
PI3K/AKT pathway with insulin had little or no effect on cell survival. After
anchorage and serum remo;al, we observed disappearance of Akt and $6
kinase. The molecular mechanisms underlying this degradataon will be
discussed.

(We/3.31352) The baculovirus antiapoptotic protein, p35, affects calpain

activity and PKCalpha proteolysis.
S. Leverrier, N. Delaunay, A. Vallentin, D. Joubert.
1NSERM U469, 141 rue de la Cardomlle, 34094 Montpellier France,

Apoptosis involves different intracellular mechanisms such as proteolysis of

specific protein kinase C (PKC) isoforms. Caspases 1 and 3, two cystein
proteases, are implicated in PKC delta and teta cleavage during apoptosis
leading to accumulation of their catalytic domain. It has also been
demonstrated that the baculovirus protein p35 protects mammalian cells
from apoptosis through inhibition of caspase activity, leading to inhibition
of PKC proteolysis. We have produced PKC alpha in the Sf9
cells/baculovirus expression system and we observed that 4-5 days
postinfection, when the viral particles are released in the medium, PKC
alpha catalytic domain is accumulated. A time dependent study of p35
expression in St9 cells indicated that p35 is expressed early after infection
and desappears at the time of PKCalpba proteolysis. The desappearence of
p35 might therefore be necessary for PKCalpha proteolysis to occur. As
p35 regulates activity of some cystein proteases, we first investigated which
protease could be responsible for PKCalpha cleavage in St9 cells. Calpain,
which is a cystein protease known to cleave PKCalpha in the hinge region
between the regulatory and the catalytic domain, was a good candidate.
When Sf9 cells were cultured in the presence of the calpain inhibitor,
calpeptin, PKCalpha proteolysis was prevented as indicated by the absence
of accumulation of the 50 kDa catalytic domain. In the presence ofa caspase
I or caspase 3 inhibitor, proteolysis was not prevented and there was no
difference with controls. We thus hypothezized that p35 could regulate
calpain activity. This was investigated by using two different substrates for
calpain: casein, a universal protease substrate, and PKCalpha. With both
substrates, the presence of p35 increased in vitro signicantly calpain
activity, which is in contrast to the kown inhibitory action of p35 on
caspase activity. The highest increase was observed in the presence of an
equimolar ratio between p35 and calpaln, which is also the case to achieve
the best interaction between p35 and caspase-1. We are currently
investigating if this observation reflects the involvement of PKC alpha or of
calpain in the apoptotic process of mammalian cells.
Keywords: apoptosis, calpain, protein kinase C

Imidacrlne induces different cell death pathways in

murine and human acute myeloid leukemia cells

A new antitumor drug, imidacrine (compound C-1311), is currently

undergoing phase I clinical trials. One of the major cellular targets of
imidacrine is DNA topoisomerase II and inhibition of this enzyme
correlates with the biological activity of this compound.
In this study, we evaluated cell death pathways induced by a sublethal
dose of imidacrine (about ECg0 concentration) in two myeloid leukemia
cell lines: human (HL-60 cells) and murine (M1 cells, clone $6).
Although both cell lines origin from different species they show striking
similarities in several biological and pharmacological aspects. HL-60
and M1 cells proliferate with the same doubling time (24 hr) and have
comparable cell cycle distribution, they do not express p53 protein
(gene deletion) and show similar sensitivity to imidacrine (EC9o
concentration equals 1 pM for both cell lines with 1 hr exposure to the
drug). Drug induced comparable levels of DNA-topoisomerase II
complexes in both cellular systems as revealed by K-SDS coprecipitation assay, but at ECgo concentration imidacrine induced a
rapid arrest at the GI-S border in HL-60 cells and G2 block in M1
cells. The kinetics of cell cycle effects was also different with GI/S
arrest in HL-60 cells being evident already at 3-6 hr and G2 block in
M1 cells at 24-48 hr. These changes in the cell cycle progression were
followed by apoptosis in human and delayed cell death in murine
cells. Cell death types were confirmed by conventional and pulse-field
gel electrophore-sis and TUNEL assay (DNA fragmentation) as well
as fluorescence microscopy (changed cellular morphology).
Our studies show that imidacrine induces different types of cell death
in human and murine myeloid leukemia cells even at comparable level
of DNA damage associated with topoisomerase II inhibition.

(We/3.3/353)

Role of the chemokine receptor CXCR4 in the control of

apoptosis induced by SDF-I in neuroblastoma SK-N-SH
A. Lombet, E. Dicou. W. Rost~ne, F. Haour
1NSERM U339, Hrpital St-Antome. 75012 Pari.~, France

The chemokine stromal cell-derived factor 1 (SDF-1) is the

biological ligand for CXCR4, a G protein coupled seven-transmembrane
receptor used as coreceptor for the entry of T-tropic strain of HIV-1 into
CD4+ cells.
SDF-1 receptors were demonstrated and characterized in the human
neuroblastoma cell line SK-N-SH. Binding studies with 1251-SDF-1 on
living ceils indicated an affinity constant of 3 nM. Various cytokines (I1-1,
TNF-alpha) and chemokines (RANTES, MCP-1, IL-8) did not compete
with the binding of 125I-SDF-1. Conversely, the gpl20 protein involved in
the entry of the HIV-1 virus into target cells, competed efficiently for the
binding of the ligand.
SDF-1 (10nM) induced a rapid and transient increase in intracellular
free calcium, detectable in Fura-2-AM treated cells. This stimulation
indicated a functional coupling between the CXCR4 receptor and
intracellular calcium mobilisation.
Continuous SDF-1 (10nM) treatment induced an apoptotic process
after 48 hrs and a massive cell death at 72 hrs. A short SDF-1 treatment (2
hrs), followed by washing, did not lead to cell apoptosis after 3 days.
At lower concentrations. (2 nM SDF-1 for 72 hours) cell death was
not total and was reversed by Na/orthovanadate, an inhibitor of protein
tyrosine phosphatase. A MEK-1 inhibitor (PD 98059) as well as inhibitors
of other types of phosphatases were unable to antagonize the apoptotic
process induced by SDF-1. Activation of tyrosine phosphatase seems
therefore to be implicated in the long-term effect of SDF-1 on
neuroblastoma cells apoptosis.
These results demonstrate the potential role of SDF-1 in human
neuronal cell death and open up new ways of research on the CXCR4
receptor as regulator of brain function in health and disease.

s368

(We/3.3/354)

Abstracts FEBS'99

Paired-box transcripion facors modulate apoptosis

in sarcomas via members of the BCL-2 family
Margue, C., Bernasconi, M., Sch~fer, B.W

(We/3.3/355)

The Reval Veterina~ and Agricultural l]nlverslty DepartmentoJ Anatomy and

Phvslotog),, Groennegaardsvej 7, 1870Frederlksberg, Denmark

l)tvtston o f ('lintcal Chemtstry atld Btochemtsu); Untversu),

('htldren~ Hospttal, Steutwlesstr 75, ('H-8032 Znrtch, Switzerland

Rhabdomyosarcoma (RMS) are characterised in large by a specific

translocation t(2;13)(q35;q14) generating a fusion protein between two
transcription factors, PAX3 and FKHR, or enhanced expression of the
PAX3 gene itself. To obtain insights into the molecular function of the
PAX3/FKHR or PAX3 in RMS, we developed an antisense
oligonucleotide (AS-ODN) strategy to downregulate these PAX proteins.
As previously shown, such treatment of RMS cells leads to specific
induction of apoptosis.
Here, we report the identification of new target genes for PAX
transcription factors m RMS We show that PAX-mediated apoptosls can
be influenced by the anti-apoptoUc proteins BCL-2 and BCL-XL
Ectopic overexpression of BCL-2 and BCL-XL can rescue RMS cells
from apoptosis induced by PAX downregulation. However, we see no
synergistic effect of AS-ODN treatment when both PAX and BCL-2 or
BCL-XL are downregulated, suggesting that these proteins lie in the
same apoptosis pathway. Northern blot experiments show that ectopic
overexpression of PAX3 or PAX3/FKHR leads to enhanced BCL-2 and
BCL-XL RNA expression, lmmunoblot experiments also show an
increased expression of BCL-2 and BCL-XL protein, when we
ectoplcally overexpress PAX3. Finally, we present ewdence with
reporter assays and band shift assays that PAX3 can transcriptionally
activate the BCL-X promoter. Our experiments suggest that at least part
of the apoptotic inhibitory actwity of PAX proteins is mediated through
BCL-2 family members, a pathway which mrght be active both m tumour
cells, and during development.

(We/3.3/356)

Apoptosis contributes to mitomycin C-hypersensitivity

in hamster cell mutants.
E. Papouti.C. Lafon,A. Valene,M. zdzienicka*, M. Defaisand F. Larminat
lnstitut de Pharraacologieet de Biologic Structurale, C.N.R.S., Toulouse,
France and ~State Universityof Leiden, Leiden, The Netherlands.

To elucidate the mechanisms of the mammalian cell defence against

cross-linking agents, we have studied the biological consequences of
MMC treatment on two MMC-hypersensitive hamster cell mutants : VH4 and V-C8, and their parental cell line V79 [1]. We have previously
shown that defect of excision repair of DNA interstrand cross-links does
not contribute to the differential sensitivity of mutants V-H4 and V-C8
towards MMC [2]. Analysis of cell-cycle progression after equimolar
MMC treatment indicated that mutant cells exhibit prolonged cell-cycle
accumulation in G2 phase, compared to their pannatal counterpart.
However, parental and mutant cell lines display equivalent accumulation
in G2/M compartment in response to equitoxic MMC concentrations,
arguing against primary defect in the regulation of G2/M transition in VH4 and V-C8 mutants. In addition, we show that mutant cells undergo
greater levels of apoptosis following MMC treatment, than parental
cells. These findings indicate that apeptosis contributes to
hypersensitivity of V-H4 and V-C8 cells to the growth inhibitory effect
of MMC. This differential apoptotic response is observed with both
equimolar and equitoxic MMC doses and is specific for the cross-linking
agent MMC. These data strongly suggest that control of the apoptotic
process is altered in both MMC-hypersensitive mutants. The defective
genes in V-H4 and V-C8 cells would then function in the regulation of
an apoptotic pathway, triggered by MMC induced damages and
independent of p53-mediated transcription. We are currently identifying
differentialy expressed genes in either parental cell line or mutant cells
that may contribute to the differential response to cross-linking agents.
[ 1] Overkamp W.J.I. et al, Somat. Cell Mol. Genet., 19, (1993), 431.
[2] Larminat F. et al., FEBS Letters, 437, (1998), 97.

Modulation of apoptosis by endogenous endothelial cell

nitric oxide synthase.
K. Mortensen,J Skouv,D.M.Hougaardand L.-I. Larsson

By reverse transcriptase-polymerase chain reaction and sequencing we show

that human breast cancer (MCF-7) cells express endothelial cell nitric oxide
synthase (ecNOS), but not other NOS isoforms. Specific inhibition ofecNOS
in MCF-7 cells leads to increased tumor cell apoptosis and this effect can be
reproduced by an NO scavenger. Conversely, low levels of NO donors inhibit,
while high levels of NO donors stimulate MCF-7 cell apoptosis. The ecNOS
promoter was found to contain a specific binding site for the apoptosisregulating protein p53. In cotrasfection studies wild-type, but not mutant, p53,
downregulated transcription of an ecNOS promoter-luciferase-reporter gene
construct. These data point to a previously unrecognized p53-dependent
regulation of ecNOS expression which may be important for regulating
apoptosis and for avoiding the generation of genotoxic quantities of NO.

(We/3.3/357)

Regulation of CD95 -induced exposure of

phosphatidyiserine at the cell surface during apoptosis.
C. Pelassy, J.P. Breitmayer & C Aussel
INSERM U343, Hrpttal de rAtchet, 06202Nlce, b)'ance

Phosphatidylserine (PtdSer), is an amino phospholipid localized in the

plasma membrane inner leaflet In mammalian cells, PtdSer is synthesized
through a Ca2+-dependent exchange of the polar head group of
phospholipids, either PtdCho or PtdEtn, by a serine. During apoptosis, CD95
induces a translocation of PtdSer from the inner to the outer leaflet of the
membrane where it participates to phagocytic recognition of the apoptotic
cell In this work we have examined 1) the effect of CD95 on PtdSer
synthesis and 2) the effect of pharmacologically-induced changes in PtdSer
synthesis on CD95-induced PtdSer exposure at the surface of Jurkat cells.
We found that CD95 induces a rapid (<15 min), strong (2.5 fold) but
transitory neosynthesis of PtdSer in Jurkat cells. PtdSer decarboxylation, a
mitochondrial process, was inhibited by CD95 suggesting that changes in
mitochondrial activity participates to the increased PtdSer synthesis. In cells
undergoing apoptosis, newly synthesized PtdSer first exposed at the cell
surface was shedded with CD95-induced plasma membrane vesicles.
Inhibition of PtdSer synthesis that follows CD3/TCR triggering or
thapsigargin treatment of Jurkat cells is accompanied by a strong reduction
of CD95-induced appearence of PtdSer at the cell surface, as monitored by
using Annexin V-FITC labelling. Conversely, increasing the activity of the
serine-base exchange enzyme system with drugs, either the K+ channel
blocker, quinine, the cationic amphiphil, stearylamine or three different
calmodulin antagonists, chlorpromazine, trifluoperazine and W7, resulted in
an increased appearence of PtdSer at the surface of CD95-treated cells. Both
PtdSer synthesis and CD95-induced Annexin V-FITC reactivity were
abrogated in ATP-depleted cells. Modifying the membrane potential with
valinomycin (hyperpolarization) or either gramicidin or KCI
(depolarization) also showed a relationship between PtdSer synthesis and
Annexin V-FITC reactivity in CD95-treated cells Our results indicate that
CD95-induced exposure of PtdSer at the cell surface is regulated by the
activity of the Serine-base exchange enzyme system.

Abstracts FEBS'99

(We/3.3/358)

s369

CROSS-TALK BETWEEN MITOCHONDRLA AND

CASPASE IN DRUG-INDUCED APOPTOSIS
S. Pervaiz, S. M. Ali, J.L. Hirpara, K. W. Loh

(We/3.3/359)

UMR-CNRS 6548, Universtt~ de Ntce-Soplua Antipolis, parc Valrose

06108 Nice cedex 2, France

Department of Physiology, National University of Singapore,

Singapore 119260

Photoprodacts of merocyanine 540 (pMC540) trigger

caspase mediated cell death in human tumor cell lines[l].
Here we have purified three active components from this
photoactivated mixture and investigated their mechanism of
anti-tumor activity.
We are specifically interested in
compounds v,~ich directly activate caspases andor trigger
mltochondrial events associated x~th apoptosis. Using two
model tumor cell lines, HL60 leukemia and MI4
melanoma, we asked three major questions, (a) if any of the
purified
photoproducts
triggered
cytochrome
C
translocation from mitochondria, (b) ff the cytochrome C
release ~as dependent upon induction of mitochondrial
permeabilit3' transition, and (c) if any of the compounds
d~rectly activated caspase protease Our results show that
all three compounds, namely CI, C2 and C5 induced
cytosolic release of cytochrome C in both cell lines and
from purified rat liver mitochondria, hox~ever, in case of
C2 this release of cytochrome C was independent of
mitochondrial swelling and induction of permeability
transition pore. Compounds CI and C2 also triggered
efficient activation of both caspase 8 and caspase 3,
whereas C5 treated cells showed minimal acti,,ation of both
caspases.
The abdit3" to actwate caspases directly
correlated with the anti-tumor actwit)" of the purified
compounds In addition, we show that mere release of
cytochrome C and reduction of permeabiliB" transttion, in
the absence of efficient caspase activation is not sufficient
for full-blown apoptosts.
[1]Perval:, S., Htrpara, ,J. L., C/dmenl, ),/-l" CasFase
proteases me,hate apoplost~ reduced by anncancer agent
preacttvated A[C 540 m human ntmor cell lines. Ca~zeer
Lea. 127, 1-12, 199.~

(We/3.3/360)

Changes of the transactivating potential

of AP-1 transcription factor during apoptosis.
B. Pyrzynska, G. Mosieniak, B. Kaminska
Nenckl Insntute of Exp Bwlog~, Warsaw, Poland

Role of CFTR in programmed cell death

C.Poujeol, M.Taue, L.Counillon,JM Blasiand P.Poujeol

The aim of this study was to investigate the role of CFTR in apoptosis
by modulating the intracellular pH Apoptosis was studied in 2 fibroblast cell
lines obtained from hamster lung : the PS 120 line which does not express the
NHE 1 isoform of the Na+/H + exchanger and the PS 120-NHE1 line which
over-expresses this isoform Each cell line was transfected with the cDNA
encoding for the human CFTR. The cell lines were incubated to undergo
apoptosis with lovastatin (30/zg/ml). Apotosis was followed I2, I6, 20, 30
and 40 hours after the beginning of lovastatin treatment by observing the
nuclear chromatin condensation after orcein staining, by double staining with
Hoeschst 33258 and propidium iodide, and by relative measurement of DNA
fragmentation. In PS120 control cells the percentage of apoptotic cells after
40 h oflnvastatin was 23 3 % (n = 7 cultures) whereas in PS120-CFTR
transfected cells this percentage was 40 4- 4 % (n= 9 cultures). Moreover the
% of DNA fragmentation was strongly increased in the presence of CFTR
(control : 53 4- 10 %, CFTR : 91 4- 20 %). In PS120-NHEI cells, the
transfection with CFTR did not modify the % of apoptotic cells after 40 h
(control : 19 4- 3 %, n= 8 ; CFTR : 17 4- 1%, n = 8). Intracellular pH was also
monitored using BCECF and fluorescence video-microscopy. In all cell lines,
the initial pH values were identical (pH = 7.46 ~: 0.04, n = 9) and treatment
with Iovastatin led to intracellular acidification. However, the pH value a~er
40 h was much more acid in PS120-CFTR tranfected cells (pH = 6 85 4- 0.02,
n=10) than in PS 120 cells ( pH = 7.15 0.03, n = 10 ). To investigate further
the origin of this increase ofintracellular acidification, the activity of the CI"
/HCO 3- exchanger was studied. PS 120 cells exhibited a CI'/HCO3 "exchanger
blocked by DIDS. The exhanger was activated by 8Br-cAMP in PS 120-CFTR
transfected cells only. In conclusion, the CFTR increased the rate ofapoptosis
induced by lovastatin in fibroblasts which did not express NHE 1. This increase
could be attributed to a better cytoplasmic acidification, due to the modulation
of the CI/HCO3 - exchanger by CFTR. In PS 120-NHE 1 cells, CFTR does not
modulate apoptosis, due probably to the fact that the over-expression of
Na*/H* antiporter limits the decrease of pHi.

(We3.3/361)

Cleavage and relocation of the tyrosine kinase p59fyn

during Fas-mediated apoptosis in T iymphocytes
J.E. Ricci, L. Maulon, F. Luciano, S.Guerin, A. Livolsi, B. Marl,
J.P. Breinmayer, J.F. Peyron, P. Auberger
CJF 96 05, 28 Av de Valombrose 06107. Nice cedex 02

Although AP-1 transcription factor is known to control cell activation and

proliferation, it is also involved in apoptosis in response to stress, DNA
damaging agents or lack of survival signals. To understand how AP-1 might
contribute to so different biological processes, we examined AP-l
composition and phosphorylation state as well as its transcriptional activity
in proliferating versus apoptotic glioma cells.
We show that the induction of DNA-binding activity of AP-1 transcription
factor composed of c-Jun, Jun B, Jun D and ATF-2 proteins preceded druginduced apoptosis. The compositional changes of AP-1 in apoptotic cells
were associated with an elevation of c-Jun and JunB protein levels and
gradual phosphorylation of c-Jun and ATF-2 proteins comparing to the
situation in proliferating cells, lmmunocytochemistry and simultaneous
examination of nuclear morphology revealed an accumulation of
phosphorylated c-Jun protein just in apoptotic cells, c-Jun expression and
transcriptional activity is known to be stimulated by phosphorylation at Ser63/73 and the activity of ATF-2 is induced by phosphorylation at Thr-71.
Since both proteins can be the substrates for c-Jun N-terminal kinases
(JNKs), we measured activity of these kinases by a solid-phase assay. We
found the prolonged induction of JNKs activity in extracts from apoptotic
glioma cells. Our results suggest an involvement of persistent activation of
JNKs and phosphorylation of c-Jun and ATF-2 proteins in the initial phase
of apoptosis.
Additionally, we show that variations in AP-1 composition and
phosphorylation state result in modification of transactivating potential of
AP-l towards different promoters. While collagenase AP-l/TRE-dependent
transcription is downregulated during apoptosis, Fas ligand promoter
becomes activated.
Our results revealed ,,double face" of AP-1 transcription factor and help us
to understand how this factor can play contradictory roles in cell growth and
apoptosis.

Ligation of Fas with its natural ligand or with anti-Fas antibodies

induces an apoptotic program in Fas sensitive cells. We report here the
identification of the tyrosine kinasc p59Fyn as a substrate for CPP32like proteinases and more particularly caspase 3 during Fas-mediated
apoptosis in Jurkat T cells. Inhibition of CPP32-1ike proteinases by AcAsp-Glu-Val-Asp-aldehyde but not by Ac-'Fyr-Val-Ala-Asp-aldehyde
prevents CPP32, PARP and p59Fyn cleavage indicating that CPP32 or
CPP32-1ike proteinases are responsible for the cleavage of p59Fyn.
Cleavage occurs in the N-terminal domain of p59fyn between Aspl9
and Gly20 and is accompanied by relocation of an active p57Fyn kinase
to cytoplasm of Fas-stimulated Jurkat cells as judged by both
biochemical and confocal microscopy experiments. Thus. p59Fyn
relocation and activity may play an important role during Fas-mediated
cell death in human T lymphocytes.

s370

Abstracts FEBS'99

(We/3.3/363)

An overexpression of bcl-2, COX-2 and iNOS accompanies

the apoptosis enhancement in ulcerative colitis in man
K. Schmehl, S. Florian, G. Jacobasch,
German Institute of Human Nutrition, 14558 Bergholz Rehbrlicke, German),

Ulcerative colitis (UC) belongs to the group of inflammatory bowel diseases

(IBD). The etiology of IBDs is still unknown. Patients suffer from
abdominal
pain,
bloody
diarrhea
and
malnutrition syndrom.
Morphologically, a destruction of the mucosal architecture with a loss of
epithelial cells, lymphocytic infiltration, oedema and a high degree of
neovascularization is seen. An increase in apoptotic activity was found in
colonic crypts from patients with ulcerative colitis (UC). On the other hand,
an overexpression of cyclooxygenase-2 (COX-2) mRNA is documented for
involved colonic tissues. Increased levels of luminal intestinal NO were
reported in patients with UC. NO is able to onset apoptosis. COX-2 and bcl2 may protect cells against nitric oxide (NO) -mediated apoptosis.
To evaluate the tissue dmtribution of COX-2, bel-2 and of the nitric oxide
synthase (NOS), tissue samples of inflamed and not affected regions from 20
patients suffering from UC were investigated histologically, and
immunohistochemically, respectively Antibodies against endothelial
(e)NOS, inducible (i)NOS, COX-2, bcl-2, and in-situ apoptosis detection kits
(TUNEL, KLENOW) were applied. Additionally, the proliferation state was
determined by PCNA and Ki-67 detection.
COX-2 - and iNOS pattern were found within whole the colonic wall of
the affected regions with a special emphasizing of myofibroblasts and
endothelial cells. Bcl-2 IR was restricted to the epithelial cells in the mucosal
crypt rests. Apoptosls tests did show an elevated activity in general.
Connective tissue cell nuclei were most strongly stained. Additionally, most
of the remaining epithelial cells showed positive IR for the proliferation
markers PCNA and Ki67.
Summarizing, it's concluded that the destruction of the mucosa m UC is
accompanied by dramatic changes in the expression pattern of the colonic
epithelial cells. Proteins related to apeptosis are co-expressed in P C N A
positive cells. We hypothesize that the increased apoptotic activity is related
to an iNOS-overexpression. An iNOS-COX-2 antagonism may play a role in
ulcerative colitis pathogenesis. Whether the inflammation itself is a
secondary effect, and which influences extracellular matrix proteins have
will be evaluated in further investigations

(We/3.3/364)

Control of apoptosis in prostate cancer cell fines.

L. Schmidt, A.W. Murray
Fhnders Umversity of South Australia, School of Btologteal
Sciences. GPO Box 2100. Adelaide S.A 5001. Austraha

PC-3 cells are an androgen independent prostate cancer derived cell line
which is highly resistant to chemotherapeutic drugs and other inducers
of apoptosis. Our laboratory is using this cell line to examine the
regulation of apoptosis in prostate cancer cells. Here, we report that
osmotic stress generated by sorbitol, at concentrations greater than
0.2M, induced apoptosis in PC-3 cells in full serum conditions after two
hours. This was confirmed by viability counts and morphological
changes using Hoechst staining. Sorbitol-indueed apoptosis also
correlated with increased acivity of the apoptosis effector enzyme
caspase-3.
A key area of interest has been the combination of various apoptotic
agents to give an enhanced response. Ghosh and Myers [1] have shown
that inhibition of the 5-1ipoxygenase pathway induces apoptosis in
prostate cancer cells. In our experiments, the general lipoxygenase
inhibitor Nordihydroguaiaretic Acid (NDGA) combined with sorbitol
produced a synergistic response in serum-free conditions. This implies
that the combination of eicosanoid inkibitors with other apoptotic agents
should be explored for its chemotherapeutic usefulness.
[1] J. Ghosh et al., PNAS, 95, (1998) 13182.

(We/3.3/365)

The adenine nucleotide translocator 1 (ANT-l)

induces apoptosis upon overexpression
A. Schubert, M.K.A. Bauer and S. Grimm
Max-Planck Institute for Biochemistry,
Am Klopferspitz 18a, 82152 Martmsned,Germany

During the last years evidence has accumulated that points towards a central
role for mitochondria in apoptosis-induction. Especially the permeability
transition (PT-)pore, an integral membrane complex, has recently been implicated in the execution of the apoptotic program. Here we describe the
isolation of adenine nucleotide translocator-1 (ANT-1), a central component
of the PT-pore, using a novel screen for dominant, apoptosis-inducing
genes. ANT- 1 overexpression led to all characteristics of apoptosis including
caspase-activation, DNA-degradation and mitochondrial membrane gradient
disruption. Although ANT-1 is known as a mitochondrial ADP/ATP-carrier,
we show that its apoptosis-inducing capacity is independent of adenine
nucleotide transport. Furthermore this apoptosis induction is highly specific
for A N T - I since ANT-2, a very homologous isoform, was unable to elicit
apoptotic changes in mammalian cells. The pro-apoptotic protein Bax, that
interacts directly with ANT-I can induce a form of cell death in S. cerevisiae
by activating the PT-pore. In contrast, ANT-I is unable to generate such
lethal effects in yeast.These data suggest that ANT-l-induced apoptosis
relies on specific protein-protein interactions.Taking into account that ANTI is already one of the most abundant proteins of the inner mitochondrial
membrane, its dominant apoptosis-induction activity must be counterbalanced by other interaction partners. We show that cotransfection of
cyclophilin D, a PT-pore associated mitochondrial matrix-protein, results in
repression of ANT-1-mediated apoptosis.Thus, ANT-1 seems to be part of a
mitochondrion-based interaction network which plays a role in the regulation
of apoptosis.

Abstracts FEB S' 99

(Wed3.3/366)

s371

Ceramides reinforce their apoptotic effects by inhibiting

the neuroprotective cAMP dependent signalling pathway.

(Wed3.3/367)

V.See, B.Koch and J.P. Loeffier.

UMR 7519 CNRS, ULP, 21 rzte R Descartes, 67084 Strasbourg, France

The pituitary adenylate cyclase activating polypeptide (PACAP) type l

receptor (PR1), a seven transmembrane receptor, is positively coupled to
both adenylate cyclase and phospholipase C. PACAP exerts neurotrophic
effects which are mainly mediated through the cAMP / protein kinase A
pathway. In this study we analyse whether such neurotrophic signals may be
blocked by the apoptosis inducing second messenger ceramide. Ceramides
have emerged recently as important mediators of several agents that affect cell
growth, viability, and differentiation. These lipid second messengers are the
breakdown products of membrane sphingomyelins. Agonists of the
sphingomyelin-ceramides pathway include membrane receptors ligands such
as Tumor Necrosis Factor c~, lnterleukin- I ~, as well as stress-inducing agents.
We show that eeramide selectively blocks PACAP-activated cAMP
production, without affecting phosphoinositide breakdown. Thus by blocking
the neuroprotective cAMP signalling pathway, ceramides will reinforce their
direct death inducing signalling. The molecular mechanisms underlying these
effects were analysed. We found that on the one hand, the reactive oxygen
species (ROS) scavenger lipoic acid, reversed the effect of ceramides and on
the other hand that H202 mimicked it. Together these data indicate that
uncoupling of the cAMP pathway by ceramides is mediated by ROS. When
we analysed the level at which ceramides and ROS uncouple cAMP
production, we found that it did not involve shortage of ATP supply or G0:s
protein function but that it rather modify adenylate cyclase function per se.
Indeed, using membranes from either ceramides or HzO2 pre-treated cells we
demonstrate altered adenylate cyclase activity "in vitro". Further, the tyrosine
phophatases inhibitors vanadate and dephostatin, but not the serine/threonine
phosphatases inhibitors okadaic acid and calyculin, prevent inhibition of
cAMP production by ROS. This suggests that H20= requires a functional
tyrosine phopsphatase(s) to block PACAP dependent cAMP production. In
summary, we show that in addition to their direct death promoting effects,
ceramides also recruit a reinforcement mechanism that blocks cAMP
dependent nearotrophic signalling.
(We/3.3/368)

Store-Operated CAP.+Current in Human

Prostate Cancer Cells LNCaP : Involvement in Apoptosis

The Ets2 transcription factor inhibits apoptosis induced by CSF-I

deprivation of maerophages through a Bcl-xL-dependent mechanism.
L.Sevilla, C.Aperlo, V.Dulic, J.C.Chambard, C Boutonnet, O.Pasquier,
P.P{~gnonec and K.goulukos..
Centre de Biochlmie, Umversttd de Nice, 06108 Nice, F~ance

Abstract
Bcl-xL, member of the Bcl-2 family, inhibits apoptosis and its
expressmn is regulated at the transcriptional level, yet, nothing is known
about the transcription factors specifically activating this promoter. The bcl-x
promoter contains potential Ets binding sites and we show that the
ti'anscnptmn factor. Ets2. first ~dentified by its sequence identity to v-ets of
the E26 retrovirus, can transacUvate the bcl-x promoter. Transient expression
of Ets2 results in the upregulation of Bcl-xL, but not Bcl-xs, an alternatively
spliced gene product which induces apoptosis. Ets2 is ubiqmtously expressed
at low levels in a variety of cell types and tissues, but as specifically induced
to abundant levels during macrophage differentiation. Since Bcl-xL is also
upregulated during macrophage differentiation, we asked whether the bcl-x
could be a direct downstream target gene of Ets2 in macrophages. BACI.2F5
macrophages which are dependent on macrophage colony stimulating factor,
CSF-1, for their growth and survival were used in these studies. We show that
CSF-1 stimulation of BACI.2F5 macrophages results in the upregulation of
expression of ets'2 and h~l-xL with similar kinetics of induction. In the
absence oI CSF 1. thesc macrophages undergo cell death by apoptosis
whereas constitutive expressmn of Ets2 rescues these cells fl'om cell death,
and bcl-xL is upregulated. These results strongly suggest a novel role of Ets2
m affecting apoptosls through its regulation of bcl-x L transcription.

(We/3.3/369) Development of,apoptosis-resistance phenotype in

epithelial tumoral A431 cells.

R. Skryma, X. F. Le Bourhis and N. Prevarskaya

S. Smida-Rezgui,S. Honore*,J.B. Rognom*,P.M. Martin, C. Penel

Laboratory o f Cell Physiology, University o f Lille 1, Lille, France

Canc~rologte Erp~rtmentale, Fac MEdecme Nord, Bd Dramard 13916 Marsellle ('edex 20

I]PRES-A, CNRS 6032, Fac. Pl~armacte, Bd Mouhn, 13385 Marsedle Cedex 09, P~an~e

Calcium (Ca2+) accumulates within the endoplasmic reticulum of cells

through function of the Ca2+-dependent ATPases family of intracellular
Ca2+-pumping ATPases. The Ca2+-ATPase pump inhibitors induce the
Ca2+ pool(s) depletion with following activation of the plasma membrane
capacitative calcium entry named store-operated Ca2+ current (IsTo~) and
induce apoptosis in variety of cell models .While the implication of Ca2+
ions in the induction of apoptosis is now generally accepted, the results
concerning the role of ISTO~ in this process are rather contradictory. Very
little also is known about these mechanisms of the Ca2+ regulation in
prostate cancer cells and their role in programmed (apoptotic) death. Ca2+dependent ATPase inhibitors, thapsigargin (TG) and cyclopiazonic acid
(CPA), depleted intraceUular Ca2+ stores and induced consecutive
extracellular Ca2+ current through plasma membrane, lsro~, in human
cancer prostate LNCaP cells. For the first time we have identified and
characterized this Isro~ in prostate cells using whole-cell, cell-attached and
perforated
patch-clamp
technique
combined
with
fura-2
mierospectrofluorimela'ical and Ca-imaging measurements. Isror~ current in
LNCaP cells has inwardly rectifying current-voltage relation, lacks voltagedepending gating and has significant unitary current noise. The unitary
conductance of Isror~ channels is 3,2 + 0.45 pS. Current has a high
selectivity for Ca2+ over monovalent cations and is inhibited by Ni2+ (1-3
mM).The treatment of LNCaP cells with TG (0.1 ~M) caused morphological
and biochemical changes within 6-12 h, arrest out of cycle in Go and
irreversible loss of their ability to proliferate. During the next 24-48 h, the
genomic DNA of the Go-arrested cells undergoes double-strand
fragmentation. Removal of extracellular Ca2+ or adding of Ni2+ augmented
apoptosis due to thapsigargin. These results indicate that in androgendependent prostate cancer cells the depletion of intraeellular Ca2+ stores
may trigger apoptosis but that it is no requirement of the activation of storeactivated Ca2+ current and the sustained Ca2+ entry in induction and
development of the programmed cell death.

A431 cells,overexpress epidermal, growth factor receptors (EGFR)

and are growth-inhibited by EGF nanomolar. We have shown that this
inhibition results from an apoptotic process.
Nevertheless, a subpopulation of A431 escapes this process. To
investigate the mechanism of apoptose-resistance, A431 cells displaying
alternate responses to EGF were selected from native A431 after several
cycles of EGF nanomolar treatment (A43 IR). We verified the resistance of
A431R for at least 6 months. A431R differ from the A431 by their
morphology. They display mesenchymatous morphology with membrane
protrusion and overexpressed actin filaments.
As compared with native A431, in A431R : Scatchard plot analysis
revealed a decreased level of EGFR (1.5 compared to 1.8 106 sites/cells)
while the affinities were unaffected. Confocal microscopy shown
internalisation and clusterization of EGFR. Autophosphorylation of the
EGFR indicated a decrease in EGF-responsive protein kinase activity. By
Western Blot, an increased (100%) MAPkinase (ERK1 and ERK2)
expression was shown, that paralleled changes in MAPkinase activation.
Single cell Ca> analysis indicated 2 signals types: spike (48%, 31%) or
spike followed by a sustained elevation (14%, 51%) for A431R and A431
respectively. Moreover, flow eytometric analysis indicated an increase of
o:2131 integrin (300%) expression during A431R cell selection, which
correlated with apoptosis resistance.
These results suggests that subsequently to the decreased activation
of the EGFR, at least 3 mediators are involved in the apoptosis-resistance
process in A431R following nanomolar EGF stimulation: these phenotypic
changes are: 1- the MAPkinase activation, 2- the calcium response by the
shil~ from a sustained elevation to a spike, and 3- the increased ot2131 integrin
expression.

s372

(We/3.3/370)

Abstracts FEBS'99

11,-4 prevents mitochondrial damages and apoptosis

without inducing switch to necrosis
V. Souvannavong, C. Lemaire, K. Andr~au, A. Adam
CNRS U3/1R8619, Universzt~Paris-Sud, 91405 Orsay, France

We previously demonstrated that the broad-spectrum caspase inhibitor,

zVAD-fmk, totally deviated apoptosis to necrosis in B lymphocytes. We
report here that, in contrast with zVAD-fmk, IL-4 protected B cells from
spontaneous and from dexamethasone-induced apoptosis and actually
maintained cell viability. This was assessed by morphological and
biochemical criteria and accompanied by the maintenance of mitochondrial
transmembrane potential (A~m) and elevated glutathione (GSH) levels.
Under these conditions, zVAD-fmk also totally inhibited apoptosis in
thymocytes, but it partly preserved cell viability with a parallel increase
in the percentage of cells exhibiting high A~m and elevated GSH levels.
Nevertheless, non-rescued cells were deviated to necrosis. Therefore, the
pathway leading to either apoptosis or necrosis appears to involve
common mitochondrial dysfunctions which could not be reversed by
caspase inhibition, suggesting that the pharmacological inhibition of cell
death should occur at an earlier stage.

(We/3.3/372)

CCRF-CEM Leukemia Cell mRNA Expression Profiling

' During Glucocortieoid-Indueed G1 Arrest and Apoptosis
M.Tonko and R.Kofler
Inst. for Gen. and Exp.Pathology, Div.of MoZPathophysiology, Universityof
lnusbruck. Austria

Glucocortieoids induce cell cycle arrest in G1 with subsequent apoptosis

in various leukemia cells including the acute lymphoblastic T-cell line,
CCRF-CEM-C7H2. Although the phenomenon is being exploited for the
therapy of this malignancy, the underlying molecular mechanisms are
largely unknown. To further address this issue, we have investigated the
mRNA profile of the CEM-C7H2 cell line and its alterations during
glucocorticoid treatment using recently developed cDNA array
techniques that allow the simultaneous analyses of hundred to thousends
of defined genes. We are currently trying to correlate these data with the
general behaviour of this leukemia cell line (as reflected in the large body
of published reports) and with glucocorticoid-induced cell cycle arrest
and cell death, in particular.

Supported by the Austrian Science Fund (F204, P11946)

(We/3.3/371) TNFcx and Fas induced changes in histone phosphorylation

H.Talasz, W. Helliger, H. Lindner, B. Sarg and B. Puschendorf
Institute of Medical Chemistry and Btochemistry, University of lnnsbruck,
Fritz-Pregl-Str. 3, 6020 lnnsbruck, Austria

In the present investigation, using NIH 3T3 cells we examined the effects
of TNFet and agonistic anti-Fas antibody on chromatin condensation and
histone phosphorylation. 2 to 3 hours after starting the treatment with
TNF~, 40% of the cells and the nuclei round up and phosphatidylserine
externalization could be measured. Later on, chromatin condensed into a
few sharply defined bodies and at the nuclear membrane condensation of
the chromatin occurred. Interestingly, using agarose gels a DNA ladder
was undeteetable. Instead during the early phase of treatment with TNFct
we found DNA of high molecular weight and, thereafter, a smear of
DNA.
During the early phase of the treatment with both TNFtx or anti-Fas
antibody the phosphorylation of all various HI subtypes and core histone
variants decreased rapidly. Somewhat later, the linker histone HI
showed a transient slight increase in phosphorylation. After 4 to 8 hours
of TNFc~ treatment phosphorylation of H2A.X increased significantly.
Simultaneous addition of PKC inhibitor staurosporine, tyrosine PK
inhibitor genistein, PKA inhibitor H89, which at higher doses also
inhibits PKG and PKC, or DNA PK inhibitor wortmannin did not result
in any suppression of the TNFct and Fas effects. The PARP inhibitor 3aminobenzamide, however, reduced markedly the induction of apoptosis.
Simultaneously, a change in the phosphorylation pattern was visible:
whereas the phosphorylation of historic H2A.X was markedly
dimimshed, the phosphorylation of the histone H3.1 was significantly
increased. The results suggest a role of both the phosphorylation of core
histone H2A.X and of H3. I in chromatin condensation.

(We/3.3/373) F-Actin is one of the target proteins for fenretinide-induced

apoptosis in squamous cell carcinoma cells
E Ulukaya and E J Wood
Umverslty of Leeds, School of BtochemistD' and .~lolecular BiologN.
Leeds. I.A~ 9J'1~, UK

Fenretinide, N-(4-hydroxyphenyl) retinamide, is a retinoid derivative with

fewer side effects compared to naturally-occuring retinoids such as all-trans
retinoic acid and 9-cis retinoic acid and has been entered into some clinical
trials in ontological diseases such as breast carcinoma [ 1,2]. Fenretinide has
also been used in a number of synergistic combinations with either some
cytokines or anti-cancer drugs in vitro, and in one study it has been
suggested that fenretinide can potentiate the effects of cisplatin in ovarian
carcinoma [3]. Although it was shown to induce apoptosis in a number of
cancer cell lines, the mechanism by which it does this is not clear yet. We
have studied the effect of fenretinide on F-actin, a major component of the
cytoskeleton. Apoptosis was detected by both hematoxylin staining to
visualize the morphological changes, and by the TUNEL assay to detect
DNA fragmentation. Cytoskeleton components were extracted as triton-X
100-insoluble proteins and then SDS-PAGE was performed. F-actin was
visualized by FITC-labelled phalloidin staining 0.5x10"5 M fenretinide
induced cell death characterized by cell shrinkage, nuclear condensation or
fragmentation, membrane blebbing, all of which are characteristics of
apoptosis. Control (untreated) cells had edge staining for F-actin and
presented F-actin stress fibres extending from one side to other side of
cells. However, when treated with fenretinide F-actin was disorganised and
damaged to different degrees depending on the stage of apoptosis. At first
stages it seemed to be like "lumps" with edge staining while at later stages
it appeared to become either thinner or dramatically disorganized without
edge staining. However, there was no evidence for a significant decrease in
the amount of F aetin according to the densitometric evaluation of actin
bands on SDS-PAGE.
[1] De Pale et al, Journal ofCelhdar Btochemist~-Supplement, 22, (1995) 11.
[2] Costa Aet al., Annals o f the New York Academy o f Sciences, 768, (1995), 148.
[3] Fonr~lli F et al., Cancer Research, 53(22), (1993), 5374.

Abstracts FEBS'99

s373

(We/3.3/374) Alkyl-lysophospholipids activate the SAPK/JNK pathway

and enhance radiation-induced apoptosis
W.J. van Blitterswijk a, G.A. RuiteP .b, S.F. Zerp~,b,
H. Bartelinkb, M. Verheij b
Divisions of Cellular Biochemistry and Radiotherapy ~, The Netherlands
Cancer Institute, Plesmanlaan 121, 1066CX Amsterdam, The Netherlands.

Alkyl-lysophospholipids (ALPs) represent a new class of anti-tumor

drugs that induce apoptosis in a variety of tumor cell lines. Although
their precise mode of action is unknown, ALPs primarily act on the cell
membrane where they interfere with the mitogen-activated protein kinase
(MAPK) signaling pathway. Since activation of the stress-activated
protein kinase (SAPK/JNK) pathway is essential for radiation-induced
apoptosis in certain cell types, we tested the effects of ALPs in
combination with ionizing radiation on MAPK/SAPK signaling and
apoptosis induction. We present data showing that three ALPs, 1-Ooctadecyl-2-O-methyl-rac-glycero-3-phosphocholine
(Et- 18-OCH3),
hexadecylphosphocholine (HePC) and the novel compound octadecyl(1,1-dimethyl-piperidinio-4-yl)-phosphate(D-21266; fromASTA),induce
time- and dose-dependent apoptosis in the human leukemia cell lines
U937 and Jurkat T, but not in normal vascular endothelial cells.
Moreover, in combination with radiation, ALPs strongly enhance the
induction of apoptosis to supra-additive levels in both leukemic cell lines.
All tested ALPs not only inhibited MAPK activity but, like radiation,
stimulated the SAPK/JNK cascade within minutes. A dominant-negative
mutant of c-Jun inhibited radiation- and ALP-induced apoptosis,
indicating a requirement for the SAPK/JNK pathway. Our data support
the view that ALPs and ionizing radiation cause an enhanced apoptotic
effect by modulating the balance between the mitogenlc, anti-apoptotic
MAPK and the apoptotic SAPK/JNK pathway. This type of modulation
of specific signal transduction pathways may provide a basis for selective
and efficient tumor cell kill.
Reference:
G.A. Ruiter et al., Cancer Research, 1999, in press.

(We/3.3/376)

Changes in composition of NFAT/AP-I transcription

factor during dex-indueed apoptosis in the thymus.
M. Wisniewska, B. Pyrzynska, B. Kaminska
Nencki Institute of Experimental Biology, Warsaw, Poland

The NFAT transcription factor (Nuclear Factor of Activated T ceils) is

involved in the inducible transcription of the interleukin 2 (IL2) and other
cytokine genes. NFAT proteins can act alone or in a complex with AP1
transcription factor. NFAT/AP1-DNA complex is more stabile then NFATDNA one. Although NFAT is studLed above all in the context of T-cell
activation, there are growing evidence of potential involvement of NFAT in
other biological phenomena, including immunodeficiency diseases,
thymocyte development and apoptosis.
Glucocorticoids appear to participate in apoptotic death of unselected
thymocytes during development. We used synthetic glucocorticoiddexamethasone (dex) to induce apoptosis in the mouse thymus in vivo and
investigate the levels of NFAT and AP-l transcription factors in thts model.
Using gel-shift method we detected NFAT DNA-binding activity in protein
extracts from the mouse thymus. The binding activity significantly
decreased during dex-mduced apoptosis. In parallel we observed changes in
AP-1 gel mobility. It suggested changes in AP-I composition after dex
administration. The observed phenomena followed glucocorticotd receptor
increase in nuclear extracts and proceeded massive DNA fragmentation.
Using westem-blnt analysis we found that the level and the pattern of NFAT
proteins were unaffected during dex-induced apoptosis. In case of Jun/Fos
proteins no changes were observed in the levels of JunB, junD and Fra2
proteins, the levels of FosB and Fral were undetectable, and the levels of
cJun and cFos proteins increased. The results show that the set of Jun/Fos
proteins is different in thymocytes from control and dex-treated mice.
The lack of NFAT DNA-binding activity is not due to the lack of
components of NFAT complex. Different set of Jun/Fos proteins can result
in different AP-1 composition. It raises possibility that altered association of
NFAT protein with AP-1 component Is responsible for the observed loss of
the NFAT DNA-bmding activity.

(We/3.3/375)

Apoptotic changes in A 72 cells infected

by canine parvovirus
M.Vuento, P. M~kinen
University o Jyv~akyla, FL 35, 40351 d~-J~skyld,
Finland

Canine parvovirus (CPV), a type member of autonomous

parvoviruses, is a small nonenveloped virus with a
5kb ssDNA genome. It enters host cells by endocytic
route involvlng at least early and late endosomes[1].
The apoptotic response of A 72 cells to infection by
CPV was studied by flow oytometry.The response was
characterized by rapid changes (from 15 min p.i.) in
membrane permeability and membrane polarization of
host cells and by a slow (from 16 h p.i.) appearance
of apoptosis markers, notably cleaved caspase substrafe (M30 antigen), cell surface exposure of phosphatidylserine, and DNA fragmentation. The results
indicate that A 72 cells infected by CPV undergo apoptosis and also suggest that CPV may affect the membrane integrity of host cells already in an early
phase of its infectious entry.
[1].M.Vihinen-Ranta

et al. J. Vir01,72,(1998),802.

s374

Abstracts FEBS'99

4.2 Macromolecular recognition and assembly

(We/4.21377)

Characterization of "I.R.G."* ot-2M in blister fluid from burned

patients
P. Chateaureynaud a, M. Clercb, M.F. Dumonb

(We/4.2/378)

H. J. Chauhan, M, J, Howard, G. J. Domingu and R. N. Perham

Department of BiachemtstrL Umpersitr of Cambrulge, 80 Te~tllls Colttt Road,
Cambrtdge, CB2 IGA. England. UK

aUMR CNRS 5.$43 et bLaboratoire de Biochimie A,Iddicale A, Universiti de

Bordeaux 11, 146 rue L#o Saignat, 33076 Bordeaux Cedex France.

IRG-~2M is a serum acute phase protein the rate related immunomodulatory

activity of which is increasing during inflammatory processes in rat and
human species.
Five isoforms of human 0t2M were revealed using iso-electric focusing 0EF).
One of them (p.i. 5.4, ionic strenght 200 mM NaCI) is IRG. This isoform is
desialilated.
We demonstrated that the incubation with neuraminidase of the different o.2M
isoforms allows them to migrate at the same p.i. as IRG. Besides, these 4
desialilated isoforms exhibited a complement inhibitory activity as IRG did.
Using IEF and immunoassays we visualized IRG in blister fluid within the 4
to 7 days after injury. The rate and rate related activity on complement of
whole blister fluid and IRG purified from blister fluid were similar to serum
IRG.
Blister fluid, in part by mean of blisler fluid 111(3, may be considered as a local
modulator of inflammation able to inhibit as well proteases as complement
activity.

Protein-protein interactions and substrate channelling

in the pyruvate dehydrogenase multienzyme complex

The molecular basis of substrate channelling in pyruvate

dehydrogenase (PDH) complexes lies in the interaction of Elp
with the lipoyl domain of the E2p component.

In Bacillus

stearothennophilus PDH, the Elp exists as a heterotetramer

(0;2[32) of sub-components El0; and Ell3.

The interaction

between E lp and the lipoyl domain of E2p has been investigated

using heteronuclear 2D NMR spectroscopy. The active site is in
the El.a component but recognition of the lipoyl domain was
found to be distributed over sites in both E 10; and El 13subunits.
The domain structure of the El0; and El,3 polypeptide
chains,

and the protein-protein

interactions

involved

in

assembly, have also been studied by means of limited

proteolysis, On cleavage of a highly conserved site in EI~, the
first catalysed reaction (oxidative decarboxylation) is markedly
enhanced, and the second leaction (reductive acetylation of the

*"IRG" : inhibiteur de rejet de greffe

lipoyl domain) is unchanged. However, on assembly of the

PDH complex in vitro with digested Elp, the overall catalytic

P. Chateaureynaud, A. Bun, G. Moureanx, R. Wasserman, R. Sanchez.

actiwty drops significantly, suggesting that active site coupling

Inhibiteur du rejet de greffe t2M. A suppressive activity on complement in

bum injury. Trauma, 1, 1999.

has been impaired by modification of the potential pathways of

(We14.2/379)

Glyeosylation of fucosyltransferase II1

Costa, T.*, Grabenhorst, E. *, Sousa, V.*, Costa, J.*
*ITQB/IBET, P-2780 Oeiras, Portugal
**GBF, D-38124, Germany

Fucosyltransferase III (Fuc-TIII) has a broad substrate specificity

preferentially transferring Fuc onto type I acceptors either sialylated or not
forming the Lewis" and sialyl-Lewisa structures; it can also use the type II
acceptors if these are highly concentrated forming Lewisx or sialyI-Lewis"
structures; it can transfer Fuc in an c~3-1inkage onto Glc residues. Fucosylated
oligosaccharides are often expressed in a regulated manner during
development, differentiation

and progression of metastasis; furthermore,

several host-pathogen interactions, e.g., Helicobacter/gastric cell, are at least

in part mediated by fucosylated oligosaccharides.
Fuc-TllI is a Golgi membrane-bound glycoprotein with the catalytic
domain protruding into the lumen of the Golgi. It has two occupied
glyeosylation sites, Asn-154 and Asn-185. Secreted forms of Fuc-TIII, the
first derived from intracellular proteolytic cleavage of the full-length form of
the enzyme expressed in BHK cells, and, the second an engineered secretory
form of Fuc-TIII (Costa et al., 1997) are differently glycosylated as detected
from different sensitivities to PNGase F and Endo H. Deletion of each or
both of the glycosylation sites by site directed mutagenesis showed that i)
there was no enzyme detected in the cells or in the medium when Ash-154
was deleted, whereas ii) deletion of Asn-185 caused no alteration in the
cellular activity.
Costa, J., Grabenhorst, E.. Nimtz. M. Conradt, H.S. (1997) J Biol Chem 272, 1161311621

domain interaction on the protein surface.

(We/4.2/380)

Mutagenesis of the E1 component of the pyruvate

dehydrogenase complex of Bacillus stearothermophilus
M. Fries ~, G. J. Domingo~, H. L Chauhan a, R. N. Perham a
a Deparmlent of Biochemisto; University of CwnbrMge,
CwnbrM,ee CB2 IGA, E~lgland

The El component of the pyruvate dehydrogenase (PDH) complex from

Bacillus stearothermophilus is a heterotetramer consisting of two cc and two
subumts. The Elc~ subunit contains the binding site for the thiamindiphosphate col:actor, whereas EI~ is responsible for binding EI to the
peripheral subunit-binding domain of the E2 component. El catalyses two
reactions, the oxidative decarboxylation of pyruvate and the transfer of the
resulting acetyl group to the lipoamide prosthetic group which is attached to
the lipoyl domain in the E2 chain.
Site-specific mutations of a solvent-exposed, conserved site on the Elot
subunit have been carried out. Three mutants were created: in the first, the
three amino acids Tyr281, Arg282 and Ser283 were changed to alanine; in
the second, Tyr281 and Arg282 were changed to serine and in the third,
Ser283 was changed to cysteine.
Kinetic analysis of the mutants revealed that the Elct281-283Ala and
Elo~281,282Ser mutants displayed up to threefold higher activity in the
oxidative decarboxylation assay compared to wild-type. The K,,, for the
substrate pyruvate for all mutants were determined using the oxidative
decarboxylation assay. Remarkably, the Elot281-283Ala mutant and the
E 10t281,282Ser mutant displayed K,, values of about an order of magnitude
greater than wild-type El. However, no difference could be observed in the
assay measuring the rate of reductive acetylation of the E2p lipoyl domain.
Most strikingly, the catalytic activity of the reconstituted PDH complex with
the Elc~281-283AIa mutant and with the E10t281,282Ser mutant was only
17% of that with El, whereas the PDH complex reconstituted with the
El~Ser283Cys mutant displayed an activity similar to wild-type.
The results suggest that the mutated site in the Elct subunit participates in
an interaction with E2 and that this site is important for the active site
coupling required for overall PDH complex reaction.

s375

Abstracts FEBS'99

(We/4.2/381) Metal ions effect on DNA structural transition in

aqueous solutions of alcohols and urea
E.V, Hackl, Yu.P.Blagoi

(We/4.2/382)

B 1 I erki;7 ln~tltule i~n Loll Temperat~we l'hl ~lc~ alTd EIl~i~Teel'll~L~.A at

4, ad o/Set ol ~ kl <n;w 4- Lenil1.4re 310164 Allat k~' ~ IxTL~INE

Using methods of IR spectroscopy and light scattering DNA structural

transitions are studied under the action of Cu -~- and Ca:- ions in aqueous
and mixed solutions containing ethanol, 1,2-propanedioI, glycerol (0+20
o6.%) and urea (0+5 M) at temperatures 29 and 450C. It is shown that on
its interactioll with Cu 2+ and Ca -~ ions in aqueous and mixed solutions
DNA transits into a compact form characterized with the ordered
morphology; the partial DNA destabilization facilitates
its
compactisation. The measure of sizes of compact particles is obtained
and the compactisation model is proposed. It is shown that DNA
compactisation is of high positive cooperativity depending on the
solution structure. DNA compactisation dependencies on tbe counterions
valency and hydration, the temperature and content of solvents are
studied. It is stated that in mixed solutions DNA compactisation depends
not only on the solution dielectric permeability but on the solution
strtlcture.
The results obtained are being discussed in the frameworks of the
theories of counterion condensation (CCT) and equilibrium binding.
Unlike CCT, the theory of equilibrium binding shown to describe
satisfactorily the DNA compactisation process under the Mt2+ ion action.

(We/4.2/383)

Structural Features of Protein.Protein and Protein-DNA

Recognition Sites
J. Janin, Laboratoire d'Enzymologie et de Biochimte Structurales,
CNRS, Gif-sur-Yvette, France, L. Lo Conte, C. Chothia, MRC
Laboratory of Molecular Biology, Cambridge, England, K. Nadassy,
S. J. Wodak, European Bioinformatics Institute, EMBL, Hinxlon,
Cambridge, England

We analyze structural features of the recognition sites seen in 75 proteinprotein and 65 protein-DNA complexes taken from the Protein Data Bank.
Conformation c h a n g e s a n d size o f the recognition site. In both types of
complexes, the size is estimated as an interface area B. It ranges between
=1200 and =5000 ~2 and shows a clear relation with the extent of
conformational changes occuring upon association. 70% of the proteinprotein complexes have 'standard size' interfaces with B in the range 12002000 A 2 and small conformation changes. In nearly all of the 27% which
have'large' interfaces burying 2000-5000 A 2, large conformation changes
are seen. Most protein-DNA interfaces have B>2000 A 2, and conformation
changes occur in the protein, the DNA or both. Many of the DNA-binding
proteins are dimers or tandem repeats which can be broken into several
binding units. In 80% of the transcription factors, individual binding units
form with DNA an interface with B in the range 1200-2000 A2 like
a'standard size' protein-protein interface. Notable exceptions are the zinc
fingers, which have B=900 A 2, an area probably too small for a single zinc
finger to achieve stable association with DNA.
Recognition sites are close-packed: the role o f water. Close-packing is

demonstrated by comparing the Voronoi volume of protein atoms at

interfaces to that of atoms buried inside proteins. The ratio of the two
volumes is 1.01_+0.05 in 95% of the protein-protein and 92% of the proteinDNA interfaces. The calculation applies to atoms inaccessible to the
solvent, which constitute only one-third of the interface atoms, but it can be
extended to the majority by taking crystallographic solvent positions into
account when evaluating Voronoi volumes. This confirms that water
molecules contribute to the close-packing of atoms that insures
complementarity between the surfaces of two proteins or a protein and
DNA. The role of solvent is also obvious in water-mediated interactions
which, at interfaces, are as abundant as direct polar interactions.

Sreening for assembly sites on p24 0tlV-1) eapsid protein

K.Hilpert, J.Behlke , Ch.Scholz, R.Misselwitz, J.SchneiderMergener , and W.Hfhne
lnstitut fib"Biochernie,Charit6 zu Berlin, D-t0117 Berlin, ~M-D-Cfiir
Molekulare Medizin, Robert-Rfssle-Stral3e10, D-13122 Berlin, Germany

Protein-protein interactions of the p24 (HIV-1) capsid protein play an

essential role in the production of infectious virus particles. To map the
putative p24 assembly site, a set of overlapping peptides spanning the p24
sequence was prepared using spot synthesis on a cellulose membrane and
probed with recombinant p24 (rp24). Three sequence regions interacting
with rp24 were identified. In the first step we investigate the influence of
these peptides on p24 dimerization. Peptides from each region were
synthesized, but only one peptide was effectively able to inhibit rp24
dimerization in solution. Amino acids being exposed in the corresponding
p24 region were mutated in rp24 resulting in a significant decrease of rp24
dimerization. All peptides are currently being tested in cell cultures to
investigate their inhibitory effect on HIV virus spreading to study the
influence of the peptide on capsid assembly. Furthermore, a p24 double
mutant is under construction to suppress dimerization even at high protein
concentrations necessary for NMR studies with the complete protein.

(We/4.2/384)

Sequence specificity of the N- and C-terminal domains of

the M.FokI DNA methyltransferase.
M. Fatemi, M. Roth, O. Leismann & A. Jeltsch
lnstitut fur Biochemie, Heinrich-Buff-Ring 58, 35392 Giessen, Germany

Type II DNA methyltransferases specifically methylate adenine or cytosine

residues within defined DNA sequences. Here we have studied the specificity
of the N-terminal and C-terminal domains of the M . F o k I DNA
methyltransferase: M.FokI(1-367) (recognition sequence: GGATG) and
M.FokI(335-647) (recognition sequence: CATCC). In this study 27
hemimethylated oligonucleotides substrates were used to measure methylation
rates at all possible FokI star sites. M . F o k ( l - 3 6 7 ) interacts very specifically
with its recognition sequence because apart from the unmethylated and
hemimethylated GGATG sites only the AGATG site is modified. The
specificity of this enzyme is comparable to that of restriction endonucleases. In
contrast, M.FokI(335-647) shows low specificity, because only two of the 12
star sites investigated (CAACC and CAGCC) are not accepted and some star
sites are modified with rates reduced only 2-3 fold. In addition, GGATGC and
CGATGC sites are modified which deviate at two positions from CATCC
demonstrating that the enzyme has a specificity for SSATSS (S = G or C). We
propose a model of the molecular evolution of the FokI
restriction/modification system in which the C-terminal domain originally had
a specificity for SSATSS and was the only methyltransferase in the cell. Later
the second domain was acquired and genetically fused as N-terminal domain
to the existing enzyme. At present, the C-terminal domain is an evolutionary
intermediate of changing its specificity to CATCC.

Abstracts FEBS'99

(We/4.2/385)

Investigation of the structure of the 1F3 module of

fibronectin and its role in fibronectin assembly
O. Lequin, J.R. Bright, J.R. Potts, I.D. Campbell

s376

(We/4.2/386)

Investigation of the structure of the 1F3 module of

fibronectin and its role in fibronectin assembly
O. Lequin, J.R. Bright, J.R. Potts, I.D. Campbell
Dept of Biochemistry, Universityof Oxford, OxJbrdOX1 3QU, UK.

Dept of Biochemistry, UniversiO' of Oxford, Oxford OX1 3Q U, UK.

Fibronectin is a large mosaic glycoprotein which is present at high levels in

most extracellular matrices [1]. It consists of multiple repeating modules
(classified into three types, I, II and III), which generally fold as autonomous
units [2]. The modules are organised into larger functional domains that
contain binding sites for a wide range of ligands in extracellular matrices.
Fibronectin exists in two forms: a soluble dimeric form and an insoluble
multimeric form in matrix that mediates most of the critical biological
properties.
Little is known about the interactions between modules in fibronectin. These
interactions are important in fibronectin self-association that occurs during the
conversion of the soluble dimeric form to the fibrillar form incorporated in the
matrix. A region encompassing the first type III module (IF3) has been
identified as playing a role in fibronectin incorporation in matrix [3].
The IF3 module has been expressed in the yeast Pichia pastoris and
structure/function studies are carried out by heteronuclear NMR. The structure
determination of the IF3 module and the identification of interactions with
other modules of fibronectin should provide a better understanding of how
fibronectin interacts with itself to form large assemblies in the matrix.

Fibronectin is a large mosaic glycoprotein which is present at high levels in

most extracellular matrices [1]. It consists of multiple repeating modules
(classified into three types, I, II and III), which generally fold as autonomous
units [2]. The modules are organised into larger functional domains that
contain binding sites for a wide range of ligands in extracellular matrices.
Fibronectin exists in two forms: a soluble dimeric form and an insoluble
multimeric form in matrix that mediates most of the critical biological
properties.
Little is known about the interactions between modules in fibronectm. These
interactions are important in fibronectin self-association that occurs during the
conversion of the soluble dimeric form to the fibrillar form incorporated in the
matrix. A region encompassing the first type III module (IF3) has been
identified as playing a role in fibronectin incorporation in matrix [3].

[ 1] Hynes, Fibronectins, New York, Springer-Verlag, (1990).

[2] Ports et al., Curr. Opin. Cell Biol., 6, (1994) 648.
[3] Chernousov et al., J. Biol. Chem., 266, (1991) 10851.

[ 1] Hynes, Fibronectins, New York, Springer-Verlag, (1990).

[2] Potts et al., Curr. Opin. Cell Biol., 6, (1994) 648.
[3] ChernOusov et al., J. Biol. Chem., 266, (1991) 10851.

(We/4.2/387)

Dynamic of the MHC class I assembly in the ER membrane~

Didier Marguet#~, E. Spiliotis, T. Pentcheva # & M. Edidin#.
# Johns HopkinsUniversity,Baltimore,USA;
Centred'Immunologiede MarseilleLuminy,Marseille,France

Major Histocompatibility Complex class I molecules (MHC class I

present antigenic peptides to effector T-lymphocytes. The mature MHC clas~'
I, a complex of a membrane-bound heavy chain, 132-microglobulin and
peptide, is assembled in the endoplasmic reticulum. Two green fluorescen
chimeras of the mouse MHC class I heavy chain, H-2L d were expressed ir
cell lines. The green fluorescent chimera H-2LdGFPin (H-2L d with theA
victoria green fluorescent protein (GFP) attached to its cytoplasmic tail

matures as the native molecule. In contrast, H-2LdGFPout (H-2L d with the

GFP attached on its extracellular domain) has slower kinetics of maturation
and does not associate with TAP complex. W e have measured the latera
diffusion of GFP-tagged MHC class I in the ER, to gain insights into thei
associations with chaperones (calnexin, calreticulin,...) and the transportel
associated with antigen processing (TAP), and consequent transient ER
retention during assembly. We found that the lateral diffusion of H-2LdGFPir
is slower than diffusion of H-2LdGFPout. To demonstrate that the former wa~'
hindered by interactions with TAP complex, we measured the lateral
diffusion of H-2LdGFPin within cells with different peptide disposability
When loaded with specific peptides, the H-2LdGFPin latteral diffusion in the
ER correlated with the affinity of the peptide used to load it. Whereas
prolongated association of the MHC class I with the TAP complex decreasec
it. We propose that he MHC class I heavy chain itself diffused at a limit sel
by the membrane lipid viscosity. The contraints reflect the association of

The IF3 module has been expressed in the yeast Pichia pastoris and
structure/function studies are carried out by heteronuclear NMR. The structure
determination of the IF3 module and the identification of interactions with
other modules of fibronectin should provide a better understanding of how
fibronectin interacts with itself to form large assemblies in the matrix.

(We/4.2/388)

Enolase isoforms in muscles: differential expression

and participation to macromolecular assemblies.
T. Merkulova and A. Keller.
CNRS UPR 9065, Collkgede France. Paris, France

Enolase is a dimeric glycolytic enzyme which, in higher vertebrates,

exhibits tissue specific isoforms. We have previously established the
spatiotemporal patterns of cx and [~ enolase gene expression from early
embryonic to adult stage in the mouse. The embryonic form, occt, remains
expressed in most adult tissues. During ontogenesis, a transition occurs
towards the specific oc13and [3[3 isoforms in striated muscle cells[l, 2].
In studies with biopsies of 7 weeks old embryos, we show for the
first time that [3 subunit expression already occurs in primary human muscle
cells. Furthermore, by in vitro cloning of these cells, we have demonstrated
that, [3 enolase is one of the few muscle specific genes to be expressed in
dividing myoblasts.
Histochemical studies with muscle sections from adult mice show that
subunit expression occurs following a gradient which parallels the
glycolytic metabolism of myofibers. Thus, it is not detectable in type I fibers
and is expressed at the highest level in type liB fibers.
The physiological significance of the existence of enolase isoforms
with very close structures and catalytic properties is not clear. It has been
proposed that isozymes differ in their ability to interact with other
macromolecules within the cell, thus segregating to different subcellular sites.
where they respond to specific functional demands. In the case of enolase, we
have shown the existence of specJfic in vitro interactions with various muscle
proteins, differing according to the isozyme examined [3]. Further
biochemical analyses indicate a differential distribution of the two subunits
between cytoplasmic and particulate fractions. The [3 predominant isoform
seems to be mostly soluble and the ot mostly bound to insoluble structures.
Immunocytochemical analyses reveal a striated pattern of cc immunoreactivity
within muscle cells. Double labelling experiments analysed by confocal
microscopy demonstrate that oc subunit is localised at the M-band. Some of
the ~ immunoreactivity appears striated and could also be visualized at the Mband. However, most of [3 immunoreactivity appears diffuse all over the
sarcoplasm. Our results support the idea that the [3[3 enolase isoform is
present in the zone of acto-myosin overlap of glycolytic fibers. There, it could
participate to multienzyme complexes bound to the contractile apparatus and
be involved with local ATP production.

otherwise mobile molecules with the T A P complex which itself is restricted

in its lateral motion. The interaction between the TAP complex and the MHC
class I is dynamic.

[1] A. Keller et al., Mech Dev, 38, (1992) 41. [2] M. Lucas et al.,
Differentiation, 51, (1992) 1. [3]. T. Merkulova et al., Biochem. J., 323,
(1997) 791.

s377

(We/4.2/389)

Abstracts FEBS'99

NMR solution study of a heparin binding t3 module

from chick embryo fibroblast type XII collagen.
Mittard V., Bright J.R., Ports J.R., Campbell I.D. Dept. of

(We/4.2/390)

[nstitute, EMBL-Outstation, Hinxton, Cambridge, England, Laboratoire

d'Enzymologie et de Biochimie Structurales CNRS. Gif-sur-Yvette. Franc

Biochemistr3", University of Oxford, South Parks Road, OXI 3QU, UK

Numerous medium to large proteins consist of repeated copies of smaller

conserved sequences, which generally fold as autonomous units. For
example, a classical mosaic protein called fibronectin comprises three types
of repeating structural units called type I modules (fl), type II modules (f2)
and type III modules (13) [1]. These modules are organized in functional
domains that contain binding sites for extracellular matrix proteins, cell
surface receptors, plasma proteins and glycosaminoglycans such as heparin.
Our current research is to study fibronectin / heparin interactions (f3 repeats)
of two modullar proteins, collagen type XII [2] and fibronectin, in the
context of other known information on protein / heparin interactions.
Heparin bindingrelies on a delicate balance between charge interactions,
hydrogen bonding and hydrophobic interactions [3]. 3D structural
information (high resolution) about these domains would provide insight into
the exposed surfaces that might be involved in heparin binding activity. A
NMR study of the heparin binding 13 module from collagen type XII 0SN
labelled sample) complexed with its ligand could allow the development of a
knowledge based approach to the more precise elucidation of heparin
binding sites. In particular, a comparison with the 1313 from fibronectin,
which will be also studied in the future by NMR, alone and complexed with
heparin, will aim to establish if there is a conserved heparin binding site in f3
modules which is not apparent from the sequence alignment. The role of
module module interactions concerning the heparin binding properties will
be also investigated in the future.
Ill Potts and Campbell, Matrix biology, 15 (1996) 313.
[2l Koch etal., J. Cell Biol., 130 (1995) 1005.
[a] Cardin et al., 1991, Methods Enzymol., 28 (1991) 557.
Virginie Minard has a long - term fellow,ship fronl Human Frontier Science Program.

Structural analysis of protein-nucleic acid binding sites

K. Nadassy~, S. J. Wodak b, J. Janinc European Bioinformatics

The atomic structure of 75 X-ray structures of protein-nucleic acid

complexes were analyzed,
Polar Interactions. The polar interactions were analyzed statistically in terms

of the number of hydrogen bonds and the type of atoms of the hydrogen
bond donors and acceptors. We fred some correlation between the number of
hydrogen bonds in the complex and its interface area and this correlation
suggest on average 125/~2 per hydrogen bond. Analysing the type of atoms
involved in these polar interactions one finds that the donor group is
contributed by the protein while the acceptor groups are from the DNA or
RNA side. The major role is played by the phosphate groups of the DNA
which is involved in over 60% of the hydrogen bonds, while on the protein
side we fred the sidechain groups of arginie and lysine to be the dominant
player.
Conformation Changes. In our dataset we found 24 complexes for which the
free form of the protein was also available. To quantify the occurred changes
we used the method of superposition. We divided the type of conformational
changes into 5 different categories defined as: disorder-to-order transitions,
quaternary changes, subunit movement within a monomeric protein or
subunit, tertiary changes and in addition to these sidechain movements were
also observed in all complexes. In 12 of the complexes we found the root
mean square distance (RMSD) values of 1-3.& involving local changes,
subunit or domain movements. There are four complexes where the RMSD
values are greater than 4 ,~ involving mostly dimers showing asymmetry in
the mode of binding. We also examined the changes occurring in the
structure of DNA by superimposing the phosphorous atoms using a
reference structure of B-DNA. Five of the complexes show a standard BDNA conformation, while half of our dataset exhibit changes of RMSD
between 1.5-3.0,~. 29 complexes (45%) of the complexes the DNA
undergoes major conformational changes with RMSD values of greater than

3.oA.

(We/4.2/391)

The assembly pathway of bacterial 20S protee

E. Seemiiller, J. Mayr and W. Baumeister
Max-Planck-hrstitutfiir Biochemie, D-82512 Martinsrted, Gerl

(We/4.21392) Binding of cis-DDP to E-tubulin site favors formation

of GDP-tubulin rings rather than microtubules
.~ A Tulub, V E Stefanox ~k -~ Kutin. B I Saprykin,
E K Skaletskii, E Bauer, and V Milligan
st Peter~burg State [ mverst 0 Nt Petersburg. 199034. Russia

20S proteasomes from the eubacterium R h o d o c o c c u s erythropolis are

built of two or-type and two [3-type subunits. Advantage was taken of this
relative simplicity in elucidating the sequence of folding, assembly and
processing events, which leads to active proteasomes. The pathway was
followed by in vitro reconstitution experiments. The first step is the
formation of ~/13 heterodimers followed by the association of half
proteasomes consisting of one o-ring and one [3-ring. Half proteasomes
dimerize to form preholoproteasomes which are converted to holoproteasomes through the autocatalytic removal of the ~]-propeptide. The
role of the propeptide is critical, though not essential: It assists the initial
folding of the [3-subunits and promotes the final maturation of
holoproteasomes. The propeptide is also fully functional when added as a
separate polypeptide. The propeptide has no detectable secondary and
tertiary structure by itself, and appears to adopt its functional fold upon
complex formation with the remainder of the subunit.
Proteolytically inactive mutants have been used to characterize processing
incompetent, fully assembled complexes, preholoproteasome
intermediates. Electron micrographs show the mass of fourteen
propeptides filling the whole central chamber and most of the two
antechambers. With wildtype proteasomes, these intermediates are
extremely short-lived and not detectable; it appears that the docking of two
half proteasomes instantly triggers the autocatalytic cleavage of the
propeptide. Further experiments show that the propeptides are
subsequently degraded in a processive manner, i. e. they undergo multiple
cleavages before the products are discharged and the inner cavities are
cleared.

GTP-tubulin under certain conditions undergoes self-assembly into

microtubules which participate in various cellular processes, including
separation of chromosomes during mitosis In present study we show
that cis-dichlorodiammineplatinum (II) (cts-DDP), a widely known
antitumor drug, interacts with GTP-tubulin in E-tubulin site [1].
Electron microscopy (in vitro experiments with negatively stained
samples of tubulin obtained from bovine brain according to WeisenbergDeitrich protocol, carbon-coated grids, simultaneous computer
processing), ~95Pt NMR spectroscopy and computer simulation have
been used Cis-DDP binding to tubulin stops an habitual process of
tubulin assembly into microtubules. Circled rings, typical for GDPtubulin assembly [2], have been formed rather than microtubules. The
binding occurs via two-step mechanism yielding a chelate with GTP [PtNT(Gua) and Pt-O(ct-phosphate) bonds] in E-tubulin site [3] First-step
binding favors GTP hydrolysis and its transition to GDP by water-ligand
shift from diaqua-form ofcis-DDP towards GTP y-phosphate
References
[1] Tulub A A , Skaletskii E K, Stefanov V E Int J Quant Chem.
V 65. (1997) P. 49
[2] Tulub AA., Skaletskii E K , Stefanov V E, Pavienko V K
Biochem Mol. Biol. Int. V 36 (1995) P. 475
[3] Stefanov V E , Tuhib A A, Kutin A A. Ragozina T N An.Quim
Int Ed. V 94 (1998) P 1

Abstracts FEBS'99

(We/4.2/393)

VE-cadherin : physiological role in endothelium

and homotypic interactions
C. Vanbelle, P. Legrand, S. Bibert, B. Morand, M. Jaquinod,
C. Ebel, M. Roth, D. Marion, T. Vemet, D. Gulino.
IBS -CEA / CNRS, 41 avenue des Martyrs. 38027 Grenoble - France.

The vascular endothelium is a semi-permeable barrier which forms an

active boundary between the bloodstream and the underlying tissues. Its
permeability needs to be modulated for allowing the transmigration of various
plasma constituents and the movement of circulating cells between blood and
inflamed tissues. Adherent junctions, which mediate the physical contacts
between endothelial cells, express a specific marker protein designated as VEcadherin (cadherin 5) [1]. VE-cadberin belongs to the superfamily of cadherin
adhesive molecules. Cadherin interacts homotypically with cadherin of
neighboring cells via their extraeellular part constituted by five modules
named Cadl to Cad5.
Although cadherins appear to be necessary for proper eell-eell
contacts, the physiological role of VE-cadberin in adult tissue has not been
clearly determined. To bring some light on this question, a human endothelial
cell culture was disturbed by a polyclonal Anti-VE-cadherin antibody. This
lead to the disruption of confluent endothelial cell monolayers and transiently
generates numerous transcient gaps at eell-cell junctions. The formation of
these gaps correlates with a reversible increase in the monolayer permeability.
The analyses of the time course for restoration of endothelial cell-cell interacts
and the VE-cadherin mRNA content give evidence for a rapid resynthesis of
VE-cadherin during the restoration of the monolayer cell integrity [2].
To evaluate the contribution of each extracellular module of VEcadherin to homotypic recognition, the single module, Cadl, and
combinations of N-terminal modules: Cadl2, Cad123 (Cadl-3) and Cad1234
(Cadl-4) were recombinantly expressed. Biophysical methods demonstrate
that Cadl, Cadl2 and Cadl-3 are unabled to give homotypic interactions. In
constrast, the Cadl-4 assembles in a Ca2+-dependent complex within an
bexameric structure.
With the aim of understanding the interaction mecanisms, structural
studies by NMR, for Cadl and Cadl-2, and X-Ray for Cadl-4, arc in
progress. We have shown that Cadl and Cadl2 are unstable as NMR spectra
display broad peaks and poor dispersion. We are producing variants of the Cterminal region with the hope of stabilizing the module. In parallel, various
additives are being tested to enhance stability. Crystals of the hexameric
Cadl-4 have been produced under various conditions, including in the
presence of heavy atomes. The diffraction data of these different crystal
forms are under investigation.

s378

s379

Abstracts FEBS'99

13.5 The multifaceted function of G-proteins

(We/13.5/394)

Trio exchange domain 1 elicits Racl and Cdc42

phenotypes through the specific activation of RhoG.
A. Bkmgy,C. Cxa~dff,E. Vignal,A. ~
S. Schrnidt and P. Fort
C.R.B.M., CNRS-UPRI086, 34293 Montpellier, France.

RhoG is a small GTPase of the Rho family, with closest similarity to Racl.
Expression of active RhoG in cells independently activates Racl and Cdc42,
provoking the apparition of membrane ruffles, lamellipodia, microvilli and
filopodia. This suggested that RhoG is part of a complex signalling cascade
involving the activation of several GTPases.
Trio is a large multidomain protein which contains two dbl-like regions
(GEF1 and GEF2), that specify nucleotide exchange in vitro on Racl and RhoA,
respectively. Given the high sequence similarity between Racl and RhoG, we
tested Trio GEF1 for its ability to induce GDP/GTP exchange on RhoG in vitro.
Our data showed that Trio GEF1 can in fact induce nucleotide exchange on RhoG
with about ten fold higher efficiency as compared to Racl. To further study the
exchange specificity of Trio GEF1, we used the yeast two-hybrid system. The
dominant negative mutant RhoG-T17N could very efficiently bind Trio GEF1,
while the similar mutation on Rac 1 did not allow its interaction with the exchange
factor. Moreover, we have developped in yeast a method to assay for GEF activity
which revealed that Trio GEFI could activate RhoG but not Rac 1.
These observations prompted us to assay whether Trio GEFI could be a
direct in vivo activator of RhoG. As previously reported in Swiss 3T3 cells,
expression of Trio GEF1 in REF-52 cells induced membrane ruffling, lamellipodia
formation, but also microvilli formation at the cell surface. These actin
remodelling activities are similar to those observed after expression of the RhoGG12V active mutant and are indicative of both Racl and Cdc42 activation.
Concomitant expression of Trio GEF1 and Racl-T17N or Cdc42-T17N indeed
resulted in the inhibition of either membrane ruffling or microvilli formation, as
previously reported for RhoG. Moreover, expression of a dominant negative form
of RhoG could inhibit both membrane ruffling and microvilli formation induced
by Trio GEFI. Finally, overexpression of a specific RhoG binding domain inhibits
Trio GEF 1 but has no effect on Rac 1-G 12V.
Taken together, these results indicate that Trio protein directly activates the
small GTPase RhoG, which in turn activates both Racl and Cdc42 through a
downstream cascade of as yet unidentified intermediates.

(We/13.5/396)

S t r u c t u r a l and f u n c t i o n a l aspects of the ARF1 S e c 7 i n t e r f a c e . S B~raud-Dufour, S Robineau, P

Chardin, S Paris, M Chabre, *J Cherfils and B Antonny.
[PMC-CNRS, Valbonne and ~CNRS-LEBS, Glf-sur- Yvette, France

ARFI, is a small GTP-binding protein involved in vesicular

trafficking. As with all G proteins, ARF1 switches between an inactive GDPbound state to an active GTP-bound state. While ARF1-GDP is quasi-soluble
ARFI-GTP is membrane bound. Activation of ARFI is catalyzed by the
guanine nucleotide factor, ARNO. ARNO is composed of a coiled-coil
domain, a "Sec7" domain which is responsible for the exchange activity, and
a PH domain which interacts with phosphoinositides and allows the
membrane recruitment of ARNO. Although both ARFI and ARNO interact
with membrane lipids, the interaction between the two proteins can be studied
in solution by using the Sec7 domain of ARNO (ARNO-SecT) and a
truncated form of ARF1 ([D17]ARF1) which are fully soluble. The crystal
structure of ARNO-Sec7 consists of 10 helices and contains a striking
hydrophobic groove. By site-directed mutagenesis we have shown that this
groove is the binding site for ARF1. Moreover, activity measurements and
gel filtration experiments with various mutated forms of ARNO-Sec7 and
[AI7JARFI suggest three contacts between the two proteins.
The E156K mutant of ARNO-Sec7 is able to form a complex with the
Mg 2+ free form of [DI7]ARFI-GDP, but is unable to catalyze the release of
GDP. Thus, Glu 156 has an essential role in the mechanism of ARNO-Sec7
catalyzed GDP dissociation and might act as a '~glutamic finger". Its
carboxylate group could point toward the bound Mg z+ and destabilize the
coordinations of Mg 2+ and the ~-phosphate hence favoring the dissociation
of the nucleotide.
The second putative contact is the insertion of the switch I region of
ARF1 into the hydrophobic groove of ARNO-Sec7 which could position the
~-phosphate and the Mg 2+ near E 156 of ARNO-Sec7. The orientation of the
two proteins was further confirmed by the formation of an intermolecular
disulfide bound between 2 cysteine mutants, (T45C)[DI7]ARF1 and
(T 197C)ARNO-Sec7.
The third contact is an ion pair interaction between D 183 in ARNOSee7 and K73 in ARFI. K73 belongs to the switch II region of ARF1 and
D183 belongs to a patch of aci&c residues in ARNO-Sec7. In addition, these
interactions promote a conformational switch in the N-terminal and
myristoylated helix of ARF1 which allows the interaction of the complex
with membrane lipids.

(We/13.5/395)

Role of the PH domains of Trio in GTPases activation

JM. Bellanger, S. Estrach, O. Zugasti, C. Astier, S. Schmidt,
S. Diriong, A.Debant
CRBM-(WRS, UPR 1086, 34293 Montpellier C&Ic'.v 5, France.

The small GTPases Cdc42, Rac and RhoA have important regulatory roles
in mediating cytoskeletal rearrangments and cell growth. Thmr activity is
regulated by guanine nucleotide exchange factors (GEFs) which accelerate
their GDP/GTP exchange rate, and thereby activate them. Rho-GEFs share a
Dll (Dbl-homology domain) domain responsible lbr the enzymatic activity,
[bllowed by a PII domain. Trio is a multifunctional protein that is
comprised of two functional Rho-GEFs domains and a serinc/threonine
kinase domain. We have shown in vitro and in vivo that the first GEl:
domain (GEFDI) activates Racl, while the second GEF domain (GEFD2)
acts on RhoA. Moreover, co-expression of both domains activates
simultaneously both GTPases. Thus Trio is the first example of a twoheaded exchangc factor, able to link Rac and Rho pathways in vivo.
Most interest has been focused on PH domains which are thought to be
involved in cellular localization and protein/protein interactions. The study
of Trio, with its two GEF domains, is appropriate for a better understanding
of the role of the PH domains in GTPase activation. We have addressed this
issue by two different approaches. First, using deletion mutants of Trio
where the PH domains have been deleted or swapped, we have shown that
the P i l l of Trio 1) is necessary for Rac activation by the GEFD1 and
cannot be replaced by the PH2 domain; 2) targets mutants to the
cytoskeleton and 3) surprisingly, mediates a Rac-dependent stimulation of
JNK activity in the chimeric construct DH2PH1. In contrast, the PH2
domain is dispensable for Rho activation by the GEFD2 domain, suggesting
that these two PH modules play a different role in GTPases activation by
Trio. Second, using a two-hybrid screen, we have shown that: 1) the GEFD1
binds the actin cross-linking protein filamin; 2) this binding is dependent of
the presence of the PH1 domain.
Taken together these data suggest that the PtlI domain is necessary for Rac
activation by Trio, probably by targeting Trio to the eytoskeleton close to
Rac and its effectors via interaction with the actin-binding protein filamin.
Those data are consistent with a role of Trio in actin cytoskeleton
remodeling.
(W~/13.5/397) Functionalproperties of the helical domain of the Gsa subunit.
EcheverrIa ,V**.,Toro, M J*. mad Olated**.*Molecular Biology
Department.Universityof Concepcion,Chile.** Biochemistry I~1
MolecularBiology Department.AlcalfiUniversity. Spain.

Gsc~ proteins from human and Xenopus laevis share 92% of

aminoacid identity. Although Gsct from X. laevis is not able
to activate Adenylyl cyclase(1). The most variable region
between both proteins correspond to the loop between etB and
a C from the helical domain(HD) that changes conformation
after GTP binding.We carried out 5/5 non conservatives
changes in this region from the human Gsct for the
homologous aminoacids present in the X. laevis Gset protein.
Wild type and mutant proteins were synthesized in E. coli,
purified, and characterized. When reconstituted with $49 cyc-(
deficient in Gs~)S49 cell membranes, the mutant G s a
proteins stimulate adenylyl cyclase activity in the presence of
GTP "yS and AIF4- to a much lower extent than does the wild
type Gset. The rate constants for dissociation of GDP,
hydrolysis of GTP and GTP'/S binding were also substantially
reduced.
These results suggest that this loop from the HD participates in
the Gsot adenylyl cyclase stimulation activity controlling G s a
guanine nucleotide binding.
1. Antonelli,M.,Birnbaumer,L.,Allendej and Olate,J.(1994).
Human-Xenopus chimeras of Gset reveal a new region
important for its activation of adenylyl cyclase .FEBS
Letter.340:249.

Abstracts FEBS'99

(We/13.5/398)

RhoG GTPase specifically binds kinectin and affects

microtubule-dependent vesicular transport.
E. Vignal, A. Blangy, C. Gauthier-Rouvi~re and P. Fort.

s380

(We/13.5/399)

Equipe 2 du Groupe de Biophysique, Ecole Polytechnique,

1=-91128 Palaiseau Cedex, France

C.R.B M., CNRS-UPRI086, 34293 Montpelher, France.

RhoG is a member of the Rho family of GTPases that shares 72% and
62% sequence identity with Racl and Cdc42Hs, respectively. RhoG protein is
mainly associated with endoplasmic reticulum membranes, with a minor fraction
located at the plasma membrane. RhoG activation elicits the formation of
lamellipodia, microvilli and filopodial extensions, resulting from the independent
activation of Racl and Cdc42Hs GTPases. In addition, microtubule
depolymerization impairs RhoG translocation and activity, but has no effect on
Racl and Cdc42Hs activities~ suggesting that a microtubule-dependent target
mediates RhoG activity. Using a yeast two-hybrid screen, we characterized the
156 kDa kinectin as a potential RhoG target. Kinectin is an integral protein of the
endoplasmic reticulum (ER), acting as a receptor for kinesin, a microtubule
motor involved in plus-end dependent forward transport. In the yeast, kinectin
binds the GTP-bound form of RhoG, and to a lesser extent of Rac 1, Rac2, RhoA
but not RhoB and Cdc42Hs. Two kinectin domains (KID) are responsible for the
interaction : KiD1 specifically binds RhoG and Rac and is located within the
central region of kinectin, which contains seven 70 a.a. coiled-coil repeats and is
involved in kinectin homodimerization. Two repeats are sufficient to promote an
efficient binding. The second domain (KID2) is located at the C-terminus and
mediates the binding to RhoA. In REF-52 fibroblasts, the kinectin co-localizes
with RhoG and RhoA, but not Rac. Expression of KiD1 and KiD2 respectively
inhibits RhoG and RhoA, but has no effect on Rac and Cdc42Hs activities. In
addition, kinectin redistributes into distinct subcellular locations depending on
whether RhoG is activated or inhibited. Kinectiu therefore fits all the criteria of a
genuine RhoG target. To address the role of RhoG in microtubule-dependent
transport, we examined the effects of RhoG activity on the exocytic pathway of
the vesicular stomatis virus glycoprotein (VSV-G). RhoG inhibition prevented
the secretion of the VSV-G protein, which redistributed with RhoG and kinectin
in particular cytoplasmic structures, distinct from ER to Golgi intermediate
(ERGIC), cis-, medial- and trans-Golgi compartments.
Our data therefore establish that kinectin behaves as a genuine RhoG
effector and suggest that RhoG promotes its effects through the control of a
microtubule-dependent vesicular transport.
(We/13.5/400)

The human [33-adrenergic receptor can activate ERK1/2

and PKB by coupling to a Gi/o protein
T. Issad, C.C. Gerhardt, J. Gros and A.D. Strosberg
UPR 415 CNRS. ICGM, 22 rue M~chain, 75014 Paris, France

Coordination of the Action of Guanine Nucleotide Exchange

Factors and Effectors on Ha-Ras p21
C. Giglione & A. Parmeggiani

Ras proteins are molecular switches in signal tranduction pathways. They

link upstream plasma membrane-bound receptors to downstream effector
molecules inside the cell. An increasing number of proteins has been
discovered to interact with Ras. All these proteins fall into two groups:
regulators (positive and negative) and effectors. From a multitude of in vitro
and in vivo studies, a common theme emerges regarding the interaction of
Ras with these ligands: the existence of coordination mechanisms, very
probably contributing to determine the physiological course of the GDP/GTP
cycle of Ras in the cell. In this context, we have investigated in this work the
influence of the molecular interactions of various ligands on the action of
guanine nucleotide exchange factors (GEFs) on the Ha-Ras activity. Using an
in vitro system with highly purified components we were able to reconstitute
the Ras/guanine nucleotide cycle. We have found that the interactions of the
GTPase activating protein pl20-GAP and GEF with Ras take place in a
coordinated and specific manner, based on a competition for binding to the
active form of Ras, the complex with GTP [1]. Furthermore, we have
provided evidence that other effectors of Ras, such as Raf-I and PI 3-kinase,
compete with RasGEF for binding to the active form of Ras [2]. As a
consequence, at a GDP:GTP ratio mimicking their molar ratio in the cell, we
have found that p 120-GAP and Raf-1 enhanced the regeneration of RasoGTP
by GEF. Our work describes a novel function for effectors of Ras (pl20GAP, Raf-I and PI 3-kinase) and suggests a mechanism by which these
effectors protect RasoGTP in vivo against unproductive exchanges, such as
that with free GTP. This would confer a dual role to the effector: as
transmitter of the downstream signal of Ras and as upstream regulator of Ras
activation. Our results are in good agreement with other functional and
structural data from the literature, that have contributed to define the regions
of Ras interacting with its ligands.
At present, we are analysing the effects of the tyrosin-kinase c-Src on p 120GAP. Preliminary results show that GAP activity is inhibited when pl20GAP is phosphorylated. Thus, the phosphorylation of regulators and effectors
of Ras add a new interesting element of regulation to the mechanisms of
activation of Ras.
[1] Giglione C. etaL Z Biol. Chem. 272, (1997), 25128
[2] Giglione C. et al. Z Biol Chem. 273, (1998), 34737

(We/13.5/401)

Differential coupling of a2A- and a2a-adrenoceptor

subtypes to AC H in cotransfected DDT1 MF-2 cell line.
R. Alard, T.W. GettysI, SM. LanierI, J.P Maltier., C.
Legrand and Limon I. ESA 7080. universit~ P. & ~ Curie. 75252
Parts, France. ~Dept. of pharraacol., MUSC, Charleston SC 29425

We present evidence that stimulation of the human 133-AR, expressed

in CHO/K1 cells, specifically activates the MAP Kinases ERK 1 and 2,
but not JNK or p38. The extent and kinetics of the ERK stimulation by
the [~3-AR are identical to those of the endogenic insulin receptor.
However, insulin augments cellular proliferation, while 133-AR agonists
inhibit proliferation due to the production of cAMP.
The pharmacological profile of the ERK activation by the 133-AR
differs significantly from its activation of adenylyl cyclase. The order of
potency and intrinsic activities of both natural ligands, noradrenaline and
adrenaline, is inversed between both signalling pathways. In addition,
BRL 37344 and propranolol, ligands that act as agonists in the
stimulation of cyclase, act as antagonists for ERK activation.
The activation of ERKI/2 is sensitive to pertussis toxin, suggesting
that the 133-AR, in addition to its interaction with Gs, can also couple to
Gi/o. The activation of ERK by the 1~3-AR is furthermore sensitive to
PD98059, wortmannin and LY294002, indicating a crucial role for M E K
and phosphatidylinositol-3 kinase (PI3K), respectively. A [~3-ARmediated stimulation of PI3K is confirmed by the observation that the
selective agonist CGP 12177A specifically activates PKB. As was
observed for the activation of ERK, the activation of PKB is inhibited by
preincubation with pertussis toxin and PI3K inhibitors, suggesting that
both are a consequence of a G~i/o-mediated activation of PI3K.

In the rat myometrium, analysis of cross-talk between a2- and 132-AR

~pathways during the course of pregnancy showed that : i) at mid
pregnancy, when ct2an)-AR subtype is predominant, stimulation of a2-AR
results in a potentiation of isoproterenol-stimulated adenylyl cyclase (AC)
II family activity, ii) whereas at term, when Ceza- expression plainly arises,
a2-AR activation inhibits stimulation of AC induced by 13-AR agonist,
Since the same AC isoforms are expressed from mid-pregancy to
parturition, the inhibition of activated AC activity observed at term was
rather unexpected.
These data prompted us to investigate whether both a2-AR subtypes
mediate different effects on Gs-stimulated AC II family activity. To
address this issue, we compared the ability of x2~vo- or a2u-AR to
potentiate isoproterenol induced activation of AC II stably coexpressed in
DDT~ MF-2 cells with one of each ~2-AR subtype. The results obtained
clearly indicated that when cross-regulation between these two
transduction pathways implied the ~2A/o-AR subtype, cX2-agonist
enhanced isoproterenol AC II activity In contrast, activation of ct2a-AR
subtype lead to a persistent inhibition As potentiation and inhibition
effects are mediated by P T X sensitive G protein, we tested the hypothesis
as to whether each subtype specific effect would be the result of a
differential coupling to Gi proteins (Gi2 and Gi3). Subtype-specific
immunoprecipitation of G protein a subunits photolabeled with
[a-32P]GTP azidoanilide, revealed that whereas a2A~-AR subtype is
coupled to both Gi proteins a2a-AR is mainly coupled to Gi2. Since Gil3y
dimers are key components to induce AC II potentiation, two major
hypothesiscould explainthe lack of cqu-mediated AC II potentiation. 1)
a2n-AR activation provide an insufficient release or" I~y dimer 2) Gi3~T
could potentiate Gs-stimulated AC II isoform, whereas Gi213y would
mediate a negative input together with Gai2 Both hypothesis are
currently under investigation in our laboratory

s3 81

(We/13.5/402)

Abstracts FEBS' 99

Roles of the PH domain and polybasic C-domain of ARNO

in binding to anionic lipids PS, PIP2 and PIP3

(Wed13.5/403)

Eric Macia, Sonia Paris & Marc Chabre

M. Meriane, P. Roux, P. Fort, C. Gauthier-Rouvi~re

CRBM, CNRS UPR1086, 34293 Montpellier, France

CNRS, lnstitut de Pharmacologie Mol~culaire et Cellulaire, 660 route des

Lucioles, Sophia-Antipolis. 06560 Valbonne. France

Efficient activation of ARFI by the the Guanoside Exchange Factor

A R N O requires its interaction with a phospholipid membrane. It is favored by
the binding of A R N O ' s PH domain and polycationic C-terminal domain to
membrane anionic lipids and phosphoinositides. We have studied the binding
of two PH domain constructs of A R N O - without or with the adjacent Cterminal domain - to artificial phospholipid vesicles doped with 10 to 30% PS
and/or 1 to 2% PIP2 or PIP3. Binding strength was assessed by varying the
ionic strength of the vesicle sedimentation medium.
Without the C-terminal domain, the PH domain alone binds weakly to
PS and more specifically to phosphoinositides (PIP3 > PIP2). Addition of the
C-terminal domain enhances much the affinity for PS and, to lesser amounts,
that for PIP2 and PIP3, without reducing much the selectivity for PIP3.
However, with 30% PS and 2% PIP2 the binding of the PH + C-terminal
domains saturates (in physiological ionic strength medium) ; it is not further
enhanced upon replacement of PIP2 by PIP3. Parallel measurements of ARF
activation by A R N O confirm that under thoses conditions, the selectivity for
PIP3 versus PIP2 is lost and PIP3 is not more efficient than PIP2 for
activating ARNO. This may explain conflicting results obtained in vitro on
the PIP3/PIP2 selectivity of ARNO.
We have further observed an effect of the specific phosphoryiation by
PKC of a serine that is located within the polycationic K K R S V K K K motif of
the C-terminal domain.This serine phosphorylation reduces about fivefold the
affinity of the PH + C-terminal domains for PS. This suggests that
phosphorylation by PKC may regulate the activity of ARNO.

(We113.5/404) _ProteinExpressiono1 RhoA..Rho-badng Preen Kinaseand MMo~!n RegulatoAy

LLahtChain2oin HumanPreqn~ Mvomelrium
Moore,F and Lopez8ernal,A., Nuffield Dept of Obstetricsand Gynaecology,Oxford
University,JohnRadcliffeHospdaLOxfordOX39DU UK

We are mveshgatmg possible mechanism of human uterine smoolh muscle contraction that may
contribute to early contractions associated with premature laboudl] One putafive mechanism is
calcium sensitization which has been demonstrated in vascular smooth muscle[2]. Calcium
sensitization is medlaled via receptor activation of the small GTPase signalling pathwayresulting
in the activationof the Rho-bindmgserine/threoniaeprotein kinase(ROK) AcfivatedROK mediates
(i) phosphorylahonof the regulatory myosin light cham(MLC2o)thereby bypassing the need for
calcmm-calmoduhn actwafionof myosin light chain kinase in smooth muscleconttaction[3]and (~l)
phosphorylation of MLCz0phosphalase Ihereby temporally sustaining MLC~ophosphorylalion[4]
Thus when smooth muscle is 'calcium sensd,zed'the calcium concentration required to initiate a
contraction rs lowered Therefore it follows that the concentrationof a calutum-moblhzmgagonist,
such as oxytocm,is also lowered
To determine whether calcium sensitization operales m myomelrial smooth muscle we first
determined the levels of RheA, ROK and MLC2oprolem expression in human nonpregnanland
pregnant myomelnaitissue. Using Western immunoblottmgwe detected Rhea protein expression
in both nonpregnantand pregnant tissue The apparent levels of expression were slmdar when
expressed per mg protein Howeverwhen expressedper mg DNA (to compensale for the known
muscle hypertrophy and hyperplasla of pregnant myomelnum) the level of Rhea protein
expression was slgmficanllyhigher m pregnantmyometnum Similarly,expressionof ROK per mg
DNA is higher in pregnant myometnum than in nonpregnant myometrium Hence m human
pregnancy there is increased expression of Rhea and ROK proteins These results are in
agreement wdh dala showing that Rhea and ROK mRNA expressionincreases in pregnantrat
myometnum[5] In addition we have found that the level of MLC~ proteinexpression is higher in
human pregnant myomeldurn than in nonpregnant myometrium The functional significance of
increased uterine RhoNROK proletn expression m pregnancy and parturillon Is under
investigation

[This work Is supported by Tommy'sCampaign]

1 Lopez 8etnaI,A el al(1997)C_h,17 in Preterm Labor, eds Eider,Lament& Romero Churchdl
Livingstone, NY.
2 UehalaM et a1(1997)Nature3~9,990-994.
3 Kimuta,K.eta1(1996)Science273,245-248
4 Kureisht,Yet a1(1997)J BioLChem 272,12257-12260,
5 NiJro,N.eta1(1997)BBRC~,356-359

RhoG, Rac and Cdc42Hs GTPases prevent

skeletalmuscle differentiation of rat L6 myoblasts.

The development of skeletal muscle is a multistep process in which pluripotent

mesodermal ceils give rise to myoblasts that subsequently withdraw from the cell cycle
and differentiate into plurinucleated myotubes The differenuauon of cultured skeletal
myoblasts is activated by growth factors withdrawal and is accompanied by
transcriptional activation of muscle-specific genes (MyoD, Myf5, myogenim and Mrf4),
growth arrest and fusion to form multinucleated myotubes.
The Rho family of Ras-like GTPases includes Rac (1,2, and 3), RhoG, Cdc42Hs,
TC10, TTF/RhoH, Rho (A, B, and C), RhoD, RhoE, and RhoL. Rho GTPases
regulate a variety of cytoskeleton-dependent cell functions and have been implicated in
various cellular events such as the control of cell morphology, cell motility, cell
polarity, cell adhesion, metastasis, smooth muscle contraction, vesicular transport and
cell diwsion
In order to examine the role of RhoG, Racl, Cdc42Hs and RhoA in skeletal muscle
differentiation of rat L6 myoblasts, we have established stable cell lines of L6 myoblasts
expressing consntutively active forms of each of these GTPases. We show that whereas
RhoA acceleratesmyogenin and Troponin T expression, RhoG, Racl or Cdc42Hs
prevent expression of the myogenic factor myogenm and of the skeletal muscle protein
Troponln T as well as myoblast to myotube transition. Such inhibition of myogenin
expression by Racl or Cdc42Hs is no longer observed with the Y40C mutants of Racl
or Cdc42Hs, which are impaired in PAK and INK activation. Expression of RaclVI2
or CdcHs42V12 leads to strong JNK activation in L6 cells. As JNK activity positively
regulates the level ofmyf5, a bHLH muscle regulatory factor down-regulated at the onset
of differentiation,we have studied myf5 expressmn in RhoG-, Racl- and Cdc42Hsexpressing cells Our data suggest that RhoG, Racl and Cdc42Hs expresston Leadsto a
sustained level ofmyf-5 protein, which prevents the differentiation of skeletal muscle
ceils.

(We/13.5/405)

Identification and characterization of cellular partners

of the Rap2 protein, member of the Ras superfamily.
V. Nancy I, R.M.F. Wolthuis2, M-F de Tand I, J,L. Bos2 and J. de
Gunzburg ~IINSERM U-248. Institut Curie, Paris. France :Laboratory
forPhysiologlcal Chemist~'. Utrecht University. The Netherlands

The Rap group of proteins belongs to the Ras superfamily and shares
approximatively 50% o f identity with the products o f Ras protooncogenes. This group consists of the 95% identical R a p l A and R a p l B
proteins which globally share 60% identity with the 90% homologuous
R a p 2 A and Rap2B proteins. To date, the role in signal transduction o f
Rap2 proteins remains unclear.
A previous two hybrid screen had led to the identification o f a putative
effeetor specific o f Rap2 that is highly expressed in brain. We have
extended our search by screening a cDNA library from the T l y m p h o m a
cell line Jurkat with the full length wild type Rap2B protein as a bait, and
have identified the three known exchange factors for the small G protein
Ral, RaI-GDS, RGL and Rlf proteins (RalGEFs) as potential effectors. In
the screen, we have isolated the C-terminal Ras/Rap interaction domain
(RID) o f these proteins which interacts in the yeast two hybrid system as
well as in vitro with the GTP-bound forms o f H-Ras, R a p l , Rap2
proteins. Such promiscuity o f interaction is not limited to the C-terminal
RIDs of the RalGEFs, since the full length forms interact also with these
three GTPases in the two hybrid system and in in vitro assays.
In viva, upon transfection o f both a RalGEF and a GTPase into HEK 293
or HeLa cells, activated form o f Ras and Rap2 can be coimmunoprecipitated with full length RaI-GDS and Rlf proteins. Moreover,
immunofluorescence experiments showed that activated form o f Ras and
Rap2 proteins recruit cytosolic RalGDS and Rlf to their respective
resident compartiment, i.e. Ras to the plasma membrane and Rap2 to the
endoplasmic reticulurn. However, in viva, only RaI-GDS and Rlf
recruitement by Ras was able to lead to the activation of their target, the
Ral GTPase. Interestingly, Rap2/RalGEFs complexes don't block the Ral
activation by Ras. Whether the recruiternent o f RaI-GDS and Rlf to the
membrane o f the endoplasmic reticulum via Rap2 triggers other cellular
responses is presently under investigation.
Reference : V. Nancy and al. (1999) J BioL Chem. 274, 8737-8745

Abstracts FEBS'99

(We/13.5/406

s382

Distribution of G.ct/G~,et Proteins in Detergent-insoluble

Membrane Domalns: Effect of Agonist Treatment.

(We/13.5/407)

Z. Pesanova~'~,J, Cernyb:, J. Novotny"b, P. Svoboda B'b

lnstttute of Physiology and ~lnstitute of Molecular Genetics, Academy of
Sciences of the Czech Repubhc, 142 20 Prague, bFacultyof Natural Sciences,
Charles Umversity, 128 O0Prague, Czech Repubhc

G proteins as well as some other signalling molecules are frequently found

in plasma membrane complexes enriched in caveolin [1]. Here we used
E2MI 1 cells (human embryonic kidney cells expressing both thyrotropinreleasing hormone (TRH) receptors and Guo~ protein in large amounts) [2]
to investigate the effect of TRH on the distribution of G~Gu protein,
caveolin and other plasma membrane proteins in detergent (1% Triton X100)-resistant membrane fragments isolated by flotation in sucrose density
gradients composed of 5, 10, 15, 20, 25, 30, 35 and 40% w/v sucrose
(centfifugation in Beckman SW 41 rotor at 36000 rpm for 24 hours).
Density gradient profiles of Gqa/Guct and G[3 subunits were compared
with those of caveolin and plasma membrane markers representing both
integral (transmembrane) and glycosyl-phosphatidylinositol (GPI)anchored proteins. The dominant portion of caveolin was detected in the
part of gradient representing 15-20% sucrose while Gqa/Gu~ proteins
were detected in multiple fractions ranging from 15 to 40% sucrose. GPIanchored proteins CD 55 and CD 59 were distributed in low density
fractions (I5-30 %) while transmembrane proteins CD 29 and M 6 were
restricted to high density fractions (about 40%) of the gradient.
Further we demonstrated that distribution of caveolin and plasma
membrane markers CD 55, CD 59, CD 29 and M 6 were not affected by
TRH treatment. On the contrary, prolonged stimulation of E2M11 cells by
TRH resulted in a marked decrease in Gqc~/G1la proteins in low density
fractions (15-30 %) as compared with control untreated cells. This
decrease was manifestant for both exogenous (mouse) and endogenous
(human) isoforms of Guct protein.
References:
[1] O. Kifor et al., J. Biol. Chem., 273, (1998)21708.
[2] P. Svoboda et al., Mol. Phannacol., 49, (1996) 646.

Expression of adenylyl eyclase isoforms in pregnant

and non-pregnant human myometrium.
SA Price, I Pochun, S Phaneuf and A Lrpez Bernal, N.jwld
I)epapttne~tt ~ Ob.~tetrits ~aul Gvmwcologr, I/mversity o) OLtbtH. John
Rad~ hide [lo~ptt.l, O~jotd, OX3 91)it, UK.

Adcnylyl cyclases (AC) catalyse the conversion of ATP to the second messenger
cAMP m response to tile actions of hormones and drugs upon cell surface
receptors. At present, ,line isoforms of AC have been described and may t~e
assigned into one of four groups according to the,r modes of regulation. Group
I ACs (types I, I11 and VIII) are stimulated by calcium and ealmodulin, whdst
Group 2 isoforms (II, IV and VII) are activated by G-protein [3y subunits and
protein kinasc C phosphorylation. Group 3 ACs (V and VI) are inhibited by low
calcium concentratim~s, whilst the Group 4 isoform AC IX is insensitive to
either calcium or [~y subumts, but is inhlbltcd by calcineurin.
T h e exact n'lechaniMn(s) necessary tol uKunlaining tile nterus ill a quiescent state

during gestation, and the transition to the contractile state required for parturition
iemam unknown, however, elevated cellulm" cAMP is known to promote
myometnal relaxatkm lhrough effects on intracellular calcium concentration and
myosin hght chain kinase. Characterisation of the AC isoform(s) present in both
pregnant and non-pregnant myometrium may allow identification of factors
inw~lved in the activation or inhibition of AC activity, and hence enable a more
efficaciotts pharmacological approach to lhe atleuuaUon of prcterm labour.
hmnunoblotting and immunocytochemistry, using a range of spectfic anubodies,
and RT-PCR were utilised to verify the presence of var,oos AC isot~arms and the
mRNA coding lot fllem m nryometrium. Messenger RNAs coding [~w AC
I~,OfOl'tI1~, I. II alld IX wclt~ presell| ill saml>le,~ o f inymneUla] tls.~lJe fron/ bolh
pregnant and non-pregnant WOlrlCn and cultured lnyolnelrltl} cell>. Western blot

aualysl~ ol plasma inembranes prepared flom pregnant and non-pregmn~t

myon'lctrltnll, aml innllunocytochconslry of cells isolated from non-pregnant
myometrium confmned the expression of the mRNA as AC protein, with ACs 1.
l], Ill, IV, V. VII and IX being identified. Differential expression of the AC
isoft)rm groups coukl be observed between pregnant and non-pregnant hssue,
wub those ACs which are stinmlated by calcium decleasing dunng gestation
whilst calcmln-mhibited ACs increased.
Hence, several factors may be
implicated m the maintenance of uterine quiescence during pregnancy and the
induction of uterine contractility at parturition via their disparate effects upon
distinct AC isoforms. The functional significance of these changes in
nayometrial AC expression during pregnancy is currently under investigation.
This work was.fitnded by 7bmmy's Campaign

(We/13.5/408)

Inhibition ofRho GTPases delineates a novel

p53-dependent apoplotic pathway
P. Roux*, P. Lassus, O. Zugasti, A. Philips, Ph Fort*, U. tlibner.
CNIIS, IGM, UMR 5535, *CRBM, UPR 1086, 1919, Route de Mende,
F-34293 Monpettier Cedex 5, France.

Rho GTPases have been implicated in many physiological processes,

such as the control of cell shape, motility, polarity and adhesion. In addition
to their role in cell morphology, Rho proteins are involved in the control of
normal and pathological proliferation. Constitutively active RhoA, Racl and
Cdc42Hs trigger entry of quiescent fibroblasts into S phase and participate
in Ras-mediated transformation. Furthermore, several lines of evidence
indicate that constitutively active Rho GTPases are also involved in the
conrol of apoplosis.
Now we show that dominant negative mutants of two members of
Rho family, Racl and Cdc42Hs are also potent inducers of apoptosis.
Contrary to cell death signalled by activated forms of these GTPases, the
NI7 dominant negative mutants have a strict requirement for wild type p53
for activating the apoptotice pathway. Moreover, their expression activates
endogenous p53, as judged by transactivation assay of p53 dependent
promoters. In contrast to the activated forms of these GTPases, which
stimulate the stress activated MAP kinases SAPK/YNK and p38, the inactive
furors of Rael and Cdc42Hs activate the extracellular signal activated
ldnases (ERKs). Simultaneous activation of p53 and ERK appears to be
necessary, but not sufficient for induction of apoptosis by tile dominant
negative forms of Rael and Cdc42Hs. Our data demonstrate the existence of
n complex set of functional interactions between GTPases of the Rho
I?~mily, p53 and ERKs, participating in the control of apoptosis.

(We/13.5/409)

Structure/function relationship of Rho-GEF domains:

example of the multifunctional protein Trio
S. Schmidt, JM. Bellanger, S. Estrach, S. Diriong, A.Debant
CRBM-CNRS, UPR 1086, 34293 Montpellier C~dex 5, France.

The Rho-family GTPases Cdc42, Rac and RhoA are involved in a wide
vari. y of physiological processes, all implying a structural reorganisation of
the :tin cytoskeleton. They are activated by guanine nucleotide exchange
fact. "s (GEFs) which accelerate their GDP/GTP exchange rate. Rho-GEFs
shar a catalytic DH (Dbl-Homology) domain, followed by a PH (Pleckstrin
Hor. ~logy) domain, which is thought to be involved in protein localisation
and r in protein-protein interactions. Recently, the three dimensional
stru ure of Rho-GEFs has been solved, and functionally important residues
of tr~ DH domain have been identified. However, the mechanistic details
dete nining the specificity of a given GEF towards its target are poorly
und stood.
Trio is a multifunctional protein that is comprised of two functional
Rho 3EFs and a serine/threonine kinase domains. It has been shown in vitro
and .7 vivo that the first GEF domain (GEFDI) activates Racl, while the
sect ~d GEF domain (GEFD2) acts on RhoA. Moreover, co-expression of
botl domains activates simultaneously both GTPases. Thus, Trio is the first
exa ,pie of a two-headed exchange factor, able to link Rac and Rho pathways
in v 'o. Trio, with its two GEF domains of different specificity, is a good
mot "1 to study the molecular mechanisms determining the binding specificity
ofa 3EF towards its GTPase, and the role of the PH domains in this context.
In t, is purpose, we have started to develop new molecular tools, peptide
inhi' itors, targeting specifically the two DH-PH domains of Trio. To date, no
Rhe GEF inhihitors exist. Using a recently developed strategy in yeast, based
on
two hybrid screen, we have already isolated peptides, which bind
spe, 'fically either Trio GEFD1 or GEFD2, and are currently testing their
inhi ,itory effects on GTPase activation by Trio in vitro and in vivo. These
pep,:de inhibitors of Trio will allow us to determine the regions and amino
acid" critical for the specificity of a GEF towards a given GTPase.

s383

(We/13.5/410)

Abstracts FEBS'99

Evidence that atypical Protein kinase C-~. and ~ participate in

Ras-mediated reorganization of the F-actin cytoskeleton.
Florian Uberall ~, Karma Hellbert~, Sonja Kampfer~, I,~arl Maly~,
Andreas VillangerI, Martin Spitaler1, James Mwanjewe 1, Gabriele
Baier-Bitterlich I, Gottfried Balerz, and Hans H. Grunicke ~
From the Institut of Medical Chemistry and Biochemistry ~and the
Institute of Medical Biology and Human Genetics :, Umversity of
Irmsbruck, A-6020 Austria.

Expression of transforming Ha-Ras L61 in NIH3T3 cells causes profound

morphological alterations which include a disassembly of actin stress fibers.
The Ras-induced dissolution of actin stress fibers is blocked by the specific
PKC inhibitor G F l O 9 2 0 3 X at concentrations which inhibit the activity of the
atypical aPKC isotypes ~, and ~, whereas lower concentrations of the inhibitor
which block conventional and novel PKC isotypes are ineffective.
Coexpression of transforming Ha-Ras L61 with kinase-defective, dominant
negative (DN) mutants of aPKC-~ and ~, as well as antisense constructs
encoding RNA directed against isotype-specific 5' sequences of the
corresponding mRNA, abrogates the I-la-Ras-induced reorganization of the
actin cytoskeleton. Expression of a kinase-defective, dominant negative (DN)
mutant of cPKC-~x was unable to counteract Ras with regard to the dissolution
of actin stress fibers. Transfection of cells with constructs encoding
constitutively active (CA) mutants of atypical aPKC-~ and ~ lead to a
disassembly of stress fibers independent of oncogenic Ha-Ras. Coexpression
of (DN) Rac-1 N I 7 and addition of the phosphatidylinositol (Ptdlns) 3-kinase
inhibitors wortmannin and LY294002 are in agreement with a tentative model
suggesting that, in the signaling pathway from Ha-Ras to the cytosketeton
aPKC-~, acts upstream of Ptdlns 3-kinase and Rac-1, whereas aPKC-~
functions downstream of Ptdlns 3-kinase and Rac-I.
This model is supported by studies demonstrating that cotransfection with
plasmids encoding L61Ras and either PKC-~, or PKC-~ results in a stimulation
of the kinase activity of both enzymes. Furthermore, the Ras-mediated
activation of PKC-~ was abrogated by co-expression of dominant negative
Rac-1 N17.

(We/13.5/411)

RhoG GTPase specifically binds kinectin and affects

microtubule-dependent vesicular transport.
E. Vignal, A. Blangy, C. Gauthier-Rouvi~re and P. Fort.
C.R B.M., CNRS, UPRI086, 34293 MontpeUier, France.

RhoG is a member of the Rho family of GTPases that shares 72% and
62% sequence identity with Racl and Cdc42Hs, respectively. RhoG protein is
mainly associated with endoplasmic reticulum membranes, with a minor fraction
located at the plasma membrane. RhoG activation elicits the formation of
lamellipodia, microviUi and filopodial extensions, resulting from the independent
activation of Racl and Cdc42Hs GTPases. In addition, microtubule
depolymerization impairs RhoG translocation and activity, but has no effect on
Racl and Cdc42Hs activities, suggesting that a rnicrotubule-dependent target
mediates RhoG activity. Using a yeast two-hybrid screen, we characterized the
156 kDa kinectin as a potential RhoG target. Kinectin is an integral protein of the
endoplasmic reticulum (ER), acting as a receptor for kinesin, a microtubule
motor involved in plus-end dependent forward transport. In the yeast, kinectin
binds the GTP-bound form of RhoG, and to a lesser extent of Rac 1, Rac2, RhoA
but not RhoB and Cdc42Hs. Two kinectin domains (KID) are responsible for the
interaction : KiD1 specifically binds RhoG and Rac and is located within the
central region of kinectin, which contains seven 70 a.a. coiled-coil repeats and is
involved in kinectin homodimerization. Two repeats are sufficient to promote an
efficient binding. The second domain (KID2) is located at the C-terminus and
mediates the binding to RhoA. In REF-52 fibroblasts, the kinectin co-localizes
with RboG and RhoA, but not Rac. Expression of KiDI and KiD2 respectively
inhibits RhoG and RboA, but has no effect on Rac and Cdc42Hs activities. In
addition, kinectin redistributes into distinct subcellular locations depending on
whether RhoG is activated or inhibited. Kinectin therefore fits all the criteria of a
genuine RhoG target. To address the role of RhoG in microtubule-dependent
transport, we examined the effects of RhoG activity on the exocytic pathway of
the vesicular stomatis virus glycoprotein (VSV-G). RhoG inhibition prevented
the secretion of the VSV-G protein, which redistributed with RboG and kinectin
in particular cytoplasmic structures, distinct from ER to Golgi intermediate
(ERGIC), cis-, medial- and trans-Golgi compartments.
Our data therefore establish that kinectin behaves as a genuine RhoG
effector and suggest that RboG promotes its effects through the control of a
microtubule-dependent vesicular transport.

Abstracts FEBS'99

s384

15.3 Plant defense mechanisms against biotic and abiotic stresses

(We/15.3/412)

INDUCTION

OF

POLYPHENOL

OXIDASE

IN

(We/15.3/413)

Protein synthesis in wheat genotypes

under water stress
J.A.Aliev

SEMPER VIVUM TECTOR UM

Veronika Abram, Marjan Donko, Andreja Stepec

Institute of Botany, Patamdar Shosse 40, 370073, Baku. Azerbaijan

Department of Food Science and Technology, Biotechnical Faculty,

University of LjubOana, Jamntkar)eva 101, 1111 Ljubljana, Slovenia
Sempervivum tectorum is an evergreen plant belonging to the large family

of Crassulaceae and exhibits crassulacean acid metabolism [I]. It is known

to survive prolonged periods of drought and is tolerant to most common
pests and diseases. This was the main reason that we found these plants
interesting and we would like to investigate and explain biochemical
grounds of such tolerance. An induction of polyphenol oxidase (PPO) was
first followed after S. tectorum plants were exposed to different abiotic
stresses (mechanical injury, drought, 10 % methyl jasmonate). PPO was
extracted from frozen homogenised fresh leaves of treated plants and its
activity was determined by the method of Sanchez-Ferrer et al. [2] with 25
mM 4-methyl catechol in 10 mM phosphate buffer, pH 6.5. Methyl
jasmonate was the most effective in the induction of PPO. Compared to
control samples the PPO activity showed a 6-fold increase after 24 hours
and a Y4-fold increase after 48 hours. SDS-PAGE electrophoresis showed
the presence of proteins with Mr of 40-60 kDa and immunoblotting with
specific antibodies showed that Mr 40 kDa belonged to PPO.

[l] Thomas, D. A. et al., Plant Physiol. Biochem., 2, (1987) 85.

[2] Sanchez Ferrer et al., Phytochemistry, 5, (1989) 1321.

(We/15.3/414)

Glucosylatlon of phenylpropanoids in tobacco cells after

elicitation is a means for their apoplastic excretion and
subsequent metabolism of hydrogen peroxide
R. Baltz, J. Chong, B. Fritig, P. Saindrenan

Photosynthetic activity of separate organs, mainly an ear, plays a key role

for the protein synthesis level of grain. Under suppression of the protein
synthesis twice as much protein-free nitrogen accumulates in grain and other
ear elements. Consequently, a low protein content in grain is connected with
deficiency in products of photosynthesis necessary for protein synthesis but
not with nitrogen deficiency. Under water stress a percentage content of
storage proteins in grain of all genotypes is increased on an average by 5%
of its absolute value. In contrast, a total content of proteins is decreased.
The losses for the numerous genotype forms vary from 20% to 70%.
Genotypes of Triticttm durum L. are more tolerant to water stress in
comparison with those of Triticum aestivum L. In milk ripeness stage
contribution of the genotypes' ear is between 30 and 60%. Subsequently,
with grain forming contribution of ear increases.
In tolerant genotypes
~4CO2 assimiliants are transported more vigorously and assimilation of
carbon dioxide by ears is twice intensively. Therefore under extreme water
supply conditions considerable protein yield is produced in grain of the high
productive intensive varieties Barakatli 95, Alinja 84 with active
photosynthetic function . In this condition the activities of C4-cycle
enzymes, such as PEP carboxylase, NAD and NADP malate dehydrogenase
and aspartate aminotransferase in leaves and ear elements increased more
significantly.
Intensity of r e a l photosynthesis and photorespiration increased
simultaneously. Therefore attempts to reduce photorespiration with the
purpose of raising productivity have been failed Both increase in the
tolerance of genotypes to water stress and strengthening of drought resulted
in a dramatic decrease in the photorespiration intensity of ear elements.
Donor properties of genotypes in relation to their tolerance to water stress
were revealed. On the base of the aboriginal tolerant genotypes new wheat
varieties capable to accumulate a great amount of protein in grain and grow
in severe soil drought conditions have been created

(We/15.3/415)

E. N. Baranova', V. A. Kadykov', T. P. Astafitrovab

~AII-Ru$~a Institute ofAgriculturaI Biotechnology,12 75_~OMoscow,Russia
bln~tute of Biology and Biophysics, 634055 TS~, Toma~ Russia

IBMP/CNRS. 12. rue du Gdndral Ztmmer, 67084 Strasbourg. France

Plant resistance to pathogen attacks is often associated with the so-called

hypersensitive response (HR) characterized by the rapid collapse of the
challenged host cells and the restriction of pathogen spread. Early events
underlying the induction of hypersensitive cell death often includes the
production of H202 reminescent of the oxidative burst during phagocyte
activation, induction of the phenylpropanoid metabolism and deposition of
related wall-bound phenolics. Recently, two early-inducible tobacco genes
(TOGT) coding for glucosyltransferases acting on hydroxycoumarins,
particularly scopoletin, and hydroxycinnamic acid derivatives issuing from
the phenylpropanoid pathway, were shown to respond rapidly to a fungal
elicitor or during the HR of tobacco to tobacco mosaic virus [1].
Glucosylation protects the highly reactive phenolics against oxidation and
renders phenolic compounds more soluble. It might also serve to produce
transport forms of phenylpropanoids into the apoplastic space providing a
means to buffer the effect of reactive oxygen species (ROS) occuring along
with the HR. We showed that elicitation of cell suspension cultures with a
fungal elicitor caused a rapid accumulation of TOGT protein which
paralleled induction of UDP-glucose:scopoletin glucosyltransferase
activity. Further, elicitation of cells induced a dramatic production of
H202 and excretion of the hydroxycoumarin glucoside scopolin and
hydroxycinnamate derivatives into the culture medium, while
accumulation of aglycone forms could be observed only in the presence of
diphenylene iodonium, an inhibitor of H202 production after elicitation.
Moreover, extracellular/3-glucosidases were shown as part of the excretion
mechanism. These results strongly support a role for TOGT proteins as
efficient intermediates in the sequence of events leading to the release of
phenolics into the apoplastic space after elicitation and point to a possible
dual role of extracellular phenylpropanoids as potent antioxidants and
precursors of the wall-incorporated phenolics.
[ 1] Fraissinet-Tachet et al., FEBS Letters, 437, (1998) 319.

Low oxygen pr--,~sure-induced changes in cells of leaves of

plant~

Leaves of maize, pea and barley plants were examined to determine the
mechanisms of influence oflow oxygen pressure and resistance to it. Structural
and functional changes in photosynthetic apparatus of mesophyl took place in
conditions of hypobaric hypoxia. The character of the response was
p~sumably dependent on dura~on of action. The assembling of photosynthetic
apparatus in the postetiolated seedlings was followed by lower of green and
yellow pigments production, low photochemical and ribulosobiphosphate
carboxylase activities, appeasence of

small cells and retardation in

development of chloroplasts granular system. The low efficiency of

photosynthesis in developing seedlings enhanced the dominate role of
respiration dunng plant adaptation to hypobaric hypoxia. Underpreasure in
enclosed space of pr~sure chamber induced the development of hypoxic state
fit leaf cells enhancing with the alteration of applied influence power and
duration of action. This phenomenon was observed not only at dark, but when
the plants were exposed to fight, and manefested by acfiv~on of the key
enzymes of

glycolysis (glyceraldehyde phosphate debydrogenase-NAD,

pyruvatekinase),

pentose

phosphate

pathway

(gluco~-6-phosplmte

dehydrogenase) and fermentation (alcohol dehydrogena~-NADH), and also by

repression of the oxidative reactions of Krebs cycle (malate dehydrogenaseNAD, isocitra~ dehydrogenase-NAD). So, in the conditions of complete
flooding or hypobaric hypoxia in mesophyl of leaf the compensatory reactions
arc based on the coupling of respiration to photosynthesis.

s385

(We/15.3/416)

Abstracts FEBS'99

Synthesis of glutathione and homoglutathiune in Medicago

C. Bladier, P. Frendo, C. Mathieu, M.J. Hernandez, G.Van
de Sype, D. H6rouart, A. Puppo
Biologic Vdgrlale et Microbiologle, ESA 6169. UNSA,06108 Nice, France

The tripeptide glutathione (7-glutamylcysteinylglycine, GSH) is a low

molecular weight thiol which is synthesised in an ATP-dependent two-step
reaction. In the first step catalysed by the 7-glutamylcysteine synthetase (TECS), y-glutamylcysteine (y-EC) is produced from L-glutamic acid and Lcysteine. In the second step catalysed by glutathione synthetase (GSHS),
glycine is added to the C-terminal end of 7-EC to form GSH. GSH is a
major component of the plant protection against abiotic stresses : it is
involved in the dctoxification of xenobiotics, in the defence against heavy
metals and in the protection against oxidative stress. In addition to GSH,
another low molecular weight thiol, homoglutathione (y-glutamyl
cysteinyll3-alanine, hGSH), has been specifically detected in legumes. The
synthesis of hGSH, instead of GSH, is determined by the amino acid
specificity (glycine or [3-alanine) of the enzymes which catalyse the
second step of the tripeptide synthesis. Little is known about the
physiological role of hGSH and similar functions to GSH have been
proposed.
GSH and hGSH were quantified by HPLC in Medicago truncatula, a phmt
model to study plant/Rhizobium interaction, hGSH was detectable only
48h after seed germination whereas GSH was present in the dry seeds.
This indicates that only GSH is used for sulphur storage in seeds pointing
to a different role between GSH and hGSH in sulphur metabolism. The
hGSH was detectable only in the underground part of mature plants
whereas GSH was present in all the organs. The differential localization of
hGSH was linked to the presence of the hGSH synthetase (hGSHS) which
was found only in roots. In contrast, y-ECS and GSHS activities were
found in both roots and leaves, eDNA encoding 7-ECS and two partial
cDNAs (gshsl and gshs2) showing high identity with GSHS were isolated
by screening of M. truncatula libraries with Arabidopsis thaliana cDNAs
as probes. High y-ECS activity was detected in protein extracts of a yECS-deficient E. coli strain expressing the M. truncatula 7-ECS mature
protein. Northern blot analysis showed that the M. truncatula 7-ECS gene
was similarly expressed in all the mature plant organs tested whereas
gshsl had an higher expression in leaves and flowers and gshs2 was motc
expressed ill roots and nodules.
(We/15.3/418)

Secondary structure and thermal stability of the extrinsic

16 and 23 kDa proteins of chloroplasts photosystem II
R. Carpentier', Y. Ishikawab, Y. Yamamoto b, H. Zhang~
"GREIB, Univ. du Quebec gt Trois-Rividres, Qua., G9A 5H7 Canada,
bDept, of Biology, Okayama Univ., Okayama 700, Japan

The chloroplasts photosystem II is a membrane-bound protein complex

reponsible for oxygen evolution. Three extrinsic polypeptides of apparent
molecular weights of 16, 23, and 33 kDa (OEC16, OEC23, and OEC33,
respectively) are associated with the oxygen evolving complex. These
polypeptides are involved in the stabilization of the manganese cluster
responsible for water splitting and in the regulation of Ca 2+ and CIrequirement in the oxygen evolving complex. In this study, the secondary
structure and thermostability of the extrinsic polypeptides OEC16 and
OEC23 have been characterized between 25 and 75C. Quantitative
analysis of the amide I band with the isolated proteins in D20 using Fourier
transform infrared spectroscopy showed that OECI6 contains 34% a-helix,
28% [3-sheet, 6% turn, and 32% disorder whereas OEC23 was composed of
5% a-helix, 37% [3-sheet, 24% turn, and 34% disorder.
Thermal
denaturation due to irreversible protein aggregation and coinciding with
unfolding of a-helical to ~-sheet structure in OECI6 and unfolding of [3sheet structure to disorder structure in OEC23 occurred with a
conformational transition at 65C for OEC16 and at 55C for OEC23.
Those transitional temperatures were considerably higher than that of
photosystem II reaction centers reported in the literature. Implication of
these results for the functions of these polypeptides in the thermal
stabilization of the oxygen evolving complex will be discussed. Further, the
large proportion of 13-sheet structure found in OEC23 may facilitate more
extensive interfacial contact between OEC23 and the lumenal side of
photosystem II membranes. This supports a structural shielding role for
OEC23 that may restrict the access of exogenous reductants other than
water to the catalytic site of the oxygen evolving complex.

(We/15.3/417)

Increased resistance to oxidative stress in tobacco plants

with bacterial serine acetyltransferase overexpression.
A B|aszczyk, R. Brodzik, A. Sirko
hTstitute of Btochemlstrv and &oplTvslcs, PAS, 02-106 l[arsaw.Poland

Sulfur metabolism in plants includes uptake of sulfate from the

environment, assimilation into cysteine and channeling into proteins and
secondary metabolic compounds like glutathione (GSH), phytochelatins and
sulfolipids [1] The biosynthesis of cysteine represents the final step of sulfate
assimilation in bacteria and plants. It is catalyzed by sequential action of serine
acetyltransferase (SAT) and O-acetyl (thiol) lyase (OAS-TL) In these
reactions, L-serine is first acetylated by SAT to form O-acetyI-L-serine (OAS)
which then reacts with free or carrier-hound sulfide to form cysteine and
acetate Both OAS-TL and SAT seem to be required in all cellular
compartments that carry out protein synthesis. They have been found in the
cytosol, chloroplasts and mitochondria from various plants [2] SAT and OAS
are the major regulatory factors in biosynthesis of cysteine in plants GSH
serves not only as the main storage form of reduced sulfur but it also plays an
important role in the control of the thiol-disulfide status of the cell, in the
detoxification of xenobiotics and finally, tn plant responses to abiotic stress and
pathogens The ascorbate-glutathione cycle has major significance as an
enzymatic scavenging mechanism for the removal of active oxygen species [3]
We have constructed plant expression cassettes containing the
Eschertchta colt cysE gene (encoding SAT) targeted to either cytosol or
chloroplasts In addition to the wild type cysE, a mutant encoding SAT which
is insensitive to the feedback inhibition by cysteine was used After the
AgT"obactermm-mediated transformation of tobacco we identified for each of
the four groups, stable transformed plants containing several-fold higher SAT
activity in comparison to the control plant. Determination of non-protein thiol
contents indicated 2 to 3-fold higher cysteine and glutathione levels in some of
these transgenic plants The maximal elevation of the cysteine level was about
fourfold while that of GSH was only about twofold higher than in the controls.
The most striking physiological consequence of that fact, however, was
several-fold increased resistance to oxidative stress
REFERENCES.
1. Brunold, C. & Rennenberg, H : Prog. Bot. 58 (1997) 164-186
2. Hell, R ' Planta 202 (1997) 138-148
3. Noctor, G. et al. J. Exp. Botany 49 (1998) 623-647

(We/15.3/419)

Sulfated oligosaeeharides mediate cross-talk between a

marine red alga and its green algal pathogenic endophyte.
K. Bouarab a, P. Potina, J- Co rrea and B. Kloareg a.
astation Biologique-CNRS UMR 1931, BP 74, 29682 Roscoff , France
bpontificia Universidad Catolica de Chile, C. 114-D, Santiago, Chile.

The pathogenic filamentous green alga Acrochaete operculata is able to

completely colonise the sporophytic phase of the red alga Chondrus crispus,
whereas it does not penetrate beyond the outer cell layers of the
gametophytic stage. Given that the isomorphic life history phases of C
crispus differ in the sulfation pattern of the extraceUular matrix (ECM)
galactans (carrageenans), we investigated whether the latter might modulate
the pathogenicity of the parasite. Consistent with the ECM composition of
the host susceptible generation, oligo-lambda--cm-rageenans induced a release of
hydrogen
peroxide,
stimulated
protein
neosynthesis,
increased
carrageeenolytic activity and elicited the induction of specific polypeptides in
the pathogen, resulting in a marked increase in pathogenicity. In contrast
oligo-kappa-carrageenans, i.e., ECM fragments from the host resistant
generation, did not induce H202 release nor significantly influenced
carrageenolytic activity, hindered amino-acid uptake, reduced the pathogen
virulence, and enhanced its recognition by the host. Moreover, C. crispus
sporophytes and gametophytes were shown to behave differently in their
initial response to challenge with cell-free extracts of A. operculata.
Gametophytes exhibited a burst of hydrogen peroxide whereas only a sma;l
release was observed in the sporophytes. The oxidative burst in C. crispu:;
garnetophytes was stronger when extracts were prepared from Acrochaete
filaments previously elicited with oligo-kappa-carrageenans. No burst was
observed upon challenging with extracts prepared from pathogen cultures
previously incubated in the presence of oligo-lambda-carrageenans. This
pathogen-induced oxidative burst was inhibited by the NADPH oxidase
inhibitor diphenylene iodonium and was shown to play a key role in the
defense system of C. crispus. This is the first report from a marine algal
pathosystem showing that, as in terrestrial plants, ECM-oligosaccharides
mediate host-pathogen interactions.

Abstracts FEBS'99

(We/15.3/420)

Insertionnal mutagenesis in the rice blast fungus

P.-H. Clergeot ~, S. Sibuet b, M.-P. Latorse b, C. Derpierre b
D. Tharreau , J.-L. Notteghem and M.-H. Lebmn"

s386

(We/15.3/421)

u Department of Plant Ph~wtolo&'~,Pohsh .4caden~v of Sctences 30-239

Krak6,a; Podht-n~t 3
zl Plctnt Protectton hlsttlut of Hztngctrtan
.4cademv of Science, BttdapeM 1022, Herman O. 15, HttngaO,

aUMR 1932 CNRS-RPA and bRhOne-Poulenc AgJv, 69009 L~on

cCIRAD. 34032 Montpellier, France.

Unravelling functions implicated in the infection process of plant pathogenic

fungi is an important challenge for crop protection. We are identifying
pathogenicity genes of the rice blast fungus, M a g n a p o r t h e grisea using
REMI insertionnal mutagenesis [1]. We have analyzed 3000 REMI
transformants for their pathogenicity defects by spore inoculation on
detached rice and barley leaves. We recovered 25 mutants either nonpathogenic (n: 8) or significantly reduced in their pathogenicity (n: 17).
Among ten mutants crossed with a compatible wild type strain, only three
(M421, M700 and M763) were tagged by one copy of the plasmid. Mutant
M700 corresponds to an insertion of the plasmid in the melanin pathway
gene B U F I . The number of lesions caused by mutant M763 was
dramatically reduced compared to wild type (-95%) and its penetration into
barley epidermal cells was impaired despite an apparently normal
appressorium. The non-pathogenic mutant M421 (no lesions) was able to
differentiate appressoria with reduced turgor [2] and was unable to penetrate
through the plant cuticle. Flanking regions to the plasmids were cloned by
inverse-PCR (M421) or by plasmid rescue (M763). Two cosmids
hybridizing with the M421 junction fragment were identified from which a
3.5 kb genomie snbclone was used to complement the M421 mutation.
Cloning and sequencing of the M421 cDNA revealed a 0.67 kb ORF
interrupted by two short introns. The 225 aa protein deduced from ORF421
is hkely to be a small membrane protein involved in apprcssorium function.
1- Sweigard et al. 1998. Mol. Plant Micr. h~teract., 5: 404-412.
2- de Jong eta/. 1997. Nature, 389: 244-245.

(We/15.3/422)

Maize H M G I/Y protein confers resistance to

nickel in Saccharomyces cerevisiae
C. Forzani 1,2, C. Loulergue, J-F. Briat Iet M. Lebrun 2
IBPMP, Agro-M/INRA/CNRS URA 2133, Place Viala, 34060 Montpellier
2Universitg Montpellier II, Place E. Bataillon, 34095 Montpellier cedex 5

Nickel is essential for plant growth but is also highly toxic upon increase in
its concentration. The molecular mechanisms involved in metal resistance are
unknown. To isolate genes involved in plant resistance to nickel toxicity, the
yeast S a c c h a r o m y c e s cerevisiae was transformed by a cDNA library
constructed from maize roots mRNA in the multicopy plasmid pYPGEI5.
One maize cDNA improved growth of transformed yeast when plated on a
modified minimum medium containing NiSO 4 0,5 raM. The resistance
conferred by this plasmid was specific to nickel, cobalt and to a lesser extent
to cadmium. This maize cDNA encoded a polypeptide of 19,9 kDa showing
homology to the high mobility group proteins of subclass HMG I/Y [1]. The
protein contains four copies of the AT-hook DNA binding motif which
mediates the interaction between HMG I/Y proteins and AT-rich sequences
within plant promoters [2]. However, the function of HMG I/Y protein
remains not well known. Western blot analysis demonstrated that the
resistance to nickel was due to the expression of the maize c D N A in yeast.
Nickel resistance in yeast was maintained even when the HMG I/Y protein
was expressed from a monocopy ptasmid. In perspective, the involvement of
the HMG protein in metal resistance in plants will be further investigated by
the overexpression of the isolated maize HMG cDNA in plant cells.
References:
1 - Nieto-Sotelo J., Ichida A. et Quail P. H., Plant Cell, 6, (1994) 287-301
2 - K.D. Grasser, The Plant Journal 7 (2) (1995), 185-192

Wheat H*-ATPase changes in response to stem rust elicitm

F. Dubert t~ . A. Skoczowski ~), B. Barna 21, E. Nlemczyk II

The plasma membrane of plant cells contains H+-ATPase consuming

energy of ATP to transfer protons from the cytoplasm to the extracellular
compartment. This provides the driving force for secondary transport of ions
or nutrients into cells. Many studies have been focused on the influence of
pathogen infections, or elicitors on the 1l+-ATPase. These indicate that treatment of plants with pathogen elicitor may increases H+-ATPase activity.
We investigated the effect of elicitor from wheat stem rust (Puccinia
gramim.~ trilici) on H~-ATPase activit~ in membranes of two near-isogenic
wheat lines with or without a single resistance gene to the pathogen.
Plants were grov,.n for 2-weeks and overground parts were taken for
isolation of plasma membranes. Rust uredosporcs were germinated on the
surface of ~ater ibr 24 h, homogenized in 70% EtOH and centrifuged 10
rain with 10000 g. The pellet was homogemzed with water, centrifuged and
supernatant '~as used as putative elicitor. Plasma membranes werc isolated
by aqueous polymer two-phase partitioning and examined for purity level
using cell organella enzyme markers.
Specific H'-ATPase activity calculated on protein content in plasma
membrane vesicles from non-treated resistant line (Ra) was significantly
higher than from susceptible (Sa) one. In the presence of small amount of
elicitor H+-ATPase activity increased significantly only in susceptible line,
but it did not reach the level of resistant one. In the resistant line significant
increase of I-t'-ATPase activity was observed only after adding the largest
dosis oi" the elicitor. Despite of these differences both lines shorted very
similar pattern of dose-responsc curves. The inf]uence of higher amounts of
elicitor on the enzyme kinetics was exactly the same both in Ra and Sa
naembrane preparations, because the relative changes of K,, and V,,~,~ values
as lnllucnced b3, elicitor were less than 1%.
We suggest that phosphorylation and dephosphorylation of plasma
membrane bound H+-ATPase may play an important role through the signal
transduction pathway in the resistant or susceptible reaction of wheat to
stem rnst.
(We/15.3/423)

A Peroxiredoxin induced by mercuric chloride in bean

G. Genot
lnstttut de Biologie Mol~culatre des Plantes/CNRS-Universtt~ Louis Pasteur,
12. rue du Ggn~ral Zimmer. 67084 Strasbourg Cedex, France

In bean, mercuric chloride induces the expression of a large array of

stress-related proteins such as pathogenesis-related proteins (glucanases,
chitinases) glycme, proline-rich protein, HSP, ubiquitins for instance.
Among these stress-related proteins we isolated a 20 kDa acidic protein.
The N-terminal amino acid sequence as well as the nucleotide sequence
of its mRNA showed a strong homology with a 2-Cys-peroxiredoxin
described in the literature [1,2].
In vitro assays showed that this 20 kDa protein prevents DNA from

oxidative burst. The regulation of the gene expression of this bean

peroxiredoxin upon heavy metal stress has also been studied.
[1] Baler, M. and Dietz, K.J. Plant Moh Biol. 31 (1996) 553.
[2] Baier, M. and Dietz, K.J. Plant J. 12 (1997) 179.

s387

(We/15.3/424)

Abstracts FEBS'99

Involvement of RNases in pathogen defense in plants

K. Hugot, R. Delbos I, A. Poupet, P. Ricci and E. Galiana

(We/15.3/425)

1NRA. Antibes, France; 1: INRA Villenave d'Ornon, France

Effects of water deficit on photosynthetic

parameters of wheat plants
M.A. lsmailov, I.S. Zulfugarov, J.A. Aliev
Institute of Botany, PatamdarShosse 40, Baku 370073, Azerbaijan

One function which is clearly identified today for plant RNases is

the inhibition of pollen tube elongation, by S-RNases, in selfincompatibility system (SI) [1]. Several studies also suggest the
implication of plant RNases in defence mechanims [2]. We have
investigated this putative role with tobacco/Phytophthora parasitica var
nicotianae (Ppn) interaction. We observed that an inoculation of Ppn into
leaves is correlated with an increase of RNase activity and with the
induction of the expression of gene coding an S-like RNase, the NE
RNase [3]. By immunoblotting, we have shown that NE RNase is
extracellular and forms dimers involving disulfide bonds. Its extracellular
localisation and its expression induced in response to fungal inoculation
allowed us to propose a possible implication of RNase NE in defence
mechanisms.
To test the hypothesis of a direct toxicity against plant pathogens,
we have expressed the NE protein in yeast. The purified protein NE has a
RNase activity and an in vitro antifungal activity against both tested
pathogens, Ppn and Fusarium oxisporum radicis-lycopersici. This effect
is cytostatic : RNase NE (50 p.g/ml) inhibited the elongation of germ
tubes. Immunohistochemistry staining allowed us to localise the NE
RNase in fungal hyphae, that supports the hypothesis of a degradation of
fungal RNAs at the origin of the antifungal activity. Based on this results,
we are investigating the role of the RNase activity and the possible
function of the dimerisation in the antifungal activity.
The demonstration of antifungal activity of RNase NE, and the
similarity of its effect with that of S-RNases in SI (cytostatic in the both
systems) support the hypothesis that S-RNases could derive from S-like
RNases implicated in defence mechanisms and specialised in
pollen/pistil interaction during the evolution [2]. The comparison of
pollen/pistil and fungus/plant cellular interactions, could allow us to
understand better the action mechanisms of plant RNases.

The effects of water deficit on photosynthesis have been a subject of

controversy and conflicting results have been reported depending on
the plant material, and the experimental procedures used for
investigations. In response to a water stress, a decrease in net CO2
assimilaton is generally observed. This effect can however result
from different events, such as an inhibition of electron transport
activity limiting the generation of reducing power or a limitation in
the metabolic activity. Classical method, based on CO2 and water
exchange measurements, supply information concerning net
photosynthetic activity. However, these informations are not
sufficient and additional techniques are required to determine without
ambiguity the effects of water stress on photosynthesis. In the present
work we have used chlorophyll fluorescence technique to screening
wheat genotypes for drought tolerance. Wheat plants were grown at
Absheron Basic Field Experimental Station near Baku both under
irrigation and drought conditions. Plants for analysis were removed
from the field along a soil drying episode in spring beginning from
earring to grain ripening.
Chlorophyll fluorescence induction kinetics, fluorescence emission
spectra at 77K and 293K, photosynthetic 02 evolution activities of
both normal and droughted wheat leaves were investigated. As a
result of our observation wide range of tolerance to water stress of
aboriginal as well as introduced wheat genotypes from various world
regions is revealed. Genotypes of Triticum durum L. are differed by
greater tolerance to water stress, than ones of Triticum aestivum I~

1 : B. McClure et al., Nature, 342, (1989) 955

2 : P. Green, Annu. Rev. Plant Physiol. Plant Mol. Biol., 45, (1994) 421
3 : E. Galiana et al., Plant Physiol., 115, (1997) 1557

(We/15.3/426)

The late embryogenesis abundant B19 genes in barley:

analysis of transcription through ABA and osmotic stress
M.S. Jorgensen, F. Haugen, K.S. Jakobsen
Division of General Genetics, Umversttyof Oslo, PO box 1031 BImdern,
0315 Oslo, Norway

The embryo-specific B19 gene family in barley (Hordeum vulgare) is

suggested to encode proteins acting as osmoprotectants during seed
dessication. These late embryogenesis abundant (Lea) genes can be induced
at the mRNA level by the plant hormone abscisic acid (ABA) and by
osmotic stress. The Lea BI9.1 and B19.4 genes are differentially regulated
by osmotic stress [IJ. By site-directed mutagenesis on different deletions of
promoter-GUS fusmn constructs of B19.1 and B19.4 we have studied cisacting elements involved in transcription control. The deletion constructs
were delivered by particle bombardment into 21 DPA embryos. The
promoter region in B19.1 conferring the ABA and osmotic stress respons
contains no G-box like ABREs in contrary to the B19.4 promoter region. In
B19.I we identified an element involved in the mannitol response but
appearently not involved in transcriptional activation by ABA or NaC1. In
B19.4, mutation in a DRE-like element showed reduction in response on
media supplied with ABA or salt, but not with manmtol. Constructs with a
mutation just upstream of the DRE-like element however, showed reductmn
in response on medm supplied with salt or mannitol, but not with ABA. At
the mRNA level m immature embryos and young seedlings following
exposure to various combinations of ABA, salt and mannitol we observed
that salt and mannitol Interact synergistically with ABA. In addition, there
seem to be minor differences in the synergistic effects of ABA and osmotic
stresses, most likely related to stage and gene family member. Our results
show that ABA-response can be mediated through classical ABRE elements
(G-box like) as well as non-G-box like elements. Furthermore, the
differential response on ABA and osmotic stress for BI9.1 and B19.4 may
be mediated through the DRE-like element in B 19.4. The synergistic effects
of osmotic stress and ABA indicates that the signal transduction pathways
are interconnected. We are now m the prosess of studying this phenomenom
of synergy using promoter-GUS fusion constructs.
[1] M. Espelund et al., Plant J., 2 (1992), 241

(We/15.3/427)

Identification of a small G protein potentially involved

in plant defense mechanisms.
F. Kieffer, T. Elmayan, F. Simon-Plas, JP. B[ein.
La&ratoire de Phytopharmacteet de Btochimiedes Into'actions
Celhdatres. G~4692. INRA, BV 1540. 21034 Dqon C~.;dex. F)'ance

When plants are attacked by an avirulent strain of a pathogen, they

exhibit an hypersensitive response (HR) which leads to rapid tissue necrosis
at the sites of infection and limits pathogen growth to a small region of the
plant. A rapid and characteristic event of the elicited cells response is the
release of active oxygen species (AOS) which is termed the oxidative burst.
This oxidative burst presents physiological similarities with the oxidative
burst of the mammalian neutrophils: both are an early and transient response
to invading organisms, and lead to a comparable rate of hydrogen peroxide
(H202) production. The neutrophil NADPlt oxidase, responsible for 02production, is composed of several cytosolic proteins which interact with a
flavocytochrome located on the plasma membrane. Racl and Rac2, 2 small G
protein of the Rho family regulate activity of this enzymatic complex.
Our laboratory, and some others, have shown physiological and
immunological similarities between animal and vegetal AOS generating
systems [1]. To further exanline if molecular similarities exist between these
two systems, we have developped an heterologous two-hybrid screening
method. A tobacco cDNA library has been used to select a plant protein able
to interact with Rho-GD1 protein of the neutrophil cell. Rho-GD1 protcin is
known to interact with small G proteins of tile Rho family.
By this technique, we have cloned a cDNA encoding a tobacco protein
homologous to Rac protein which interacts with the neutrophil Rho-GDI
protein. The different components of the animal NADPH oxydase will be also
used as bait-protein to identify by the two-hybrid screen new tobacco
proteins involved in the AOS generating system in plants. Moreover,
characterization of the gene(s) encoding tobacco protein homologous to Rac
could allow the cloning, by the two-hybrid system, of the over components
of this system which interact with these small G protein.
[1] VSeffcretd, Tohea:oa:tlsmr~m aprotc'~i t r r n m o l ~ rehtedto the r't~utrophilsmallG
protehaR,w2md ~.volved~adicitorinducedo:~htiveb~'st.FEBSLetto~,'403(1997) 149-153.

Abstracts FEBS'99

(We/15.3/427)

Cold induced protein kinase(s) in rice seedlings

S. Komatsu, W. Li, R. Rakwal

s388

(We/15.3/428)

National Institute of AgrobiologicalResources, Tsukuba, lbaraki, Japan

Calcium-dependent protein kinases (CDPKs) play an important role in plant

signal transduction. Protein kinase(s) activities induced by 5C cold stress
in rice seedling were investigated. No significant differences were found
from the in-gel assay patterns under light and dark condition. Protein
kinases induced in three rice seedling tissues investigated, namely, leaf,
stem and root show similar results in an early (upto 45 min) and late (upto
12 hr) response study, where the leaf had 38, 48, and 55 kDa activities, the
stem 25, 37, 47, and 68 kDa activities, and the root showed protein kinase
activities of 52 and 57 kDa only in the membrane fractions. A 16 kDa
protein shows constitutive kinase activities in the rice seedling tissues
examined. It was further identified that the 47 kDa protein kinase activity in
both cytosolic and membrane fi'actions of stem tissues was Ca2+-dependent
and cold stress induced a cytosolic and membrane-associated increase in
this kinase activity. However, except for a higher protein kinase activity in
5-week old seedlings than in 8-day-old seedlings, the CDPK showed similar
patterns in the stem tissues. CDPK activities between intact seedling and
stem segments were identical, though reached to a maximum at different
time periods. Incubation with the Ca2+ ionophore A23187 increased this 47
kDa activity in stem segments whereas the potent protein kinase C inhibitor
staurosporine completely inhibited this activity in vitro. The Ca 2+ channel
blocker, lanthanum, and the Ca2+ chelator, EGTA decreased the CDPK
activity, whereas the protein phosphatase inhibitor okadaic acid increased
this activity. This CDPK activity was also induced by salt and drought stress,
and by the phytohormone ABA.

(We/15.3/429)

Characterisation of the high affinity binding site of

cryptogein on tobacco plasma membrane.
A. Lebrun-Gareia, S. Bourque, M-N. Binet, A. Pugin.
UMR 1NRA/Universit~BBCM-IPM, BV 1540, 21034 Dijon cedex, Franc,

Secondary metabolites production from licorice

Glycyrrhiza glabra L. transformed cells
P G Kovalenko
Department of Chemzcal Engineering, Inst. of Btoorganic
Chemtst~, )/lurmanskaStr 1. Klev-9g. 253660, ~.Tcrame.

The pharmaceutically important plant, licorice (Glycyrrhiza glabra L ),

has been used as a sweetener and anti-inflammatory drug. 'This plant
contains a large amount of saponins and fiavonoids. For this purpose
usage of genetic engineering manipulation of licorice will be very
important to improve quality of secondary metabolites production. In
order to increase biotechnological potential in Glycyrrhiza glabra L., the
cultural conditions were established for the efficient initiation of
suspension culture from callus. Electroporation was used to evaluate
parameters important in transient gene expression in G. glabra L.,
suspension protoplasts The both chimaeric plasmids, pCaMV35S-nos
and pDNt23-cat were used. (1) pCaMV35S-reporter enzyme activity,
chloramphenicol acetyl transferase (CAT) coding region under the
control of the CaMV 35S promoter, nopaline synthase (nos) terminator,
and (2) plasmid pDNt23 (root specific promoter) [1] Factors influencing
the transient expression of the introduced foreigns DNA in licorice
suspension protoplasts were determined by assaying CAT activity and
study cell selection of transgenic cell lines on glycyrrhyzin productivity.
In order to increase the level of secondary metabolites in obtained cell
lines, Agrobacterium rhizogenes (15834) inoculation was used. The level
of secondary metabolites of double transformed cell lines has been 2fold higher than in non-transformed cells
Reference
1. Yefimenko I., Kovalenko P., Medvedeva T., et al. 1995.
Organ-specific gene expression in transgenic potato. Biopolymers
and cells 11:96-103

(We/15.3/430)

Elicitor-induced protein phosphorylation and MAPK

activation in tobacco cell suspensions
F. Ouaked, D. Lecourieux, A. Chiltz, A. Pugin,
A. Lebrun-Garcia
UMR lNRA/Umversit~BBCM-IPM, B V 1540, 21034 Dijon Cedex, France

Elicitins are 98 amino acid proteins, secreted by Phytophthora species, that

elicit plant defence reactions in tobacco. The mode of action of these elicitors
was mainly described for the elicitin cryptogein using tobacco cell
suspensions [ 1]. Specific binding of cryptogein on high affinity binding sites
on tobacco plasma membranes was reported (Kd=2 nM; number of binding
sites: 220 fmol/mg proteins) [2]. Comparison of binding properties of four
elicitins showed that they bound to the same binding sites with the same
affinity, although they induced different levels of calcium influx, active
oxygen species production and extmcellular alkalinisation.
Biochemical characterisation of the cryptogein binding sites revealed that they
corresponded to a plasma membrane glycoprotein with a N-linked
carbohydrate moiety involved in eryptogein binding. Indeed plasma
membrane treatment with proteases or N-glycosidase suppressed cryptogein
specific binding.
Radiation-inactivation experiments performed on tobacco plasma membrane
preparations using glucose-6-phosphate dehydrogenase and plasma
membrane H+-ATPase as markers of molecular mass indicated that eryptogein
specifically bound to a plasma membrane component with an apparent
functional molecular mass of 193 kDa. Moreover, using the homobifonctional
cross-linking reagent disuccinimidyl suberate and tobacco plasma membranes
incubated with 125I-cryptogein, we identified after SDS-PAGE and
autoradiography, two 12 15,_cryptogein linked proteins, a N-glycoprotein of
about 160 kDa and a 50 kDa polypeptide. These results fitted well with
radiation-inactivation results and led us to propose a model in which the
cryptogein binding sites were constituted of a 160 kDa N-glycoprotein
associated with a 50 kDa protein. Similar results were obtained using
Arabidopsis thalicma and Acer pseudoplatanus plasma membrane
preparations. However, cryptogein did not induce any effects on the
corresponding cell suspensions, suggesting that the binding sites in
Arabidopsis or Acer, are non functional homologues for cryptogein signal
transduction.

Cryptogein, a proteinaceous elicitor of defense reactions induces in tobacco

cell suspensions (Ntcotiana tabacum var. Xanthi), phosphorylation events
that control downstream elements of the signal transduction including
calcmm influx, activation of a plasma membrane NADPH-oxidase which
activity is responsible for active oxygen species production (AOS), external
medium alkalinization and cytosol acidification [ 1].
Therefore, we analyzed by 2D electrophoresis the in rive phosphorylation
pattern of proteins after treatment of tobacco cells with cryptogein and/or
wlth inhlbitors of protein kinases (PKs) or protein phosphatases (PPs).
Results showed that many proteins had enhanced levels of phosphorylation
after cryptogein treatment. Interestingly, the treatment of tobacco cells by
calyculin A, an inhibitor of phosphatases 1 and 2A, produced a similar
pattern of phosphorylation, which is in agreement with experiments
showing that inhibitors of PPs mimicked early events induced by elicitors
(extmcellular alkalinization, Ca2 influx, AOS production...).
We showed that protein kinases involved in cryptogein-signal transduction
pathway include homologs of mitogen-activated protein kinases (MAPKs):
two homologs of MAPKs are rapidly activated after cryptogein or
nligogalacturonate treatments of tobacco cells [2]. However, kinetics and
intensities of activation depended on the elicitor. Regulation of the MAPK
activation was analyzed in regard of known responses induced by
cryptogem. Results demonstrated that MAPK activation was: (i) dependent
on both Ca 2+ influx and upstream phosphorylation events, (ii) independent
on AOS production, (iii) not mediated by salicylic acid.

[ 1] Pugin et al., Plant Cell, 9, (1997), 2077.

[2] Wendeherme et al., FEBS Lett., 374, (1995), 203.
[31 Bourque et al., Plant Physiol., 118, (1999), 1317.

[1 ] Pugin A. et al., Plant Cell, 9, (1997) 2077-2091.

[2] Lebrun-Garcia A. et at., Plant J., 15, (1998) 773-781.

s389

(We/15.3/432)

Abstracts FEBS'99

Antioxidants from Sah,ia officinalis

(We/15.3/433)

Nihan Ozdalyan, Selma Turkay, Artemis Karaali

Istanhul Techmcal Umverslt); 80626 lstanbul Turkex

Transcriptionnal derepression of the ZmFerl maize

ferritin gene in response to iron.
JM. Petit, S. Lobr6aux et JF. Briar
Blochimle & Physiologle Moleculatre des Plantes, AGRO-M/INRA/CNRS,
URA 2133, Place Vtala, 34060 Montpellier cedex I

Spices and herbs have been used in seasoning of foods since very ancient
times, to enhance savoriness of foods, and are valued both as flavoring
agents and for properties like stimulation of appetite by increasing
salivation, their carminative, preservative and antio\idant action in some
tbods, delaying the onset of lipid oxidation and thereby food deterioration
Since the use of synthetic antioxidants like BHA and BHT is graduall~
decreasing because of fears of their possible mutagenity and consumer's
rejection of synthetic chemicals, and also in parallel with the growing
consumer interest in "'natural" products, there is a growing demand for
natural antioxidative substances to replace the conventional commercial
antioxidants
The spices from the Labiateae family, notably Salvia offictnah~(sage), are
especially well-known for their antioxidative properties
Major
antioxidants identified in sage are the phenolic compounds like rosmarinic
acid, carnosic acid and its derivatives(carnosol,rosmanol isomers,
rosmadial and methyl carnosate) Rosmarinic acid and carnosic acid are
very active antioxidants and are greatly responsible for the antioxidative
efficiency of sage
This study was undertaken to investigate the possibilities of preparation of
an odorless and flavourless natural antioxidant extract from a natural
Turkish spice, sage, using supercritical fluid extraction(SFE), as well as to
evaluate the antioxidative power of this extract by applying a rather
unconventional
a n a l y t i c a l technique, differential scanning
calorimetry(DSC) A high recovery of a natural product with significant
antioxidative activity was obtained which was used as an antioxidant in
treating sunflower seed oil for delaying the onset of its induction period,
thus extending its shelf-life.

(We/15.3/434)

A Lipid Phosphate Phosphohydrolase gene is induced by

ionizing radiations in A. thaliana.
O. Pierrugues a, P. Thuriaux b and M. Kazmaier a.
LR V/DEVM/DSV/CEA Cadarache, 1=-13108St Paul lez Durance.
~SBGM/DSV/CEA Saclay, F-91191 G(sur Yvette.

To identify components induced by genotoxic stress in A. thaliana cell

cultures, we used differential display to screen for mRNAs which rapidly
accumulate after exposure to ionizing radiation (IR). A 45 l-base pair cDNA
fragment, hybridizing to a 1,5 kb mRNA rapidly induced by IR, was used
to isolate the 1351-base pair corresponding cDNA from an A. thaliana
cDNA library: ATGR5 (A. t_haliana Gamma-ray Response). The IR-induced
kinetics of ATGR5 mRNA accumulation in cells and plants reveal a
transient and dose-dependent effect, with a maximal stimulation (6 fold) 30
min after irradiation. The deduced amino acid sequence revealed the
membership of the ATGR5 gene product to the very recently defined Lipid
Phosphate Phosphohydrolase (LPP) superfamily of related proteins [l]. The
precise biological functions of these phosphohydrolases are still unknown,
but some members of this family of proteins are implicated in phospholipids
signal transduction pathways [2]. Among the potential substrates of the LPP
family is phosphatidic acid. However, in plants we did not observe any
change in the phosphatidic acid level and phosphatidic acid
phosphohydrolase activity after irradiation. Gene disruption of the 2 yeast
LPP homologues, very close to ATGR5 gene product, suggests that yeast
LPPs are not involved in response to genotoxic stress. Therefore, other
phospholipids pathways, such as the sphingolipid or ceramide pathways,
that are involved in radiation induced stress response in mammals [3], will
be examined in view of cross-talk between signaling pathways involving
lipids mediators.
[1] Brindley and Waggoner, J. Biol. Chem., 273, (1998), 242581.
[2] Kanoh et al., Biochim. Biophys. Acta, 1348, (1997), 56.
[3] Haimovitz-Friedman, Radiat. Res., 150, (1998), S 102.

Iron homeostasis needs to be controlled tightly in living cells because this

element is both essential for important metabolic processes and potentially
toxic. Ferritins are involved in iron storage and detoxification because of their
ability to store iron in a soluble, nontoxic and bioavailable form [1]. Iron
overload induces mRNA accumulation from the Z m F e r l maize ferritin gene
[2]. In contrast antioxidant treatment has been shown to inhibit its
accumulation in maize suspension cells [3]. In order to characterize promoter
sequences involved in the iron dependant regulation of the Z m F e r l gene
expression, chimeric genes were constructed, fusing differents deletions of the
Z m F e r l gene promoter to the GUS reporter gene. Transient expression assays
in BMS maize cells enabled to describe a DNA region localized between -154
and -94 base pairs responsible for the repression of the Z m F e r l : G U S gene
expression under low iron conditions. Site directed mutagenesis of putative cis
regulatory elements has been perlbrmed on a -154 bp deleted promoter. Then
transient expression of a fusion between this promoter fragment and the
luciferase gene has been analysed in response to iron treatments. These
experiments revealed that a 15 base pairs region, located between -113 and -98
bp, is implicated in the ZmFerl gene transcription derepression in response to
iron overload. New deletions experiments are in progress in order to localize
the promoter region involved in the redox regulation of this gene.
References :
1- Briat JF, Lobr6aux S, Iron transport and storage in plants. TIPS, 2, (1997)
187.
2- Fobis-Loisy I, Loridon K, Lobr6aux S, Lebrun M, Briat JF, Structure and
differential expression of two maize ferritin genes in response to iron and
abscisic acid. Eur J Biochem, 231, (1995) 609.
3- Savino G, Briat JF, Lob6aux S, Inhibition of the iron-induced Z m F e r l
maize ferritin gene expression by antioxidants and serine/threonine
phosphatase inhibitors. J Biol Chem, 272, (1997) 33319.

(We/15.3/435)

Cryptogein, a fungal elicitor, induced

depolymerizatlon of microtubules in tobacco cells
A. Pugin, C. Humbert, M. Vantard and M.N. Binet
UMR INRAIUnlver,rlt~, BBCM.IPM, BV 1540, 21034 Dijon Cedex, France

Cytoskeleton has an important role in the regulation of several

signal u'ansduction pathways in both animal and plant systems. Relatively
little is known regarding its involvement in the signal wansduction pathway
iaidated by plant-pathogen interactions and leading to plant dcfence.
In this work, we studied the involvement of microtubule, a major
eytoskdetal componem in the signal transducdon pathway induced by a
fungal elicitor, cryptogein. Cryptogein is a 10 kDa protein produced by
Phytophthora cryptogea which causes hypersensitive like response and
induces systemic resistance against pathogens in tobacco plants. Using
Nicotfana tabacwn vat Xanthi ccU suspensions, some of the cryptogcininduced signaling events were characterized : specific recognition of
crypmgcin by high affinity plasma membrane sites leads to a rapid cascade
of reactions including protein phosphorylation, a large influx of calcium,
K* and Cr cffluxcs, plasma membrane dcpolarisation, activation of
mitogcn-actlvated protein kinase and activation of NADPH oxidase which
in turn produces active oxygen species and gives rise to exWaccUular
alkalinisafion and cytosol acidification.
The microtubular response to cryptogcin was studied by
immanocytochcmistry in tobacco cells. We demonstrated that cryptogcin
induced a specific depolymerization of microtubulcs : a beaded bundled
dcpolymerization form progressed during cryptogcin treatment. After 60
minutes, cortical microtubules disappeared and pcrinuclear micrombular
morphology was observed. Using a calcium-chelator, in addition to
cryptogcin, the cryptogein-induced microtubulcs disruption was blocked
suggesting that this event is calcium-dependent. DqDolymedzation induced
by oryzalin, a microtubde disrupting agent, differs in morphology to the
one triggered by cryptogein. In addition, the effect of the microtubulc
stabilizing agent, taxol, reduced the severity of the depolymerization
induced by cryptogein but did not suppress it.
The dynamic behavior of microtubules in viva is modulated by a
variety of proteins such as microtubule-associated proteins which promote
polymerization and other proteins which promote disassembly of
microtubules. Our focus is to identify the regulators of the eryptogeininduced depolymerizadon and to understand the possible role of
mierotubules disruption in the hypersensitive response.

Abstracts FEB S' 99

(We/15.3/436)

Salt tolerant

s390

t r a n s g e n i e tobacco generated f o r

(We/15.3/437)

synthesis of compatible solute: ectoine

H. Rai, H.J. Bohnert, Dept. Biochemistry,
University of Arizona, Tucson AZ 85721-0088

Rapid accumulation of transcript of a plant cytoehrome

P450 gene involved in the synthesis of cutin monomers
R. Le Bouquin, F. Pinot, F. Durst, I. Benveniste, and J.-P. Salatin
Institut de Biologie Mol~culaire des Plantes/CNRS-Universitd
Pasteur. 28 rue Goethe, F-67083 Strasbourg Cedex, France.

Louts

A number of transgenic plants have been generated with the ability to

overproduce indigenous compatible solutes or novel osmolytes not previously
reported in plants. The results suggest that such metabolites can provide some
protection to osmotically stressed plants. Encouraged by these reports, we
generated transgenic tobacco plants containing a novel compatible solute, ectoine,
which is increasingly being viewed as an excellent universal osmoprotectant [1].
Ectoine has been shown to be distributed widely in nature, especially in
halotolerant eubacteria and has been shown to confer protection on various nonhalotolerant eubacteria by a novel mechanism, involving enhanced synthesis of
endogenous compatible solutes such as N-acetyl glutaminyl glutamate, glutamate
and trehalose. Ectoine itself is not accumulated at osmotically significant levels
from the medium in these cases. It has tentatively been assigned the role of a
regulatory molecule functioning in osmoprotection. Precursor for synthesis of
ectoine is aspartate semialdehyde, an intermediate of amino acid biosynthesis in
all organisms. Sequences of the genes comprising the ectoine biosynthesis
pathway from Marinococcus halophilus are available [2]. The operon of three
genes has been shown to be induced under salt stress by complementation in
E.coli .The three genes are: ectB - coding for L-2,4,diaminobutyric acid
transaminase; ectA - coding for L-2,4,diaminobutyric acid acetyl transferase and
ectC - coding for L-ectoine synthase. We cloned each of these genes under control
of a constitutive plant promoter and polyadenylation signal. The constructs were
transferred into Agrobacterium- based plant vectors and introduced into tobacco.
Transgenic plants, selected on double selection medium (kanamycin and
hygromycin) showed positive PCR, RT-PCR and northern blotting for each of the
three genes. Ectoine was detected in the transgenic plants by HPLC, NMR and
Mass Spectroscopy. Transgenic tobacco plants show constitutively higher
potassium (10-25%) and sugar levels (glucose, fructose and sucrose). Seeds from
transgenic plants exhibit enhanced germination at NaCI concentrations ranging
from 50-200mM in comparison to wild type. Differences are particularly
pronounced at 150raM and 200mM NaCI. Further analysis of transgenic plants is
underway.

Plants are subjected to chemical, physical and biological stresses. As a first

physical defence barrier, the plant is protected from outer environment by
cuticular layers and epicuticular waxes. Elongation of the cutin matrix involves
the esterification of a carboxylic function by the terminal hydroxyl group of
fatty acids (FAs), that conferring a pivotal role in cutin synthesis to
cytochrome P450s hydroxylating the omega carbon position. In addition,
recent reports indicate that cutin monomers may protect the plant against
fungai infection by inducing the plant defence mechanisms. The plant
antiauxin 2-(p-chlorophenoxy)-isobutyric acid (clofibrate) , which is also an
human hypolipidemic drug, and most of peroxisome proliferators (i.e. the
plasticizing agent DEHP (di(2-ethylbexyl)-phthalate) and the herbicide 2,4-D)
strongly induce the cytochrome P450-catalyzed activities of fatty acid omegahydroxylation in microsomes from Vicia sativa seedlings. Recently, a cDNA
coding for a plant fatty acid omega-hydroxylase (CYlX)4A1) was isolated and
expressed in yeast. This P450 catalyzes the omega-hydroxylation of a large
range of saturated and unsaturated fatty acids (C10 to C16 and C18:1, C18:2,
C18:3). Northern blot analysis of RNAs from clofibrate-treated Vicia sativa
seedlings revealed a very rapid (after 20 min) and large accumulation of the
CYP 94A1 transcripts (1). These results suggest the involvement of receptors
in the signal transduction that should be similar to the peroxisome proliferator
activated receptors (PPARs) found in mammals. To assess the mechanism of
regulation of CYP94A 1 by elofibrate, and the possible involvement of PPARs
in this regulation, a promoter sequence of CYlX)4A1 (600 bp upstream the
ATG translation start) has been isolated. Search for key regulator elements
similar to PPRE conserved motifs was unsuccessful. In addition, a study of
peroxisome proliferation in Vicia sativa in response to clofibrate, allow us
isolation of a cDNA coding for Acyl CoA oxidase (ACO) which is a marker
enzyme of peroxisomes. Preliminary results show that ACO seems not to be
induced by clofibrate in V. sativa at the transcriptional level.
Although clearly CYP94A1 catalyzes the formation of most of cutin and
suberin monomers needed for repair and defence, its involvement in plant
defence process such as the production of secondary messenger and its role
for fatty acid detoxification through the peroxisomal machinery are currently
underway.

[ 1] Ono et al., J Bacteriol., 181, (1999) 91.

[2] Louis P & Galinski EA, Microbiology,143, (1997) 1141.

1) Tijet N., Helvig C., Pinot F., Le Bouquin R., Lesot A., Durst F., Salatin J-P.,
and Benveniste I. (1998) Biochem. J. 332, 583-589.

(We/15.3/438)

Inhibitors of the trans-einnamate 4-hydroxylase: a new

tool to control the flux of metabolites in the phenylpropanoid pathway
G. Schoch, M. Morant, M. Schalk, P. Saindrenan, D. Werck
IBMP/CNRS-Umversit6
Strasbourg, France

Louts Pasteur, 28, rue Goethe, 67083

The CYP73A subfamily of plant P450s catalyse trans-cinnamate

4-hydroxylation, the second step of the general phenylpropanoid pathway
and branching point where the main pathway diverges from the synthesis
of salicylic acid (SA). SA is a phytohormone involved in defense
mechanisms against pathogens. Its accumulation is essential for systemic
acquired resistance. Our aim was to test the possibility of diverting the
main flux of metabolites to the synthesis of SA. We thus designed a set of
cinnamate analogues bearing activable functions. Incubated in vitro with
yeast-expressed CYP73A 1. several of these molecules behaved as potent
mechanism-based irreversible or quasi-irreversible inactivators. In vivo, in
normal or elicited tobacco cell suspensions, such inhibitors induced a
sharp decrease in 4-coumaric acid concomitant to cinnamic acid and SA
accumulation. Potential applications of such inhibitors are 1) elucidation of
the SA biosynthetic pathway, 2) chemical induction of plant defense.

(We/15.3/439)

Kinetics of the catalase activity in NaCI treated

M e s e m b r y a n t h e m u m crystallinum L. plants
A. Skoczowski, Z. Miszalski
Pohsh Academy of Sctences, The Franctszek G6rski Department of Plant
Phvstolo~,. ul. Podlulzna 3, 30-239 Cracow, Poland

Irrigation of M e s e m b r y a n t h e m u m crystallinum plants with NaCI leads to

expression of crassulacean acid metabolism (CAM). It has been shown that the
full expression of CAM state in the A~ crystallinum causes dial oscillation of
the catalase (CAT) activity [1]. The aim of the present studies was to
investigate changes in the kinetics parameters of CAT activity during initial
period of salt treatment.
Crude extracts were obtained from leaves harvested between 8:15 and
10:15 (30-minute time intervals after switching on light at 8:00). The CAT
activity reaching 0.58 ~tmol(H202).mg'~(protein).min"1 at 8:15 decreased
slowly to 0.28 ~tmol(HzO2).mg-Z(protein).min "t at 9:45. These two samples
were used for estimation of kinetics parameters of CAT.
Simultaneously with decreasing of the CAT activity significant increase in
K,~ value (from 34 to 60 mM(H202)-dm -3) was observed; whereas Vma~ values
were similar. Increase in K,, value connected with decrease in total CAT
activity was obtained after purification of crude extracts on Superose-6 FPLC.
Both samples showed very similar dependence of CAT activity on pH (pH
range from 5.0 to 11.5), whereas their responses to pH changes differed
significantly especially in pH range of 5.0-8.0.
We can conclude that strong changes in the biochemical properties of CAT
take place during the investigated time period.
[I] Niewiadomskaet al., In: Abstract Book of Winter Meetingof the Society for Free Radical
Research, Grenada, Spain, December 17-19,1998, p. 146.

s391

Abstracts FEB S' 99

(We/15.3/440)

Plant ferritin - the protective protein.

J. Sm61, T. Twardowski

(We/15.3/441)

lnstmtte of Bwor~;amc Chemlst~' PAS

.Voskowsktego 12 14, 61-704 Poznan, Poland

Ferritin is an iron keeping protein found in many tissues. It is a

multimeric spherical protein assembled from 24 subunits which
define a shell surrounding a central cavity which is able to
accommodate up to 6000 iron atoms per ferritin molecule (in the
condition of a strong environmental stress).
The key function of this protein is regulation of iron concentration
in plants and protection of the cell against toxic concentration of
Fe +2. The ferrous ion can be toxic to the plant because iron
catalyses the production of free radicals which are responsible for
degradation of RNA and DNA. Thus iron needs to be stored in a
safe form inside the ferritin molecule. This protein is known to
sequester and de-toxify iron taken up by cells, which is not utilized
for metabolic requirements. The stored iron ions can be used by the
cell in case of shortage of these ions according to the needs of the
cell such as oxygen transfer, nitrogen fixation.
It has been pointed out that ferritin successfully absorbs cadmium
and lead as well as iron from the environment We expect that
ferritin is able to chelate also other metals.
In light of the aforementioned findings, we would recommend that
belts of protective flora characterised by high levels of ferritin
capable of capturing harmful ions should be planted on the sides of
busy roads (motorways) situated in the vicinity of farmlands

(We/15.3/442) Mitoehondrial plasmid-like DNAs polymorphisms in Tunisian

date-palm varieties and ba~coud disease
M. Trifl a, A. Rhoumaa, A. Rode, M. Marrakchia
Facult~ des Sciences de Tunis, 1060 "Cunts, Tunisie, b 1BP, Bat 630,
UPS~Centre d'Orsay, 91405 Ors~. . France

In North Africa, date-palm groves are currently menaced to be

completely destroyed by a vascular fusariosis called <<bayoud~ disease and
caused by an imperfect fungus (Fusariura oxysporum fsp albedinis) Due to
its rapid propagation into the eastward, Tunisian plantations are continuously
threatened by this disease. Studies aiming at the identification of early
molecular markers, associated to the fusariosis, have been investigated over the
world Up to day, three plasmid-like circular DNAs, isolated from
mitochondria of date-palm, have been reported as potential markers [1].
However, the correlation between the presence of such molecules and the
phenotype of trees has not been clearly established
In order to elaborate a preventive fighting strategy, our investigations
were performed to molecularly characterize Tunisian date-palm varieties with
respect to these mitochondrial plasmid-like DNAs Starting from a set of
accessions, the following groups have been identified: the first one is
composed of varieties exhibiting exclusively the S or the R mitochondrial
plasmid, whilst a second one regroups accessions sharing simultaneously both
of S and R plasmids. Hypothesis based either on intramolecular or
intermolecular recombination events involving a nuclear genetic control were
advanced to explain this polymorphism. Reports strongly supporting these
hypothesis have been described [2,3]
Here we report how the u ~ of PCR procedure could provide a rapid
and efficient screening method to identify the date-palm's mitochondrial
plasmid setting. Moreover, we exhibit how the presence of either of the S or R
plasmid can be considered as suitable molecular marker of resistance/sensitive
to the plague. Our data provide the evidence of common features between
plasmid-like DNAs and the main mitoehondrial genome
References
[1] Benslimane et al., Curr. Genet., 26, 1994, 535
[2] Flamand et al., Nucl. Acids Res., 21, 1993, 5468
[3] Benslimane et al., Curr. Genet., 29, 1996, 591

Identification of mRNAs with modulated expression in

dehydrated bean roots using differential display RT-PCR
G. Torres, C. Lelandais-Bri~re, M.-F. Jubier, C. Mazubert, F.
Corre, A. Rode, C. Hartmann
Laboratoire de Physiologte Cellulaire et Moldculaire du Stress chez les
Plantes; IBP; UMR 8618;Universitd Paris XI, Orsay, France.

When subjected to environmental stresses, plants respond by metabolic and

gene regulation changes. The water deficit is one of the most important
among these abiotic factors which determines great damages to plant
productivity. By using differential display RT-PCR, we performed a
comparative analysis of the expression pattern of bean (Phaseolus vulgaris
L.) roots under dehydration conditions. Accumulation of transcripts
corresponding to four differentially expressed cDNAs cloned from bands
isolated from ddRT-PCR gels was studied. Sequence analysis and database
searches revealed significant homology to known sequences for two of them.

(We/15,3/443)

How do aphids metabolise plant alleiochemicals?

A. Urbanska, B. Leszczynski, H. Matok
Department o f Biochemistry, Agricultural & Pedagogic
Untverslty. uL B. Prusa 12, 08-110 Siedlce, Poland

Phenolics are principal chemicals of cereal defence against aphids, since they
are high toxic and reduce their growth and reproduction [1]. However, the
aphids are able to survive and reproduce on cultivars with relatively high
content of these allelochemicals.
The present paper relates the grain aphid Sitobion avenae (Fabr) and the bird
cherry-oat aphid Rhopalosiphum p a d i (L) performance on cereals to activity
of their detoxifying enzymes including several oxidoreductases and 1I phase
transferases.
The aphids' polyphenol oxidase was able to oxidise most of the cereal
o-dihydroxyphenolics e.g. gallic acid, caffeic acid, protocatechuic acid,
chlorogenic acid, (+) catechin and quercetin [2]. Complementary peroxidase
beside listed o-dihydroxyphenolics oxidised also methoxyphenolics such as
ferulic acid and sinapic acid [3]. Both oxidoreductases act mostly in plant
tissues due to the action of secreted aphids' saliva as well as within their
alimentary canal. Among the second phase transferases UDP-glucosyl
transferase that conjugates some of the o-dihydroxyphenolics and also
monophenolics e.g. p-hydroxybenzoic acid and p-coumaric acid not oxidised
by the oxidoreductases was found. In addition, highly active
sulfotranspherase and phosphotransferase were detected in cytosol of the
aphids' midgut.
In summary, oxidation and conjugations to glucose, sulfate and phosphate are
the major steps in the metabolism of the plant phenolics by the aphids.
The significance of these defensive mechanisms for the natural cereal
resistance to aphids is discussed.
[1] Leszczynski B et al , Insect Sci Appl., 6, (1985) 157.
[2] Urbanska A. et.al., EntomoL Exp. Appl., 86, (1998) 197.
[3] Urbanska A. et.al., In: Aphids in natural and managed ecosystems
(J.MNieto Nafria and A F.G Dixon, eds), Le6n (Spain), (1998) 119

Abstracts FEBS'99

(We/15.3/444) Elicitins trap and transfer sterols f r o m m i c e l l e s ,

liposomes a n d plant p l a s m a m e m b r a n e s .
S Vauthrin, V Mikes, M-L Milat, M Ponchet, B Maume, H
Osman and J-P Blein.
Laboratoire de Phytopharmacie et de Biochimie des Interactions
Cellulaires, INRA, BV 1540, 21034 Dijon Cedex

Using elicitins, proteins secreted by some phytopathogenic fungi

(Phytophthora), known to be able to transfer sterols between

phosphoIipid vesicles, the transfer of sterols between micelles,
liposomes and biological membranes was studied.
Firstly, a simple fluorometrie method to screen the sterol-carrier
capacity of proteins, avoiding the preparation of sterol-containing
phospholipidic vesicles, is proposed. The transfer of sterols between
DHE-micelles (donor) and stigmasterol- or cholesterol-micelles
(acceptor) was directly measured, as the increase in DHE fluorescence
signal. Results obtained with this rapid and easy method lead to the
same conclusions as those previously reported, using fluorescence
polarization of the mixture of donor and acceptor phospholipid
vesicles, prepared in the presence of different sterols. Therefore, the
micelles method can be useful to screen proteins for their sterol carder
activity.
Secondly, elicitins are shown to trap sterols from purified plant plasma
membranes and to transfer sterols from micelles to these biological
membranes. This property should contribute to understand the
molecular mechanism involved in sterol uptake by Phytophthora. It
opens new perspectives concerning the role of such proteins in plantmicroorganism interactions.

s392

(We/15.3/445)

Nitric Oxide Signaling in the Tobacco Defense Response

D. Wendehenne, J. Dumer, R. Navarre, D. Kumar, R. Noad
and D. F. Klessig
Waksman Institute, Rutgers, the State University of New Jersey,
190 Frelinghuysen Road, Piscataway, NJ 08854-8020, USA

Over the past decade, nitric oxide (NO) was found to have important signaling
functions in a number of mammalian physiological and pathophysiological
processes including inflammation, immune regulation and programmed cell
death. NO signaling studies in plants are now generating a wealth of
comparative information [1,2]. We demonstrated that infection of resistant, but
not susceptible, tobacco with tobacco mosaic virus (TMV) resulted in enhanced
endogenous NO synthase (NOS) activity. In addition, TMV induction of the
defense-related gene PR-1 was blocked by L-NMMA, a NOS inhibitor.
Furthermore, administration of NO donors or recombinant NOS to tobacco
suspension ceils or leaves triggered expression of the genes encoding PR-1 and
phenylalanine amonia-lyase (PAL), the first enzyme of the phenylpropanoid
pathway. Interestingly, NO-activated PR-1 expression was salicylic acid (SA)
dependent while PAL induction was not. These genes were similarly induced
by the second messengers of mammafian NO signaling cascade, cGMP and
cADPR. Consistent with a role of cGMP in cell signaling, NO treatment
resulted in a dramatic and transient increase in endogenous cGMP levels.
Furthermore, cGMP induced PAL expression while NO-induced PAL
activation was suppressed by the guanylate cyclase inhibitors LY 83583 and
ODQ. In addition, experiments with the calcium channel blocker ruthenium-red
suggest that cADPR mediates cGMP action through a Ca2 release mechanism
and/or that cGMP and cADPR act in parallel to produce a synergistic effect on
gene expression.
In animal cells, besides guanylate cyclase, cytoplasmic and mitochondfial
aconitases are important cellular targets of NO and NO-derived species. Both
enzymes are particularly sensitive to inactivation by NO produced during
various pathological conditions. We found that tobacco aconitase, like its
mammalian counterpart, is inhibited by NO and may be a key redox sensor in
plants. Moreover, the pathogen and SA-activated tobacco MAP kinase, SIPK
(SA-Induced Protein Kinase) was also activated by NO in a SA-dependent
manner.
In summary, we conclude that important components of animal NO signaling
are also present in plants.
[1] Delledonne et al., Nature, 394, (1998), 585.
[2] Durner et al.., Proc. Natl. Acad. Sci. USA, 95, (1998), 10328.

s393

Abstracts FEBS'99

18.2 The cytoskeleton in rnorphogenesis and signalling

(We/18.2/446)

Stathmin slows down GDP dissociation from tubulin :

Implication in the regulation of microtubule dynamics
P. Amayed ~, P. Currni b, A. Sobel b, D. Pantaloni ~ and
M.-F. Carlier*

(Wed18.2/447)

lnstztutfur Bmchemie der Medizmischen Fakult/it,

Universit?~t Wren. Dr Bohrgasse 9/3. A-I030 Wlen, Austria

"LEBS, CNRS, Gtf-sur-Yvette, France and ~INSERM U440, Paris, France.

Stathmin is a phosphorylation-regulated protein which plays an

essential role in the control of cell cycle by regulating the dynamics of
mitotic microtubules. We previously found that stathmin inhibited
microtubule assembly by sequestring 2 tubulin molecules in a ternary
complex (T2S). We examined here how nucleotide exchange on dimeric
tubulin, which is an important kinetic step in the control of dynamic
instability, was modified by stathmin. Using a stopped-flow apparatus,
we mesured the role of GDP dissociation from tubulin using S6-GDP
which, in binding to tubulin in the GDP site, quenches the tryptophane
fluorescence of tubulin. In 80 mM PIPES, 0.5 mM MgCI2, pH 6.8, at
20C, GDP dissociated from tubulin at a rate of 0.12 s t . A 24-fold
slower value was found for the tubulin-stathmin complex. The GDP
bound to the two tubulins in the T2S complex dissociated at rates that
were identical (0.005 s ~) or too similar to be kinetically distinguished. At
substoichiometric stathmin:tubulin ratios ( [S]/[T] < 0.5), the kinetics of
GDP dissociation were biphasic. The two phases had rate constants of
0.12 s -t and 0.005 s~, and amplitudes that reflected the proportions of
tubulin in the free and complexed forms.These results indicate that
stathrnin is in slow association-dissociation equilibrium with tubulin. By
slowing down the regeneration of polymerisable GTP-tubulin, stathmin
may regulate microtubule dynamic instability.

Desmin and LIF both influence cardiomyocyte

development in vitro
A. Bader, S. Puz, H. Al-Dubai and G. Weitzer

Embryonic stem cell (ES cell) technology and the generation of transgemc mine
have become a widespread tool to investigate gene functmn in animal models. In
parallel, m vitro differentiation of ES cells in embrymd bodies has been proven to
be a powerful model system, somehmes giving the possibdity to reveal
phenotypes which were hidden in vivo [1]. In embryoid bodies ES cells
differentmte into a variety of cell types, including all three muscle types (skeletal,
smooth and cardiac muscle), fibroblasts, neurones or endodermal cells.
We focused our interest on the development of cardiomyocytes in embryoid
bodies, particularly on factors leading to the commitment of cardiomyocytes and
maintenance of the cardiac phenotype. One of these proteins is desmin, a class III
intcrmedmte filament protein which is one of the earliest myogcmc markers with
expressmn starting at 7.25 days post coitum in the developing heart of murme
embryos. This suggests a possible role of dcsmm in cardmmyocyte development.
To address this issue ES cells were generated, expressing desmm ectoptcally
under control of the rous sarcoma vires promoter. Embryold bodies made from
wild type and transgemc ES cells were compared for development of beating
cardmmyocytes. Desmin expression led to an increased formation of
cardmmyocytes.
In previous experiments we had further observed that the presence of feeder cells
expressing leukaemm inhibitory factor (LIF) [2] somehow influences the
development of beating cardiomyocytes. LIF is a member of the IL-6 cytokme
family activating internal signals via binding to a heterodlmer consisting of the
LIF-receptor and the transmembrane protein gpl30 [3]. To elucidate the role of
this cytokine, recombinant L1F, feeder cells expressing LIF and antl-LIF
antibodies respectively have been added to the culture medium of embryoid
bodies. These experiments demonstrated that LIF promotes survival of
cardmmyocytes leading to an increased longevity of cardmmyocytes in vitro
[1] Weitzer, G. et al., Developmental Biology, 127, (1995) 422; [2] Gearing, P.D.
et al.EMBO Journal, 6, (1987) 3995; [3] Dam, Ch. et al., Developmental Biology,
203, (1998) 149

(Wed18.2/448)

ERK and JNK pathways are required for IL-I synthesis

upon ml'crotubule disruption in HL60 cells.
B. Cambien, M-A. Millet, H. Schrnid-Antomarchi,
N. Brossette, B. Rossi and A. Schmid-Alliana.
INSERM U364, Avenue de Valombrose, 06107 Nice Cedex 02. France

Microtubule reorganization is often observed during intercellular

contacts that are associated to IL-1 production. Here, we show that in
HL60 cells, vincristine a microtubule disrupting agent that induces a
strong production of IL-1, triggers the activation of both ERK and
JNK1. While ERK activation is rapid and transient, peaking at 10
minutes, the JNK1 activation is delayed and more sustained reaching a
maximum at 2 hours. ERK activation was blocked by CPl18556
indicating it is regulated by a Src-like kinase, while JNKI was inhibited
by piceatannol revealing an upstream regulation by Syk. Each kind of
the non-receptor tyrosine kinase blockers efficiently inhibits the
vincristine-induced IL-I production and diminishes the level of IL-1
transcripts, indicating that the ERK and JNK pathways act
coordinately to elicit the transcription of the IL-1 gene. Furthermore,
we found that pertussis toxin a blocker of Go/Gi proteins abrogated
the vincristine-induced Src and Syk activation. Our data support a
model where the status of microtubule polymerization influences the
activity of Go or Gi proteins that control Src/ERK and Syk/JNK1
cascades that are both necessary to sustain IL-1 synthesis.

[1] Sehmid-Alliana et al., J. Biol. Chem., 273, (1998) 3394.

[2] Mani6 et al., J. Biol. Chem., 268, (1993) 13675.
[3] Allen et ah, Am. J. Physiol., 26l, (1991) L315

(We/18.2/449)

Molecular and tubulin binding properties of the

~tathmin-like domains of the stathmin family proteins
E. Charbaut. S. Ozon. P. Ctwmi. S Lachkar. A. Sobel
I \SERM ~ 44# IP M

I -, mtc du Fer ~1 ~touhn. -5005 Payl~ t:~a~c

Stathnam is a ubNuitous 19 kDa cytosolic phospboproteln, most

abundant in the ner~ ous system. Its expression and phosphor? lation on tour
serines are regulated in a large set of situations, including cellular responses
to gro~ah and differentiation [:actors. or through the cell cycle. Stathmin
possesses a phosphorylation-dependent destabilizing acti~ ity on
microtubules tn vitro and in cultured cells, but the wa3 it proceeds remains
uncertain. It may displace the tubulir~microtubule equilibrium by
sequestering free tubulin, since it interacts with two a[3 dimers of tubulin in
vitro : it ma~ also act through a direct microtubule catastrophe-promoting
activit),.
Five stathmin-related proteins have bccn identified which are. unlike
stathmin, specific of the nervous system and membrane-associated.
specifically at the Gotgi apparatus. SCGI0. SCLIP, RB3 and its two splice
variants RB3" and RB3"" have, like stathmin itself, the capacity to
depolymerize microtubules v~hen transfected in cells. Their different
expression patterns, as well as the presence of specific additional domains.
suggest that the 5 may play partially different, most likely complementar3~
roles. (see Ozon et al.)
In order to investigate the similarities as well as the differences of
these related proteins, we have compared the structurally similar stathminrelated domains of SCGI0. SCLIP. RB3 and RB3'. We found that the main
biochemical features of stathmin are conserved, namely high solubility.
heat-stability, and shape-asymmetry revealed by an abnormally large Stokes
radius. Using gel filtration chromatography, we found that the five stathminlike domains arc able to form a complex with tubulin. Four of them display
a Stokes radius similar to that of the stathmin-tubulin complex, which
suggests thin the? also haxe a 21 stoechiometry : the RB3'-tubulin complex
displa3s a much smaller Stokes radius, closer to that of tubulin alone.
Moreover. surfhce plaslnon resonance allox~ed us to compare the respective
binding kinetics and to point out that the RB3-tubulin complex dissociates
more slox~b than the others. Altogether. this stud 3 suggests that despite a
global sunilarity between the different domains, some differences exist
x~hieh ha'< to be investigated to assess their physiological relevance.

Abstracts FEBS'99

(We/18.2/450)

CD82 induces morphological changes and co-activation

that are dependent upon small G protein.
Delaguillaumie A., Lagaudri~re-Gesbert C.. and Conjeaud H.

s394

(We/v/451)

h V S E R M u477. Hdpital Cochin,27 rue du F g St .Jacques.

75014 Paris, France

CD8Z a widely expressed molecule, belongs to the tetraspan superfamily

(TM4SF, CD9. CD37. CD53. CD63, CD81). On hematopoietie cells.
CD82 surface expression increases upon activation/differentiation. We
previously shown that on T cells, engagement of CD82 induces
association of CD82 with the cytoskeletal matrix and actin
polymerisation followed by cell spreading and development of
membrane extensions [l]. When associated with suboptimal TcR
stimulation. CD82 provides costimulatory signal leading to high cytokine
production [2]. To get further insights on the role of the cytoskeleton in
the CD82 costimulatory activity we studied the effects of various
cytoskeletal inhibitors on the CD82 induced morphological changes and
costimulatory activity. We show that depolymerizating agent
(cytochalasin). inhibition of all the small G proteins (toxin B from C.
difficile,) or specific inhibition of Rho (exoenzyme C3) inhibit the CD82
induced morphological changes, whereas only cytochalasine disrupts the
cytoskeletal association of CD82. Similarly, both cytochalasine and toxin
B inhibit primary (tyrosine phosphorylation) and late events of activation
(induction of cytokine mRNA), indicating that .the CD82 induced
cytoskeletal mobilisation plays a major role in its co-activation abilities.
Lagaudriere-Gesbert,C., LebeI-Bmay,S. Hubeau, C., Fradelizi, D and
Conjeaud. H, Eur J Immuno11998. 28' 4332
LebeI-Binay, S, Lagaudnere, C.. Fradelizi, D and Conjeaud, H. dlmmunol
1995. 155 101.

(We/18.2/452)

Cell adhesion modulates association between

talin and phosphoinositides.V. Martel, C. RacaudSultan, F. Paulhe, M.R. Block, and C. Albig~s-Rizo.
LEDAC, UMR 5538, lnstitut Albert Bonniot, Facultd de Mddecine de
Grenoble, 38700 La Tronche, France.

Talin is a key protein localized in adhesion plaques and appears to link actin
filaments to integrins. Its interaction with lipids could be of importance for
talin dependent actin nucleation. PIP2 is involved in the organization of the
actin cytoskeleton by regulating several actin-binding proteins. We provide
evidence that the binding of PIP or PIP2 on talin induce a dramatic
conformational change of the protein. This effect might be relevant to a
physiological process since both phosphoinositides become associated with
talin upon platelets adhesion on fibrinogen. Furthermore, talin is able to
coprecipitate with PIP once HeLa cells are in suspension, whereas it
associates transiently with PIP2 after ten minutes of adhesion on fibronectin,
a starting-time for HeLa cells spreading. Finally upon clustering of c~,f~,, the
interaction of talin with the cytosolie tail of the integrin seems to be enhanced
with the presence of the phosphoinosJtides. Our results support the
hypothesis that interaction between talin and phosphoinositides could be
involved in the regulation of actin cytoskeleton by integrins.

Proteases implication in the bronchial epithelial cell

migration process.
C. Legrand, J.M. Tournier, J.M. Zahm, M. Polette, and
P. Birembaut.
INSERM U.514. IFR n~53, 51092 Retnls, France.

Matrix metalloproteinases (MMPs) play a crucial role in the

migration process. We previously demonstrated that epithelial cell-produced
MMP-9 actively contributes to the in vitro wound-repair process of the
respiratory epithelium [ I ].
We used an in vitro wound-repair model and videomicroscopy
techniques to study cell migranon concurrently to the activation of MMP-9
by the plasmin system. Indeed, plasmin system has been shown to be
involved in the activation of some MMPs and in the migration of several
cell types.The conversion of inactive plasminogen into plasmin is known to
he mediated by plasminogen activators, specially urokinase-type
plasminogen activator (uPA).
We observed that uPA is only located in focal contacts of migrating
cells. Moreover, the incubation of migrating cultures with increased
plasminogen concentrations leads to a decrease or a total inhibition of cell
migration correlated to an increasing secretion of activated MMP-9. Our
results suggest that uPA is implicated in the migration of human bronchial
epithelial cells and that a balance deregulation of MMPs leads to an
inhibition of migration.
[1] Buisson A.C. et al., J. Cell. Physiol, 166, (1996), 143.
C. Legrand is the recipient of a fellowship from the Ministrre de
FEnseignement Suprrieur et de la Recherche, France.

(We/18.2/453)

Identification and functional properties of the neural

proteins of the stathmin family.
S. Ozon ". O. Gavet", S. El Mestikawy b A. Sobel "
"INSERM U440, IFM. 17 rue du Fer dl Mouhn,, 75005 Parts, France
I~INSERM [,'288. Facult~ de Mddecme Pitid-SalpdtrtOre, Parts'. France

Stathmin is a 19kDa ubiquitous cytosolic phosphoprotein, proposed to

be an intracellular relay integrating diverse intracellular pathways. Moreover,
stathmin controls microtubule dynamics by sequestering free tubulin and
possibly also increasing the rate of catastrophes.
Two neural stathmin-like proteins have been identified previously:
SCO10 which is a neuronal differentiation marker and XB3, a xenopus neural
protein. In a project aiming at finding novel members of the stathmin family,
we identified various proteins. RB3 is the mammalian homologue of XB3,
RBY and RB3" being two spliced variants derived from the same gene.
SCL1P is a mammalian protein very similar to SCG10 (70% amino acid
identities). All these proteins contain a well conserved stathmin-like domain
of 134 amino-acids conferring common properties, as well as additional NH2terminal domains responsible of the localization of the proteins at Golgi
membranes.
Whereas stathmin is ubiquitously expressed, the other stathmin related
naammalian proteins are exclusively expressed in the nervous system.
Northern blot and in situ hybridization experiments show that the respective
developmental expression patterns and cellular distributions of the various
stathmin-related mRNAs are different in rat brain, suggesting a specific role
lbr each protein. Their localization is not associated with a particular neuron
phenotype such as cell shape or neurotransmitter synthesis. Moreover. they
are all expressed by neurons but, for stathmin and RB3, also by glial ceils.
To lhrther investigate the role of each of these neural proteins, we
analyzed their action on microtubule dynamics. We showed that all stathmin
related proteins depolymerised microtubule arrays when overexpressed in
HeLa cells. Moreover the stathmin-like domain of each protein is able to
interact, like stathmin, with free tubulin (see Charbaut et al. ).
Altogether, our results suggest that the neural phosphoproteins of the
stathmin fanfily, through mechanisms involving at least in part their
subcellular action on microtubule dynamics, are playing diverse role in the
lbrmation, development and activities of cells of the nervous system.

s395

(W~18.~454)

Abstracts FEBS'99

The r o l e o f e y t o s k e l e t o n
in formation of terminally
differentiated uroeplthelial cells
P. Verani~, R.Romih, K.Jezernik, M.Peni~nik
Institute of Cell Biology, Medical Faculty,
Lipi~eva 2, 1000 LJublJana, Slovenia

The o r g a n i s a t i o n
of a c t i n f i l a m e n t s and e x p r e s s i o n
of
cytokeratins
w e r e s t u d i e d in rat u r i n a r y b l a d d e r
during epithelial
cell d i f f e r e n t i a t i o n
which followed
the d e t a c h m e n t
of c e l l s i n d u c e d by e y c l o p h o s p h a m i d e
(CP). It was e s t a b l i s h e d
t h a t in b a s a l and i n t e r m e d i ate c e l l s the a c t i n f i l a m e n t s
surrounded
the w h o l e
c e l l s b e n e a t h the p l a s m a m e m b r a n e ,
w h i l e in t e r m i n a l l y
differentiated
superficial
c e l l s the a c t i n f i l a m e n t s
w e r e f o u n d in l a r g e a m o u n t s o n l y at the b a s o l a t e r a l
membrane.
A f t e r s h e d d i n g of e p i t h e l i u m
c a u s e d by CP
the r e m a i n i n g
basal cells recovered
a normal urothel i u m in 7 to 8 days. D u r i n g r e p a i r we c o u l d f o l l o w
the r e a r r a n g e m e n t
of a c t i n f i l a m e n t s
in c e l l s at the
l u m i n a l s i d e of u r o t h e l i u m
f r o m a s t a g e w h e r e the
actin filaments
equally surrounded
the w h o l e c e l l s to
a final stage where they disappeared
f r o m the a p i c a l
p a r t of the c e l l s . T h i s p r o c e s s was a c c o m p a n i e d
by a
c h a n g e in a set of c y t o k e r a t i n s ,
a l s o . The c y t o k e r a t i n
17 w a s r e p l a c e d w i t h e y t o k e r a t i n
20, w h i c h c o u l d be
f o u n d o n l y in s u p e r f i c i a l
cells.
Our r e s u l t s p r o v e d that in a p r o c e s s of d i f f e r e n t i a t i o n of s u p e r f i c i a l
urothelial
c e l l s the m a j o r c h a n g e s
t o o k p l a c e in the s t r u c t u r e of a p i c a l m e m b r a n e w h e r e
the a s y m m e t r i c a l
unit membrane
(AUM) is f o r m e d s i m u l taneously
w i t h the d i s a p p e a r a n c e
of a c t i n f i l a m e n t s
b e n e a t h it. The r e a r r a n g e m e n t
of o y t o s k e l e t a l
elements
therefore
a p p e a r to be s t r o n g l y r e l a t e d to the s t a t e
of c e l l d i f f e r e n t i a t i o n .

(Wed18.2/456)

ATPase kinetic characterization of mutant human kinesin

motors defective in microtubule-based motility
Takashi Shimizu j, Aaron Ruby 2, and Ronald D. Vale 3'2
INatl. Inst. Biosci. Human-Technol., Tsukuba 305 Japan, and 2Dept.
Pharmacot., Univ. Calif. San Francisco, and 3Howard Hughes Medical
Institute, San Francisco, 94143 U. S. A.

Conventional kinesin is a microtubule-based motor protein that provides an

excellent model system for understanding mechanochemical transduction.
The crystal structures of motor domains from kinesin as well as two kinesinrelated proteins have been obtained. However, the structural bases of how
microtubule stimulate kinesin's ATPase rate as well as how ATP turnover is
coupled to force-production are still unknown. As tools for investigating
these questions, Woehlke et al. [1] generated 35 alanine mutations in solventexposed kinesin motor domain residues. In addition to mapping the kinesin
residues involved in microtubule binding, this study also identified three
mutants (Y 138A, loop 11 triple (L248A/D249A/E250A), and E311 A) that
exhibited -3-fold reductions in both microtubule gliding velocity and
microtubule-stimulated ATP activity. Here, we investigated the underlying
defects in these mutants by presteady state kinetic analysis. All mutants, as
well as a loop 12 triple mutant that is severely defective in microtubule
binding, exhibited wild type second-order ATP binding kinetics, indicating
correct folding of the active site. The Y138A and loop 11 triple mutants
exhibited defects in nucleotide hydrolysis (>5-fold decrease in rate), even
though the mutated residues are not located within the active site. These
mutants also exhibited 2--4 fold reductions in microtubule-stimulated ADP
release rates, while the rates at which they released ADP in the absence of
microtubules were normal. These findings suggest that Y138 and loop 11
may participate in the allosteric pathway in which microtubule binding leads
to accelerated product release from the active site. Finally, with the E311A
mutant, we found that neither the rate of nncleotide hydrolysis nor ADP
release can account for its slow ATP turnover and gliding velocity. A step
subsequent to nucleotide hydrolysis but before ADP release (either phosphate
release or a conforrnational transition) appears to be rate-limiting in this
mutant. We speculate that E311, a highly conserved residue in the kinesin
superfamily, plays an important role in force production.
[1] Woehlke et al., Cell 90 (1997) 207.

(We/18.2/455) MECHANISM OF CONTROL OF ACTI]~IF1LAMENT LENGTH AND

TLrRNOVERBY ACTIN DEPOLYIVIERIS1NGFACTOR(ADF/COFILII~

Fariza Ressad, DominiqueDidry, DominiquePantaloniand Marie-FranceCarl
Dynamiquedu cytosquelett,Laboratoired'Enzymologieet BiochimieStructurales
CJg.R.S.. 91198 Gif.sur-Yveae, France

The rapid turnover of actin filaments drives actin-based motility processes

such as the forward movement of the leading edge of the lamellipodium in
locomoting cells or the propulsive movement of Listeria and Shigella or the
movement of actin patches in yeast.
Actin-binding proteins of ADF/cofdin family have recently been
demonstrated to enhance the turnover of F-actin by increasing the rate of
depolymerization from pointed ends.
In the present work, the goal is to understand the molecular mechanism by
which ADF enhances filament turnover, is it the consequence of the
increased number of filaments due to the severing action of ADF, or of the
enhanced dynamics of individual filaments, or of both.
We show that ADF increases the steady-state concentration of filaments by
measuring the number of filaments in a solution of F-actin used as seeds in
seeded polymerization assay.
The behavior of plant and human ADF were compared. The maximum
increase in number of filaments reached at saturation is about 6-fold for
plant ADF and 4-fold for human ADF. This corresponds to less than 2fold at physiological ADF/actin ratio.
ADF appears to change the steady-state length distribution of actin
ftiaments in a manner dependent on saturation of F-acfin by ADF.
However, the results indicate that ADF does not increase the number of
filaments shorter than llam. Indeed no barbed ends were created by ADF
when filaments were capped by gelsolin at a gelsolin:actin ratio of 1:300.
ADF increases the turnover of actin filaments in an end-dependent fashion,
in the presence of ATP or ADP under conditions where the flux of
subunits from the pointed ends to the barbed ends cannot occur, all barbed
ends being blocked by gelsolin at a gelsolin:actin ratio of 1:300.
We propose that the cooperative nature of ADF binding to F-actln
generates two populations of energetically different filaments: ADFdecorated filaments depolymerize rapidly while more stable bare filaments
grow. In the presence of gelsolin, a rapid flux of subunits is driven from
the pointed ends of ADF-decorated filaments to the pointed ends of bare
filaments.
The ADF concentration dependence of the filaments turnover rate in ATP
and ADP displayed a bell-shaped curve.
The turnover of actin filaments capped at both ends by gelsolin and Arp2/3
complex is also increased by ADF. It is shown that Arp2/3 generates
barbed ends in solution of gelsolin capped filaments.

(We/18.2/457)

Cortical actin dual regulation by Ca z+ / Calmodulin and Rho

R.Sullivan, A.Koffer
Dl,pt t'h~ wolo,vl. UJlil el~i 0 College L~mdon. London WCIE 6J,I, I/K

The cortical actin cytoskeleton of rat peritoneal mast cells (RPMCs) is

regulated by a variety of signal transductlon pathways. We have
investigated its regulation by Ca > via the small GTPase Rho using
streptolysin-O permeabllised RPMCs [1]. Achvat~on of RPMCs with
Ca > MgATP induce', cortical actin disassembly [2]. In the presence of a
constitutively active mutant of Rho, VI4RhoA there is synergistic
enhancenaenl of disassembly. By itself V 14RhoA only induces - IO- 14~{
cortical disassembly. Inhibition of endogenous Rho by the exotoxln C3transferase partial inhibits Ca2+.MgATP-induced actin disassembly
(-10%). We have found that novel peptide mhlbitors of CaM (based on
the CaM binding domains of MLCK and cNOS) prevent Ca>.MgATP induced disassembly, but not that due to V 14RhoA. Likewise
reconstnuting permeabilised RPMCs with exogenous CaM promotes
Ca-"MgA'FP-mduced dlsassembly. We propose a novel mechamsm of
c{wtlcal actm regulation by Ca~*/CaM and Rho which act on a common
downstream target hmial evidence suggests that this target is
Ca>/('aM-dcpendent myosin light chain kmase. Cortical disassembly
ma~, in part, be regulated by acto-myosm-based contractility.
[ 1] Lmdau M. et al Biochem. Biophys. Acta 107 I, ( 1991 ), 429.
[2] Koffer A. el al J Cell B i o l . l l l , (1990), 919

Abstracts FEBS'99

s396

19.2 Molecular mechanisms in infectious diseases

(We/19.2/458) Evaluation of two amplification technologies

for the

detection of M y c o b a c t e r i u m tuberculosis
V. Baumanis*, I. Jansone*, O. Marga**. L.Broka***,
T. Pan~aka*, I.Mandrika*
*-B~omed Res.& Stud)" Centre Unlv of Latvia, Ratsupaes ],Rtga,
Latvta, L V1067, **MedAcadLatvia, *** -Tub.Center of Latvia(TC)
The rapid detection of :klycobacterium tuberculosis from
respiratory specimens and bronchial washings is critical for
optimal treatment of patients. Incidence of tuberculosis and its
drug resistance has been increased in Latvia from 27.4 per
100.000 population in 990 till 74 in 1998. In TC amplification
of IS6110 fragment by in-house polymerase chain reaction (PCR)
, ligase chain reaction of 38kDa protein gene fragment by Abbott
amplification kit LX (LCR) was introduced therefore and results
of both amplification methods analysed and compared with the
BACTEC cultivation , smear microscopy, clinical findings as
well. IS6110 PCR in 95-100% cases confirmed other three
methods , but was the most sensitive likely due to presence of
more than one copy of IS6110 in the clinical isolates studied.
Detection of rifamicin resistance of cultivated bacteria was
screened by the single strand conformation polymorphism
analysis and confirmed by Inno-LIPA test (Murex). Missence
mutations in Asp516
and His 526 positions were found.
Determination of drug resistance in the clinical material by costeffective molecular methods needs to be improved however.

(We/19.2/460)

Complementation and localization studies o f

BNYVV TGB1 protein fused to eGFP
M. Erhardt, D. Gilmer.
IBMP/C.RS, 12, rue du gdn~ral Zimmer. 67084 Strasbourg
cedar. France.

Beet necrotic yellow vein virus is the causative agent of sugar beet
rhizomania and belongs to the benivirus family. The virus requires at least
three proteins for its cell-to-cell movement. These proteins, (42, 13 and 15
kDa), are encoded by a triple gene block cluster present on RNA-2.
Independent expression of movement proteins is possible by using
replicon RNA-3, a viral vector derived from RNA-3. Such replicons
expressing movement proteins are able to complement defective BNYVV
RNA-2 mutants.
Enhanced jellyfish fluorescent protein (eGFP) was fused either to the Nterminal or C-terminal extremities of wild type P42 B N Y V TGB1
protein. In order to study their cellular localization, transcripts of RNA 3
replicons carrying each contruct were inoculated together with RNA I and
wild type or TGB defective RNA 2 into Chenopodium quinoa protoplasts
and plants. Fluorescence was observed both in protoplasts and plants but
complementation occured only if eGFP was fused to the N-terminus of the
protein. Microscopy studies revealed no particular localization of GFP-P42
fusion protein in C. quinoa protoplasts. On the contrary, fusion protein
was localized in the cell wall of plant cells when expressed by the virus
during the infection cycle. Electron microscopy studies localized the GFPP42 fusion protein within the plasmodesmata. In the absence of P13
and/or P15, GFP-P42 protein lost its capability to localize within the cell
wall, suggesting a possible interaction with the two other TGB proteins.

(We/19.2/459)

Cytotoxic necrotizing factor 1 (CNF1) endocytic and

membrane transloeation mechanisms.
S. Contamin and P. Boquet
INSERM U452, NICE

The cytotoxic necrotizing factor (CNFI) elaborated by certain strains of uropathogenic

Escherichla coli is a toxin of 110 kDa. CNF1 induces on cultured cells a massive
reorganization of the actin cytoskeleton consisting in accumulation of stress fiber,
membrane folding and filopodia. CNF1 has been shown to activate by an intracellular
catalytic process the small GTP binding protein Rho and also probably Cdc42. These two
GTPases are pivotal regulatory proteins involved in the control of the actin cytoskeleton
orgamzation. CNF1 is a deamldasewhich specifically acts on glutamine 63 of Rho (or 61
of Cdc42) transforming this residue into glutamic acid. Deamidation of glutamine 63 to
glutamic acid block the intrinsic and GAP-mediatedhydrolysis of GTP into GDP by Rho
thereby activating permanently the regulatory molecule. CNF1 is divided into three
polypeptidic domains encompassing each Ca 300 amino acids. The N-terminal domain
contains the binding site of the toxin for its cell receptor. The carboxy-terminaldomain
contains the enzymatic activity whereas the mid-domain is probably involved in the
transport of the toxin catalytic activity across the cell membrane. In the work presented
here we have examined the entry mechanismof CNFI into HEp-2 cells. Using transiently
transfected cells with dominant negative mutants of proteins involved in the formation
(dynamin 2) or control (Epsl5) of coated-pas, we were able to demonstratethat CNFI uses
a clathrin-dependant pathway of endocytosis to be taken-upby HEp-2 cells. After receptor
-mediated endocytoslsCNF1 is transported to early endosomeswhere most likely the toxin
(or its enzymatic fragment)is transferredinto the cytosol This Is shown by the facts that I/
brefeldin A which blocks the transport from endosomes to the trans golg, network (TGN)
2/That nocodazole which disrupt microtubules, structures required to transport endocytic
carrier vesicles from early to late endosomes3/that dominant negativeform of Rab7 which
impairs transport from early to late endodomes and homotypic fusions of these
compartment as well as those with lysosomes are all without any activaty on CNFI
cytopathogemc effects (measured quantitatively by cell multinucleatlon), tnhthitors of
endosomes acidification such as monensin and bafilomycinAI block totally the cellular
effects of CNFI. Furthermore, incubating HEp-2 cells with CNFI at different pH (ranging
from 7,5 to 4) m the presence of bafilomycin (to block the normalentry pathway of CNFI)
allows entry of the toxin directly from the cell membrane.Translocation starts when the
external pH applied was 5,3, as visualized by the Rho induced shift of molecular weight
due to deamidation of its glutamine in posthon 63. These data demonstrate that CNFI
translocates from early endosomesinto the cytosol by an acidic pH dependent step. Finally,
using a non-toxic mutant of CNFI (mutated on cystem 866 to serine 866) which fully
competes on CNFI cell receptors for the wild-typetoxin, we have estimatedthe affinity of
CNFI for its cell receptor by the Schild plot analysis method.

s397

(We/19.2/462)

Abstracts FEBS'99

Langerhans Cells and Cytokine Expression in HPV

Associated Cervical Lesions
S. L. Giannini. H. Piron, J. Boniver and P. Delvenue
University of Li/~ge,Departmentof Pathology
B-4000 Li~,ge,Belgium

The chronic infection of mucosal epithelium keratinocytes of the

cervix by some types of human papiUomavirus (HPV) is associated
with the development of squamous intraepithelial lesions (SIL) and
cervical cancer. Within the epithelium, Langerhans ceils are
important for the immuno-surveillance against mucosal infections
and cancers, due to their superior capacity to present antigen. The
aim of our study has been to access the density and function of
Langerhans ceils in the region most sensitive to cervical
carcinogenesis the transformation zone (TZ), and in SILs. In
addition, we investigated if the production of cytokines within the
micro-environment of the TZ and SIL may influence their
accumulation and function during the development of HPVassociated SIL.
For our investigation, we used a combination of immunohistochemistry, semi-quantitative RT-PCR and a mixed
lymphocyte epithelial response assay (MELR). We have
demonstrated that there is a diminished density of CDla+
Langerhans cells in the TZ and most SILs compared to paired
normal exocervix biopsies. In addition, we also found that the
epithelial cells derived from TZ and most SILs were less efficient
in inducing an allogeneic response as assayed by MELR. Since the
depletion and the function of Langerhans cells could be influenced
by local cytokines we evaluated the relative expression of several
cytokines known to influence their migration and accumulation
(GM-CSF, TNFot, IL-II] ) and function (ILl0). Interestingly, we
observed that the TZ and SILs are associated with higher average
levels of the immunosuppressive cytokine ILl0. Our preliminary
results indicate that the expression patterns of GM-CSF, TNFo~,
IL-11] do not correlate with the reduced density of Langerhans
cells.

(We/19.2/463)

Characterization of interaction domains of URE2p and

analyze of mutant alleles of the URE2 gent.
E. Fernandez-Bcllot,E. Guillemet,A. Baudin-Baillieu,
A. Komarand C. Cullin. CGM,, CNRS, Gif-sur-Yvette, France.

In the yeast Saccharomyces cerevisiae, the non-Mendelian inherited

genetic element lURE3] behaves as a prion.[ 1,2]
A hypothesis has been put forward which states that lURE3] arises
spontaneously from its cellular isoform Ure2p - the product of the URE2
gent - and propagates through interactions of the N-terminal region
domain of the protein, thus leading to its aggregation and loss of function.
Various N- and C-terminal deletion mutants of Ure2p were constructed
and their cross-interactions were tested in vitro and in vivo using affinity
binding and a two-hybrid analysis. We show that the self-interaction of the
protein is mediated by at least two domains, corresponding to the first
third of the protein (the prion forming-domain) and the C-terminal
catalytic domain.[3]
Moreover, we performed a random mutagentsis of the URE2 gent. The
expression on a monoeopy plasmid of one mutated allele showed a
dramatic increase of the [ URE3] phenotype. The analysis of the mutations
of this allele showed that mutations in the prion domain and in the
catalytic domain could increase the frequency of appearance of lURE3].
The mutations in the prion forming-domain and in the catalytic domain
have been studied separately, and their effect suggests that the C-terminal
part of Ure2p plays an important role in the prion formation.
[1] Aigle, M. and L,acroute, F., Mol. Gen. Genet., 136, (1975), 327
[2] Wickner, R. B. Science, 264, (1994), 566
[3] Femandez-Bellot, E. et al., Biochem. J., 338, (1999), 403

The augmented expression of ILl0 and the relative deficiency in

Langerhans cells may interfere with an efficient induction of an
efficient immune response and contribute to the predisposition of
the TZ to cervical carcinogenesis and to the progression of S1Ls.

(We/19.2/464)

Tumor necrosis factor cc induction in murine macrophages

by CoxieUa burnetii strains and their lipopolysaccharides
E. Gajdo~owia, M. Kube~b,V. Mucha ~, E. ~kult~ty a, R. Tomana
Dept. of Rickettsiology. ~Dept. of Immunology, CDept. of ViralStructure
Inst. of Virology, Slovak Academy of Sciences, BratiMava, Slovak Repubhc

Lipopolysaccharide (LPS), a major component of the outer envelope of

Gram-negative bacteria, has many biological responses. They are
mediated by cytokins among which tumor necrosis factor a (TNF-ct) plays
a prominent role. TNF-a is a pivotal cytokine in inflammation and is
considered to be a main endogenous mediator of septic shock. The present
study was undertaken to investigate the ability of various strains of C.
burnetii and their LPSs to stimulate murine peritoneal macrophages to
produce TNF-ct. After induction of macrophages with corpuscles and/or
LPSs, we observed considerable differences in the TNF-et production in
dependence on the strain applied. The most effective, in order from the
best, were corpuscles of "S", Priscilla, Lugs, and Henzerling I strains.
Both strains "S" and Priscilla are considered to be associated with chronic
form of Q-fever. With some C burnetti strains, a prolonged TNF-tx
production (after 15, 18, and 20 h) was obtained. After induction of
macrophages with LPSs, we observed as the most effective in the TNF-ct
production, in order from the best, strains "S", Priscilla, Nine Mile II, and
Luga. The highest values of TNF-a were obtained in the three hour
interval of production and then, after 6 h, a sharp decrease was noticed.

(We/19.2/465) Structural studies of PBP2x from Streptococcus pneumoniae.

E.J. Gordon, N. Mouz, A-M. Di Guilmi, L. Martin, E. Duee,
T. Vernet and O. Dideberg
LCM/LIM, lnstttut de Biologie Structurale, 4L rue Jutes Horowltz,
38027 Grenoble Cedex 1 France

Penicillin binding proteins are the primary targets of I~-Iactam antzbiotics.

Inhibition of these enzymes arrest the critical step of
transpeptidation/cross-linking of the cell wall and result in cell death. In
pathogenic strains of Streptococcus pneumoniae, drug resistance is due to
modification of several of the essential PBPs. In particular, PBP2x has
been shown to be involved in both low level and in high level drug
resistance.
The aim of the study is to understand how antibiotics are recognised and
react with PBP2x
and how acquisition of resistance changes these interactions. The structure
of a sensitive PBP2x has been determined to 2.4~, and the structure
complexed with a therapeutically important antibiotic has been determined
to 2.8A [1].
Naturally occurring mutants from isolated clinical drug resistant strains
have up to 100 changes in their amino acid sequence. The two most
frequent substitutions, T338 and Q552, are probably the most important
in terms of acquisition of resistance. We have used crystallographic and
site directed mutagenesis techniques to demonstrate the structural
importance of these substitutions [2,3]. Structural and biochemical data
will be presented.
Structural studies ofa PBP2x from a clinical resistant isolate are underway.
Data have been collected and refinement is in progress.
[1] Gordon et al.. in preparation
[2] Mouz et aL (1998) PARAS 95, 13403.
[3] Mouz et al. (1999) J.B.C in press

Abstracts FEBS'99

(We/19.2/466)

s398

Clostridiumperfringens (x-toxin, a bacterial model

of proteins-Ca -phosphollplds interactions.

Guillouard 1.~, Alzari p.b, Alape-Giron A.~and Cole S.T.a

Molecular analysis of the interactions between aphids

and plant viruses
P. Irving*, P. Seddas*, A. Seddas**, E. Herrbach*

"UJ*IlOde GdnOttque ~loleculalre Baetdrtenne ~and Unll~ de Immunologic

Gendrale lnstttut Pasteur, 28 rite du doeleur Roux, 75 "24 PARIS ~de~ 15
Instttuto Clodomlro Ptcado, Utuverstdad de Costa Rtca San Josd Costa Rtca

* INRA, 28 Rue de Herrhsheim, Colmar 68021, France. ** Laboratolre de

Production des Anucorps Monoclonaux, LNPV, 28 Rue de Herrlisheim, Colmar
68021, France

2+

Clostridium perfringens (x-toxin, the major lethal virulence

factor of human pathogenic strains, is known to play a prominent role
in gas gangrene pathogenesis. This phospholipase C can hydrolyse
two essential glycerophospholipids of cellular membranes
(phosphatidylcholine and sphingomyelin). It causes cytotoxicity for
erythrocytes, fibroblasts and muscle cells and can aggregate blood
platelets in vitro. (z-toxin is also known to activate the inflammatory
process by inducing the arachidonic acid cascade.
Structure-function relationships reveal that c~-toxin is
composed of two domains. The N-terminal fragment retains
lecithinase, sphingomyelinase and hemolytic activities. By means of
site-directed mutagenesis experiments of this module, we show that
the catalytic site of (x-toxin is composed of amino acids implicated in
zinc coordination (histidines in position 11, 68, 126, 136 and 148;
tryptophane 1), acidic residues (aspartate 56, 130 and glutamate 152)
and other residues including phenylalanine 69 [ 1].
The C-terminal domain retains no enzymatic activity in vitro.
We identified a low affinity calcium binding site composed of
aspartates 269, 273 and 336. It was also shown that this C-terminal
domain is required for the cytotoxic effect of the (z-toxin in UDPglucose-deficient cells [2]. We suppose this C-terminal domain of
being a C2-type domain responsible for the interaction of the protein
with membrane phospholipids by the intermediate of calcium ions [3].

[1] Guillouard et al., Infect. and Immun., 64, (1996), 2440

[2] Flores-Diaz et al., JBC, 273, (1998). 24433
[3] Guillouard et al, Mol. Microbiol., 26, (1997), 867

(We/19.2/468)

(We/19.2/467)

The dimerizalion of HIV-I genomic RNA: Importance of

RNA loop structure and magnesium binding
F. Jossinet, J.C. Paillart, E. Skripkin, C. Ehresmann, B.
Ehresmann, and R. Marquet
"IPR 9002 CNRS, IBMC, 15 rue Ren~ Descartes, F~7084 Strasbourg

Retrovimses encapsidate their genome as a dimer of homologous RNA

molecules non-covalently linked close to their 5' ends in a region called the
dimer linkage structure (DLS). The dimerization initiation site (DIS) of human
immunodeficicncy virus type 1 (HIV-1), the essential part of the DLS, is a
hairpin structure located upstream of the major splice donor site that contains
in the loop a six-nucleotide self-complementary sequence flanked by two 5'
and one 3' purmes. Not only the self-complementary sequence, but also the
flanking purines are crucial for dimerization of HIV-I RNA, which is
mediated by formation of a "kissing loop" complex between the DIS present
on each monomer. Here, we used interference of chemical modifications of
the phosphate backbone and bases on the dimerization process, and leadinduced cleavage of the dimers to compare dimerization of subtype A and B
HIV-1 RNAs. The DIS loop sequence of these RNAs is AGGUGCACA and
AAGCGCGCA, respectively. Our results demonstrate the importance of the
loop structure in the dimerization process. Furthermore, they reveal an
unexpected difference existing in the dimerisation mechanism of these RNAs.
Unlike subtype B, subtype A requires binding of a divalent cation in the loop
to promote RNA dimerization. This difference must be taken into account in
the design of antl-dimerization molecules aimed at inhibinng H1V-1
replication.

The luteovirus Beet western yellows virus (BWYV) is obligately

transmitted by aphid vectors tn a circulalave and specific manner. In these
vectors, transmission reqmres the viral particles to cross at least two epithelial
membranes and the specificity of transmission implies the involvement of
receptors on these membranes. In order to establish the nature and location of
such receptors, studies have been carried out on the mteractions between
BWYV and its main vector, Myzuz" persicae (Sulz.).
Aphid proteins which are able to brad to BWYV may be active in virus
acquisition. To identify such proteins aphid extracts were electrophoretically
separated in one dimension, blotted and probed with an anti-BWYV antiserum,
'after incubation with viral particles. However, preliminary experiments, using
the available anti-BWYV polyclonal antiserum, showed high levels of nonspecific binding to whole aphid proteins. To address this problem, monoclonal
antibodies (MAbs) have been raised against purified BWYV particles. For a
more precise identificanon of the binding proteins, the aphid extracts are now
being separated by two dimer~s~onal electrophoresis prior to tar western blotting
with the virus and MAbs.
As the first epithelial barrier encountered by viral particles is in the aphid
digestive tract, a second area of the study has been the membrane proteins of
these tissues, lnitmlly the digestive tracts from 500 adult M. persicae were
isolated by dissection and proteins extracted from these digestive tracts were
used as antigens for MAb production. The pattern of binding to gut proteins
has been established tot each of these MAbs. On western blots, where a MAb
recogmzes a receptor protein, binding may be prevented by incubation of the
blotted proteinb with the specific ligatc before addition of the amtbody.
Identlficataon of wral receptors could therefore be achieved by looking for
BWYV interference of the MAb interaction wuh aphid proteins.
Concurrent with the production of monoclonal anubodies has been the
completion of a complemental3, DNA library. Messenger RNA, extracted from
entire adult M. persicae, has been used to make a high titer eDNA library in a
Lambda ZAP expression system. The MAbs raised against digestive tract
proteins are now being used to interogate the library.

(We/19.2/469)

Differential regulation of arginase I and II

in rat alveolar macrophages.
S. Klasen, R. Hammermann, K.F. Beck*, J. Pfeilschifter* K. Rack~
Inst. Pharmacol. & Toxicol, Univ. Bonn, Reuter-Str. 2b, D-53113
Bonn; *Inst. Pharmacol. & Toxicol., J.W. Goethe-Univ. Frankfurt

L-Arginine is the substrate of nitric oxide synthase (NOS) and arginase. In

rnacrophages (M~), including alveolar M~ (AM~), both enzymes can be coexpressed and compete for the common substrate, eventually resulting in a limitation of L-arginine utilization by either pathway [1,2]. As little is known about
the regulation of the expression of the two arginase isoforms in M~, the effects
of lipopolysaccharides (LPS) and interferon-7 (IFN-7), two potent inductors ot
iNOS in AMq% was studied.
Rat AM4>, freshly prepared or cultured for various times in the absence or presence of 1 gg/ml LPS or 500 U/ml IFN-7 were used to isolate total RNA and
to extract cellular proteins, mRNA for arginase I and II, iNOS and 13-actin
were determined by semi-quantitative RT-PCR and protein levels of arginase I
and II, iNOS and (x-tubulin were measured by immunoblotting.
In freshly prepared AMe; mRNA for both isoforms of arginase as well as the
corresponding proteins were detected. At~er a 20 h culture period mRNA for
arginase I1 was markedly reduced. Presence of LPS during the culture period
prevented the decline in arginase II mRNA and enhanced arginase I mRNA
above initial levels. This was accompanied by an increase in protein levels of
arginase I and II after 20 h culture in the presence of LPS. INF-y, which like
LPS caused a marked induction ofiNOS mRNA and protein, had no effect on
the expression of arginase I, but enhanced both the mRNA and protein levels
of arginase II. Time course experiments showed that an inductive effect of
LPS on arginase I mRNA was present after 5 h, whereas an inductive effect of
LPS on iNOS mRNA was already seen after 2 h. Dexamethasone (10 gM),
known to induce the expression of arginase I in the liver [see 3], had no such
stimulatory effect in AM4~, on the contrary, it opposed the LPS induced upregulation of both arginase isoforms as well as the LPS-mediated induction of
iNOS.
In conclusion, rat AM~ coexpress arginase I and II, but their expression is differentially regulated. The levels of both argirmse isoforrns are enhanced by
LPS, but via different mechanisms. IFN-y, like LPS an inductor ofiNOS, only
enhanced arginase II, excluding a simple link between the expression of iNOS
and the arginases.
[1] Hecker et al., FEBS Lett., 359, (1995) 251
[2] Hey et al., Br. 3. Pharmaeol., 121, (1997) 395
[3] Wu et al., Biochem. J., 336, (1998) 1

s399

(We/19.2/470)

Abstracts FEBS'99

Rapid spectroscopic analysis of bacterial cells

(We/19.2/471)

H. Lamfarraj, G. D. Sockalingum, M Manfait

Umt~ Mb.DtAN, UFR de Pharmacie, Untverslt~ de Reims ChampagneA rdenne, 51 rue Cognaeq Jay, 51096 Reims, France

Fast and exact identification of a great number of microorganisms i

becoming a serious challenge. Vibrational spectroscopic techniques (Rama,
and infrared) can be complementary and useful methods in this field due t~
their rapidity, "fingerprinting" capabilities, and the molecular information tha
they can provide Using surface-enhanced Raman scattering (SERS) at silve
colloids, we have conducted pilot studies to rapidly detect and identit~
bacterial clinical strains. The use of metal colloids allow to gain in sensitivit:
and circumvent fluorescence. P s e u d o m o n a s aerugtnosa and Escherichia col
clinical strains, responsible for nosocomial infections, have been our first tes
samples Strains ,,;'ere grown in presence of Ag colloids and direct on-plat
analysis was performed SERS Spectra recorded with a Ramm
microspectrometer, equipped with a He/Ne laser and a CCD detector, wer.
reproducible, with diminished fluorescence, and reveal characteristic cellular
level information As a bacterium is a complex and real biological system, it
Raman or SERS spectrum is expected to be also complex with most of th,
vibrational modes mixed From our experiments we found that bacteria
systems exhibit strong C-H stretching bands in the 3000 cm q region and C-t
deformation (1448 cm "t) bands most probably originating from the fatty aci~
chains of the different amphiphilic compounds such as phospholipids anq
from amino side-chains of proteins The region comprising 1173-1340 c m "1 i
characteristic of amide Ill and that at 1640-1700 cm q of amide l Othe
characteristic cellular component bands are those of phenylanaline at 100,
and 1034 cm q, originating from C-C and C-H modes. In addition, the SER~
spectra of bacteria show a series of quite resolved peaks which we hay
tentatively assigned to particular bacterial substructures as the RNA/DN:
oligo- and polynucleotides base-ring vibrations of guanine, thymine, adenine
and cytosine; and the amino acid side-chain vibrations (tryptophane, tyrosin
and phenylalanine) of the proteins. Our data indicate that SERS spectra ar
rich in molecular information that could be used to identify and charaeteriz
micro-organisms in general and the establishment of a solid data bank couh
be complementary to classical methods

(We/19.2/472)

Prion protein expression during the bioaminergic

differentiations of a neuroectodermal progenitor - S.
Mouillet~Richard~, M. Vidaud b, JM. Laanay ~, O. Kellermann~,
JL. Laplanche~ - "CNRS URA 1960, btstitut Pasteur, 75724, ~JE351,

S. aureus adherence to airway epithelial cells, a

E. Mongoda~,D. Gaillard~,Q. Bajolet,J21.K1."~ssekc, J.P. ~ 1 ,
E. Puct~le", S. de B e n ~ w .
"INSERMU514.I'CHRRobertDd~rd51092Rebns;'CHUZ Bel,tcod86021Poiiie~,~,
'tH&pitcdBrc~sc~ 75014Panx Frore

Staphylococcus aureus is a major human pathogen involved in genetic or in

acquired bronchial diseases. Ulrich et al. (Am J Resp Cell M o l Bioi(1998) 19,
83) recently showed that in vivo, S. aureus mainly adheres to mucus

components, the adherence to the bronchial epithelial cells being negligible.

Surprisingly. in this study conducted on cystic fibrosis (CF) bronchial tissues,
no remodeling or damaged epithelial structure was described. Until now, the
adherence of S. aureus to airway epithelium, in relation with its remodeling
state, has never been investigated. In order to study the mechanisms of
staphylococcal adherence and the receptors involved, we developed an
immunolabeling method based on the affinity of staphylococcal protein A
(SPA) for the Fc region of IgG. This technique allowed us to detect most of
the S. aureus strains in light fluorescence microscopy and scanmng or
transmission electron microscopy, in a cell culture model as well as in lung
tissues obtained from CF patients, bl vitro studies showed that S. attreus
mainly adhered to the mucus layer present at the surface of airway epithelial
cells, but no bacteria was found to adhere to ciliated differentiated airway
epithelial cells. In contrast, S. aureus adhered mainly to poorly differentiated
and migrating epithelial cells, as well as to the collagen I matrix on which
ceils were seeded. In vivo we observed a great heterogeneity m the
remodehng state of CF-infected airway epithelium. When the CF airway
epithelium wa~ tight and pseudostratifted, S. attreus was trapped by the
mucus and kept away from the epithelial cells, hut we could detect bacteria in
remodeled areas, especially in enlarged ctliated ducts. In these areas, where
the airway epithelium is partially desquamated or the basal lamina denuded,
bacteria were detected in between the epithelial layer, in contact with
columnar and basal cells, as well as beneath the basement membrane and in
the connective tissue underlying the airway epithelium. Our results suggest
that S. aureus mainly adheres to remodeled airway epithelium, in vitro as
well as m vivo, and is able to migrate across this epithelium to invade the
connective tissue.
E. Mongodm is a doctoral tcllow of the DRET/DGA.

(We/19.2/473)

cyclic AMP-dependent protein kinase A (PKA) type I

contribute to CD4 T and B cells deficiency in AIDS
S.Rahmouni, J.Boniver and M.Moutschen
Laboratory of Pathology, University of Liege, 4000 Likge, Belgtum

Facultd de Phatwlacie, 75006, 'HOpttal LariboisiOre, 75010 Paris, France

The biological function of the cellular prion protein PrPc is currently

unknown. PrPc is a nearly ubiquitous glycoprotein anchored to the plasma
membrane, most abundantly expressed in neurons. We took advantage of the
bipotential neuroectodermal 1 C l l cell line to monitor PrPc expression
during its bionminergic differentiations. The Fg-derived 1 CI 1 precursor cell
line displays a stable and immature phenotype in the absence of extracellular
signal and has the capacity to acquire upon induction a complete
serotonergic or noradrenergic phenotype, the two pathways being mutually
exclusive.
We developed a real-time quantitative PCR assay to assess PrPc gene
expression at definite times of the two programs that correspond to the
sequential acquisition of neurotransmitter-specific functions. IC11 cells and
their differentiated progenies express significant amounts of PrP transcripts
and of the corresponding protein, while the level of PrP gene expression is
much fainter in F9 and other multipotential cells. Entry of 1CI 1 cells into the
serotonergic - but not the noradrenergic - pathway is accompanied by a
decrease in PrP gene expression. Besides, NGF treatment results in but a
slight, significant downregulation of PrP mRNAs in the serotonergic cells.
Our in vitro study accurately establishes that PrP gene expression (i) is
strongly upregulated concomitantly with cell fate restriction of multipotential
cells towards the neural lineage; (it) is differentially regulated according to
the serotonergic versus noradrenergic differentiation program of a unique
neuroectodermal progenitor.

Infection of C57BL/6 mice with the retroviral preparation

RadLV-Rs causes a lethal immunodeficiency syndrome which
bears some similarity with human AIDS. The disease is
eharacterised by the loss o f T and B cell functions.
cAMP-dependent protein kinase A (PICA) type I is an important
inhibitor of lymphocyte proliferation, with few exceptions all
known actions of cAMP are mediated through PKA. Enhanced
cAMP levels completely abolish early tyrosine phosphorylation
following engagement of the Ag receptor as well as T cell
proliferation induced through the TCR/CD3 complex. We
therefore investigated the possibility that activation of PKAI
may be involved in the process of immunodeficiency observed
in MAIDS. Our results demonstrated that CD4 T and B cells, but
not CD8 T cells, from infected mice have increased levels of
cAMP compared to control cells, and that CD4 T cells are more
sensitive to inhibition by cAMP analogs than are normal CD4 T
ceils.
No difference have been observed concerning the expression of
protein kinase A type I studied by western bloting in the cells
from infected compared to healthy mice.
These data suggested that an upregulation of cAMP and
increased activity of protein kinase A type 1 could participate in
the anergy involving CD4 T and B cells in MAIDS and that
PKA I may be a potential target for immunomodulating therapy.

Abstracts FEBS'99

(We/19.2/474)

s400

(We/19.2/475)

mivity ofFe 011)immporphymIX andie symhak

F.Omot~ Sa~N.Basilic~, DA4orJi,P.OIli~,D.T~mndli

Relationships between cell tropism,

cytopathology and clinical features of HIV-1 isolates
from CIS./I.Sakhuria, M.Shchelkanov, E.Karamov /
]vanovsky Institute of Virology, Moscow, Russia

The degradation of haemoglobin is a crucial event for the survival of the

intraerythrocytic malaria parasites. The reaction occurs in the acidic digestive
food vacuole, primarily during the trophozoite stage. Ferrous haem molecules
are released and in the presence of oxygen, rapidly oxidised to ferric haem -Fe
(III)PPIX (All)- with the generation of reactive oxygen species. All is
definitively detoxified by polymerisation into inert malaria pigment
(haemozoin). The reaction is reproducible in vitro in acidic conditions and
leads to the formation of ~-haematin (BH), a synthetic polymer, structurally
identical to the native haemozoin.
We show that accumulation of either AH or BH inside peritoneal mouse
macrophages (pMO) impair TNFo~ and nitric oxide (NO) production in
response to LPS. Such inhibition appears to be related to the haematin-induced
oxidative stress. In fact i) addition of sulphydryl group donors or GSH partially
restores pMO functions, ii) increased levels of lipid peroxidation and free
radicals production and loss of polyunsaturated fatty acids are observed.
Other phagocytes, e.g. microglia (MG) cells, maintain their capability to
synthesise eytokines a~er BH ingestion and are resistant to AH and BHinduced oxidative stress. The higher susceptibility of pMO compared to MG
cells is apparently related to the different GSH content and to the different
lipid composition of the cell membrane.
Further studies performed on arachidonic acid micelles and phospholipid
liposomes in different experimental conditions allow the following
conclusions: a) maximal prooxidant activity is exerted by AH when present at
concentrations <_2 ~tM (monomeric form). BH activity is lower than that of AH
monomers, but higher than that of AH aggregates. In contrast to All, BH
prooxidant activity is linearly related to its concentration and inhibited, rather
than enhanced, by chloroquine b) All as well BH-induced peroxidation is
promoted through the decomposition of pre-existing hydroperoxides c) free
radical reaction chain occurs within the lipophilic compartment, d) hydroxyl
radicals, superoxide anion, H202, as well as free iron possibly released from
the haem moiety, are not involved in the peroxidative reaction.

(We/19.2/476)

BLOOD
STAGE
MALARIA
INFECTION
INTERLEUKIN-I DEFICIENT MICE
R. S.-P. TAN~, Y. MATSUMOTO ~, A. U. KARA a

~l~lt~l Parasttot Lab. l)ept of BtoL Sct. Natzonatl lmv r f ,S'mgapore. ,S~gainwe.
~l)epl ofMo! lmmunol. ,~'h. of'AgncTdtureand Ltfe Sct. linty ofT?~k5,o"Japan

Malaria continues to be one of the most devastating infectious diseases known

to mankind. During the course of infection, parasitised erythrocytes stimulate
macrophages to secrete a host of cytokines that eventually induce nitric oxide
(NO) which can kill the parasite. NO is a short-lived biological mediator that
has been shown to be induced in various cell types and is able to cause many
metabolic changes in target cells. It has been shown that the bioactivity of
lnterleukin-I (IL-1) can be modulated by NO in murine macrophages.
However, the role of IL-I in mediating NO production has never been clearly
ascertained.
IL-1 was originally identified as an endogenous pyrogen, but was later shown
to display pleiotropic activities. IL-1 plays a key role in the defense response
to various infections by generating fever, activating lymphocytes and NO
dependent functions are regulated by the activation of the macrophage.
Recently, mice deficient in both the IL-I genes (IL-la and IL-113) were
produced by successive homologous recombination in an embryonic stem cell,
thus allowing for directly assessing the role of this cytokine in the immune
response In this study, we used these double knockout mice in blood stage
Phzsmo~hum herghet ANKA infection. We show here that the IL-1-/- mice
exhibit higher mortalities with an accelerated course of infection compared to
the wild type controls, "with pathological studies revealing gross dysfunction
in the visceral organs of the infected IL-I-/- mice. The IL-1-/- mice are
impaired in NO production with reduced iNOS expression in both the spleen
and liver upon infection. We demonstrate that IL-1, the major
proinflammalory cytokine is a regulator of the production of the free radical,
NO. The administration of the receptor antagonist IL-1Ra prevented the
release of NO from the macrophages obtained from the infected wild type
mice. However, the addinon of recombinant IL-I was able to reconstitute the
release of NO production in the macrophages from the IL-I-/- mice in a
concentration-dependent manner. Our results using double IL-I knockout
mice in a blood stage malaria infection provide the first experimental evidence
for a new activity of IL-I.

To investigate relationships between cell tropism, cytopathology and

clinical features for HIV-I isolates, 36 HIV-1 isolates were derived
from infected patients (29 - Moscow region (B serotype); 7 Svetlogorsk (Gomel region, Byelarus) outbreak (A/C serotype, A
genotype) among IVDUs) according to the standard procedure
based on co-cultivation of HIV-infected patient PBMC with
blast-stimulated (PHA/IL-2) donor PBMC. To estimate cell
tropism a set of HIV-permissive cell lines (H9, MT-4, CEM,
CEM SS, Sup T 1, THPI, U373-CD4-CXCR4, U373-CD4-CCR5)
and blast-stimulated donor PBMC were acutely infected, and virusinduced cytopathology process, syncytium-induced activity and p24
supematant concentration were monitored during 24 days with the 4
day intervals.
Comparative analysis of the rate and the level of virus
reproduction reveals the existence of rapid/low, slow/high and a
set of spacing HIV-1 variants in addition to accepted rapid/high
and slow/low phenotypes. Two types of HIV-induced
cytopathological forms were detected: closely packed syncytia
with high optical density and large transparent balloons
(simplasts). Rapid/high isolates were recognized to be simplastinducing (Sml); slow/low - both syncytium-inducing (SI) and
Sml; in the consequence rapid/low-spacing-slow/high - only SI
with monotonic increasing of virus reproduction level being
observed. All isolates from Svetlogorsk outbreak were
established to be phenotypically similar being slow/low and bot
non-SI and non-Sml.HIV-1 phenotype taxonomy seems to be
more complicated. Our results confirm the hypothesis about the
existence of single epidemic source in Svetlogorsk outbreak
which was proposed previously on the base of serological and
genetic data.

(We/19.2/477)

Slructuralcharacterization of
Saccharomyces cerevisiae priori-like protein Ure2

C. Thual*, A. Komar", L. Bousset*, E. Fernandez-Bellot +.

C. Cullin~and R. Melki*
*LEBSand ~CGM.CNRS.qt1198Gif-sur-Yvettecedex. France

Sacchromyces cerevisiae prion-like protein Ure2 [I] was

expressed in E. coli and purified to homogeneity. We show in this
study that Ure2p is a soluble protein that can assemble into fibers that
are similar to those observed in the case of PrP in its Scrapie Priori
Filaments (SPF) form [2] and that can bind Congo Red. Ure2p self
assembly is a cooperative process where one can distinguish a lag
phase followed by an elongation phase preceding a plateau.
A combination of size exclusion chromatography, sedimentation
velocity and electron microscopy demonstrate that the soluble form of
Ure2p consists of at least three lbrms (monomeric, dimeric and
tetrameric) whose abundance is concentration dependent.
By the use of limited proteolysis, intrinsic fluorescence and
circular dichroism measurements we bring strong evidences for the
existence of at least two structural domains in Ure2p molecules : Ure2p
NH2-terminal region is found poorly structured while its COOHterminal domain appears to be compactly folded.
Finally. we show that the Ure2p assembly into insoluble high
molecular weight oligomers involves slight conformational changes
that affect essentially the COOH-terminal part of the molecule. Our
finding is in favor of a mechanism of assembly similar among all
prions,

[ 1] Wickner et al., Armu. Rev. Genet., 30, (1996) 109.

[2] Dearmont et a/.,Cell, 41, (1985) 221.

s401

(We/19.2/478)

Abstracts FEBS'99

PFGE for epidemiologic analysis of bacterial

N.Vavatsi 1, E.Kaitsa 2, P.Kazila 2 , O. Manou 2

strains

LDept. of Biological Chemistry, School of Medicine, Aristotle

University of Thessaloniki, 2. "Theagenion" Cancer Hospital of
Thessaloniki, Greece.

Molecular typing of bacterial strains is currently used for the

epidemiologic study of an infection, as more accurate and specific, than
methods based on the phenotypic characteristics of bacteria. Molecular
methods analyse the bacterial DNA and among them PFGE (Pulsed Field
Gel Electrophoresis) is preferred as simple and reliable[l]. PFGE is an
agarose gel electrophoresis in which the electric field alternates at a constant
angle to the direction of migration and makes possible the separation of long
DNA molecules, after restriction enzyme digestion. In order to avoid
sheafing of DNA molecules during isolation, bacterial chromosomal DNA is
prepared by in situ lysis of bacteria embedded in agarose and subsequent
digestion with restriction enzymes, that have infrequent recognition sites.
Thus total genomic DNA can be resolved into a limited number of fragments
with distinct electrophoretic mobility.
We have used PFGE to type 34 methicillin resistant Staphylococcus
aureus (MRSA) strains isolated during a year from cancer patients with
infection, at the Cancer Hospital of Thessaloniki. The purpose of the study
was to find out whether these M R S A strains with similar biotype and
susceptibility tests had the same genetic origin. The bacteria from each strain
were embedded in inCert agarose 1.5%, lysed with lysostaphin, and their
DNA digested with restriction enzyme Sinai. PFGE was perfomed in 1%
SeaKeam agarose, TBE buffer, 200 Volts, 13oc, 120 angle, initial switch 2",
final switch 45" for 24 hours in a Rotaphor V (Biometra) apparatus. Lambda
ladder DNA concatemers were used as molecular weight markers. The gel
was stained with ethidium bromide and photographed under UV illumination.
PFGE typing showed two groups of MRSA strains (30/34 and 4/34) with
common clonal origin [2] and these results suggest that most of the infections
from M R S A were endonosocomial infections caused by the same genetic
clone of the bacterial strains.
[1] Arbeit R, Manual of Clin Microbiol, 1995, p 190
[2] Tenover F, et al., J. Clin. Microbiol. 33 ; 1995, p. 2233

(We/19.2/480)

Role of Rho proteins in control of the actin cytoskeleton and

VE-cadherin localization in HUVEC
v. Vouret-Cravtan,P. Boquet*.D. Grail.J. Poayssegur. and E. Van
Obberghen-Schilling. Centre de Biochimie. CNRS UMR 6543 a,d
*INSERM U452. Nwe. France

Integrity of the vascular endothelium is largely dependent on endothelial

cell shape and establishment of intercellular junctions. We have analyzed
the involvement of p21 Rho family proteins in the contol of the actin
cytoskeleton and monolayer integrity of endothelial cells using pathogenic
bacterial toxins. We report here that Escherishia coli cytotoxic necrotizing
factor 1 (CNF1) activates Rho in human umbilical vein endothelial cells
(HUVEC). In confluent monolayers, CNFI treatment induces prominent
stress fiber formation without significantly modifying peripheral
localization of VE-cadherin, a specific marker of vascular endothehal cell
adherens junctions. Further, Rho activation with CNF1 blocks thrombininduced redistribution of VE-cadherin staining and gap formation in
HUVEC monolayers. Rho inhibition by prolonged treatment of cells with
C3 exoenzyme (Clostridium botulinum), eliminates actin stress fibers
without disrupting the continuity of VE-cadherin staining, indicating that
Rho-dependent stress fibers are not required for maintaining this adhesion
receptor at sites of intercellular contact.

Lethal toxin (Clostridium

sordellii), an inhibitor of Rac as well as Ras and Rap, potently disrupts the

actin microfilament system and monolayer integrity in HUVEC cultures.

Downstream signaling events of p21GTPases will be discussed.

(We/19.2/479)

Baculovirus-host interaction: study of the hr viral

sequences
R. Vincent, M.-H. Ogliastro, M. Cerrutti, G. Devauchelle
Station de Pathologic Compar~e INRA]CNRS URA2209, 30380 StChrislol-lez~Alds, Franre

Baculoviruses, which are lepidopteran viruses, have dynamic genomes that are
capable of rearrangements and recombination during propagation in their hosts.
Nonhomologous recombination with sequences originating from the host
genome occurs under normal propagation conditions. This unique and
particular feature among eukaryotic viruses will offer an advantageous model to
study the molecular mechanisms of the virus-host interaction. Among those
viral sequences which should have derived from the host, the hr (homologous
region) sequences are particularly interesting. Indeed, they should be
implicated in the viral replication as well as in viral enhancer activities. The hr
sequences contain potential sites for the binding of host factors. Thus, in order
to study the part of the host in the viral cycle, we searched for specific
interactions between the hr sequences and host-cell factors. Electrophoretic
Mobility Shift Assay experiments were done with nuclear extracts from various
lepidopteran cell lines. Comparisons were done between infected (AcMNPV
baculovirus) and non-infected cells. Our results suggest specific interactions
between host factors and the hr sequences. The role of these binding in the
baculovirus cycle needs to be further determined.