Chapter

3-I – Protein Structure and Function

GBME, SKKU
Molecular & Cell Biology

H.F.K.
Chapter 3 – Protein Structure and Function
 Chapter 3 - Protein Structure and Function

3.1 Hierarchical Structure of Proteins

3.2 Protein Folding
3.3 Protein Binding and Enzyme Catalysis
3.4 Regulating Protein Function
3.5 Purifying, Detecting, and Characterizing Proteins
3.6 Proteomics
 Protein?
• One of the final products in cell
• Various functions
1. Structure
 Protein Structure and Function
3.1 Hierarchical Structure of Proteins
• Protein sequence specifies folding into secondary and
tertiary structures that either are functional units or
can interact with other peptides to form quaternary
structure functional units.
• Homologous proteins evolved from a common
ancestor, have similar sequences, structures, and
functions, and can be classified into families and
superfamilies.
Four levels of protein hierarchy.
• (a) Primary structure – linear sequence of amino acids
linked together by peptide bonds
• (b) Secondary structure – Folding of the polypeptide
chain into local α helices or β sheets
• (c) Tertiary structure
• structure of a peptide composed of secondary
structural elements and various loops and turns
• may form distinct, independently stable domains
• (d) Quaternary structure – some functional proteins are
composed of more than one polypeptide
 Primary structure
Beta
Alpha

Structure of a polypeptide.
• Proteins – unbranched polymers constructed
out of 20 amino acids with different R group
side chains.
• Amino acid side chains determine the
distinct properties of individual proteins.
 Secondary structure
The ! helix, a common secondary structure in
proteins.
• Secondary structures – stable spatial
arrangements of polypeptide chain segments
held together by hydrogen bonds between
backbone amide and carbonyl groups
• ! helix:
• Polypeptide backbone (ribbon) folds into a
spiral/helix with 3.6 amino acids per turn
(0.54 nm).
• Helix is stabilized by hydrogen bonds
between backbone oxygen and hydrogen
atoms (more bonds-more stable).
• R groups project outward from the surface
of the helix – determine chemical nature
of helix faces.
• Prolines – can’t participate in hydrogen
bonding and usually are excluded from an
! helix. Do you remember the proline structure?
Please check it!
 Alpha helix

https://www.youtube.com/watch?v=eUS6CEn4GSA
 Secondary structure

The " sheet, another common secondary

structure in proteins.
• β sheet – laterally packed β strands, each of
which is a nearly fully extended polypeptide
segment
• (a) Three-stranded β sheet – antiparallel β
strands with connecting loops (top view):
• Stabilized by hydrogen bonds between
backbone oxygen and hydrogen atoms
in amino acids on different strands
• (b) Antiparallel β sheet (side view):
• ! carbon bond angles produce a
pleated polypeptide backbone contour.
• Alternate R groups project above and
below the plane of the sheet.
• (c) Parallel β strand sheet: same N-to-C
strand orientations with connecting loops.
Beta sheet

https://www.youtube.com/watch?v=wM2LWCTWlrE
 Secondary structure

Structure of a " turn.

• Composed of four residues.
• Reverses direction of a
polypeptide chain (180° U-turn).
• Cα carbons of the first and fourth
residues are usually less than 0.7
nm apart and linked by a
hydrogen bond.
• β turns facilitate the folding of
long polypeptides into compact
structures.
• Glycine (smallest R group) and
proline (built in bend) are
commonly found in β turns.
Think about the reason in discussion!
Protein folding?
 Three models of protein folding
• Framework model
• Nucleation model
• Hydrophobic-collapse model
No single mechanism…

https://www.youtube.com/watch?v=B275XtegrJ4
Easy example…

The oil drop model of protein folding.

• Tertiary structure – stabilized
primarily by hydrophobic interactions
between nonpolar side chains and
hydrogen bonds involving polar side
chains and backbone amino and
carboxyl groups
• Hydrophobic residues (blue): cluster
together like drops of oil in the folded
protein core, driven away from the
aqueous surroundings by the
hydrophobic effect.
• Charged and uncharged polar side
chains (yellow): form stabilizing
interactions with surrounding water
and ions on the protein surface.
Four ways to visualize protein structure.
• Ras, a monomeric (single polypeptide chain) protein that binds to guanosine diphosphate
(GDP, in blue).
• (a) Cα backbone trace – depicts how the polypeptide is tightly packed into a small volume
• (b) Ball-and-stick representation – reveals locations of all atoms
• (c) Ribbon diagram – emphasizes how β strands (light blue) and α helices (red) are organized
in the protein
• (d) Water-accessible surface model – reveals protein surface topology with positive charge
(purple) and negative charge regions (red)
 How to find?
• http://www.uniprot.org/
Find structure of CREB!
 Similar functions by similar structures!!!

• Structural motifs:
• Regular combinations of secondary structures usually
with a specific type of function
• Can be encoded by a highly conserved sequence motif
 Motifs of protein secondary structure.

• present in many
• A type of helix- DNA-binding
loop-helix motif in proteins that help
many proteins, regulate
including many transcription
• Two α helices wound calcium-binding
around each other and DNA-binding
regulatory proteins
 The Alpha-Helical Secondary
Structure of Myoglobin (Mb)
• These helical segments are labeled A-H and
span the following residues of this 153 amino
acid residue polypeptide chain of Mb.
Segments (residues): A (3-18), B (20-
35), C (36-42), D (51-57), E (58-76), F (83-
95), G (100-118), & H (124-149).

https://biosci.mcdb.ucsb.edu/biochemistry/tw-prt/myoglobin/alphahelixf.htm
 Zinc finger domain

https://www.youtube.com/watch?v=WyU2v7HT6bw
Leucin zipper domain

https://www.youtube.com/watch?v=2-qFLfVymnw
Tertiary and quaternary levels
of structure.
Tertiary and
quaternary
levels of
structure.

Protein quaternary structure is the

number and arrangement of multiple
The tertiary structure will have a single folded protein subunits
polypeptide chain "backbone" with one or
more protein secondary structures, the
protein domains.
 Modular nature of protein domains.

• Epidermal growth factor (EGF) precursor: generated by proteolytic

cleavage generates multiple EGFs (green)
• Neu: EGF domain plus other domains
• Tissue plasminogen activator (TPA): EGF domain plus other domains
Several proteins work together in Cell!
A molecular machine:
the transcription
initiation complex.

Supramolecular complexes
Molecular evolution?
 Evolution of the globin protein family.

Myoglobin (symbol Mb or MB) is an iron- Leghemoglobin (also leghaemoglobin or

and oxygen-binding protein found in legoglobin) is a nitrogen or oxygen carrier and
the muscle tissue of vertebrates in hemoprotein found in the nitrogen-fixing root
general and in almost all mammals. nodules of leguminous plants.
 Protein Structure and Function
• 3.2 Protein Folding
• Protein amino acid sequence determines its 3D
structure and function.
• ATP-dependent molecular chaperones and
chaperonins assist protein folding in vivo.
• Misfolded/denatured proteins can form well-
organized amyloid fibril aggregates that can cause
diseases, including Alzheimer’s disease and
Parkinson’s disease.
 Think the protein folding!

What’s the important physical movement

for the folding?
Rotation!
Rotation between planar
peptide groups in proteins.

Polypeptide backbone
steric restraints and
amino acid side chain
properties severely
restrict Cα–amino nitrogen
bond (the Φ angle) and
Cα–carbonyl carbon bond
(the Ψ angle) rotations.
 What are the trans and cis?

limit the shapes into which proteins can fold.

• Planar peptide bonds

Which R group of amino acid has the tras- and cis-forms?

 Proline cis/trans isomerizations
influence protein folding and
structure.

• Cis/trans isomerization of a single

proline:
• Alters structure of a protein SH2
domain.
• Can influence a protein’s activity.
• Proline isomerases may act as
switches that regulate protein
activity.
Hypothetical protein-folding pathway

primary (a) → secondary (b–d)

→ tertiary (e) structure
 But,
the folding is not always
‘easy-going’ process
Molecular chaperone–mediated protein folding.
Chaperonin-mediated protein folding.
It’s a protein chamber…
Chaperone and Chaperonin

https://www.youtube.com/watch?v=d1QIEQEyYRo
Misfolded proteins
can form ordered
amyloid aggregates
based on a cross-"
sheet structure.
 Protein Structure and Function
3.3 Protein Binding and Enzyme Catalysis
• Protein function depends on binding other molecules
(ligands).
• Enzymes accelerate rates of cellular reactions by
lowering activation energy and stabilizing transition-
state intermediates.
• Enzymes often use acid-base catalysis mediated by
one or more amino acid side chains.
• Metabolic pathway enzymes may be associated as
domains of a monomeric protein, subunits of a
multimeric protein, or components of a protein
complex assembled on a common scaffold.
Protein-ligand binding of antibodies.
• Ligand: molecule to which a protein binds
• (a) IgG antibody (ribbon model): two identical heavy chains (light and dark red) and two
identical light chains (blue) covalently linked by disulfide bonds
• CDR (complementarity-determining regions): six highly variable loops form the antigen-
binding sites
• (b) Hand-in-glove fit: molecular complementarity between the antibody CDR and the site to
which it binds (epitope) on its target antigen
Remind the role of enzyme
Active site of the enzyme trypsin.
• Enzymes (proteins or RNAs) catalyze making or breaking substrate covalent bonds.
• (a) Trypsin (serine protease) active site:
• Substrate-binding pocket – binds specific substrate
• Catalytic site – contains side chains of the catalytic triad Ser-195, Asp-102, and His-57 that
breaks peptide bonds.
• In some enzymes, the catalytic and substrate-binding sites overlap; in others, the two regions are
structurally distinct.
Schematic model of an enzyme’s
reaction mechanism.
 Next class…
• Real enzyme action!
• Structural change by ligand binding & tech.
The Role of ApLLP in Synaptic Plasticity
713

• Practical protein works learned by text (?!?!)

What’s this?

Kim et al., Neuron 2006

 Discussion with friends
• Think about what cause misfolding of proteins.
• Then discuss how the chaperone and
chaperonin help the proper folding of proteins
(mechanisms).
• Practically protein misfolding can be a big
problem in protein production in basic
research field and industry. How can you solve
this misfolding problem? Please find plausible
ways except the chaperone and chaperonin.
 Further questions
• How do the chaperon and chaperonin
recognize the misfolding proteins?
• Can the human chaperon be used in other
animals such as rat and mouse?
 Chaperonin
• GroEL belongs to the chaperonin family of molecular
chaperones, and is found in a large number of bacteria.
To function properly, GroEL requires the lid-like
cochaperonin protein complex GroES.
In eukaryotes the proteins Hsp60 and Hsp10 are
structurally and functionally nearly identical to GroEL
and GroES, respectively.
• Unfolded substrate proteins bind to a hydrophobic
binding patch on the interior rim of the open cavity of
GroEL, forming a binary complex with the chaperonin.
 Conformational specificity of the chaperonin
GroEL for the compact folding intermediates of
a-lactalbumin
GroEL binding
Manajit K.Hayer-Hartl, for bacteriophage capsid assembly (Coppo et al., 1973;
Jonathan J.Ewbankl'2, Thomas E.Creighton1 Georgopoulos et al., 1973). It interacts with newly synthe-
and FUirich Harti sized polypeptides in the bacterial cytosol and is required
vol.13 no.13 pp.3192-3202, 1994
for their folding (Bochkareva et al., 1988; Goloubinoff
The EMBO JournalHoward Hughes Medical Institute, Cellular Biochemistry and et al., 1989a; Horwich et al., 1993). The GroEL homo-
Biophysics Program, Memorial Sloan-Kettering Cancer Center, 1275 logues in chloroplasts and mitochondria are involved in
York Avenue, New York, NY 10021, USA and 'European Molecular
Conformational specificity of the chaperonin
Biology Laboratory, Postfach 10.2209, Meyerhofstrasse 1, 69012
Heidelberg, Germany
protein
ive
folding and assembly reactions within the respect-
organelles (Barraclough and Ellis, 1980; Cheng et al.,
GroEL for the folding intermediates of
compact
2Present address: Department of Biology, McGill University, 1205 Dr
Penfield Avenue, Montreal, PQ, Canada H3A lBI
1989; Ostermann et al., 1989).
The basic function of the Hsp6Os is to assist the folding
a-lactalbumin Communicated by W.Neupert of monomeric polypeptide chains (Ostermann et al., 1989;
Laminet et al., 1990; Martin et al., 1991; Viitanen et al.,
The chaperonin GroEL binds unfolded polypeptides, 1991; Zheng et al., 1993). Hsp6O binds a partially folded
preventing aggregation, and then mediates their folding polypeptide, preventing aggregation (Buchner et al., 1991),
in an ATP-dependent process. To understand the struc- and releases it in an ATP-dependent reaction that can
Manajit K.Hayer-Hartl,
tural features in non-native polypeptides for
recognized bacteriophage
result capsid
in folding assembly (Coppoetetal.,al.,1989b;
(Goloubinoff 1973;Martin et al.,
Jonathan byJ.Ewbankl'2,
GroEL, we have Thomas E.Creighton1 (aLA) as a
used a-lactalbumin Georgopoulos et al., 1973). It interacts with newly
1991, 1993a). This process depends on the co-chaperonin synthe-
and FUirich model substrate. aLA (14.2 kDa) is stabilized bysized
Harti fourpolypeptides
HsplO,in GroES the bacterial cytosola and
in E.coli, singleis required
heptameric ring of
disulfide bonds and a bound Ca2+ ion, for
offering their
the folding
-10 (Bochkareva
kDa subunits et al.,
that 1988;
forms Goloubinoff
a complex with GroEL
Howard Hughes Medical Institute, Cellular Biochemistry and
possibility of trapping partially folded disulfide al., 1989a;(Chandrasekhar
etinter- Horwich et al.,et 1993). al., TheViitanen
1986; GroEL homo- et al., 1990; Saibil
Biophysics Program, Memorial Sloan-Kettering Cancer Center, 1275 mitochondria are involved
mediates
York Avenue, New York, NYbetween
10021, USAtheandnative andMolecular
'European
logues in chloroplasts
the fully unfolded et al., 1991; andLanger et al., 1992a; Landryinet al., 1993).
Biology Laboratory, The conformers
state.Postfach of aLA 1,with
10.2209, Meyerhofstrasse 69012high affinity protein
for foldingGroES regulates reactions
and assembly the ATPase within the respect-
activity of the GroEL sub-
Heidelberg, Germany
GroEL are compact, containing up to three disulfide ive organelles (Barraclough
units, coordinating and Ellis,
their 1980;
action Cheng
to allow et al.,
the release of
2Present address: Department ofhave
bonds, and Biology, McGill University, 1205 Dr
significant secondary 1989;
structure, but Ostermann
bound et al., 1989).
polypeptide in a manner productive for folding
Penfield Avenue, Montreal, PQ, Canada H3A lBI The basic function the Hsp6Os
lack stable tertiary structure and expose hydrophobic (Gray andofFersht, 1991;isBochkareva
to assist theetfoldingal., 1992; Jackson
Communicated by W.Neupert
surfaces. Complex formation requires almost theLaminet monomericet polypeptide
ofcom- al., 1993; Martinchains (Ostermann
et al., 1993a,b; et al.,Todd
1989;et al., 1993).
plete aLA sequenceunfolded and is strongly dependent on salts et al., 1990; Martin et al., 1991;
During folding, the polypeptide is maintained Viitanen et al., in a shielded
The chaperonin GroEL binds
that stabilize hydrophobic interactions. polypeptides, 1991;
Unfolding ofZheng et al., 1993). Hsp6O binds a partially
environment (Martin et al., 1991), apparently provided by folded
preventing aggregation,
aLA to an and then mediates
extended state astheir wellfolding
as the burialpolypeptide,
of the central aggregation
preventing cavity of the (Buchner
Hsp6O al., 1991),
etcylinder (Langer et al.
in an ATP-dependent
hydrophobic process. To understand the struc-
surface upon formation of ordered and releases it
1992a; in an ATP-dependent
Braig reaction
et al., 1993; Saibil et al., that 1993).
can
tural features in non-native
tertiary polypeptides
structure prevent recognized
the binding Inter-in foldingWhile
to GroEL.result (Goloubinoff al., 1989b;
there haset been Martin progress
considerable et al., in under-
by GroEL, estingly,
we haveGroEL used a-lactalbumin (aLA)
interacts only with a specific as a 1991,
subset of 1993a). This process depends on the co-chaperonin
standing the mechanistic principles of Hsp6O-mediated
model substrate.
the many (14.2 kDa)folded
aLA partially disulfideby intermediates
is stabilized four HsplO, of GroES folding,in E.coli, a singlefeatures
the structural heptameric ring ofby the chap-
recognized
disulfide bonds and a bound
aLA and thus may influence Ca2+ ion, offering the -10
in vitro the kinetics of kDa subunits that forms a
eronins in unfolded polypeptides arecomplex with GroELstill undefined.
possibility ofthetrapping partially
folding pathways folded disulfide inter-
that lead to disulfide bonds with (Chandrasekhar et al., 1986; Viitanen et al.,
Although GroEL binds an amphiphilic N-terminal 1990; Saibil peptide
mediates between the native andWethe
native combinations. fully unfolded
conclude et al., 1991; ofLanger
that the chaperonin rhodaneseet al.,with
1992a;lowLandry
affinity, al., 1993). it in an a-
et stabilizing
state. The conformers
interacts with of aLA with high affinity
the hydrophobic surfacesfor exposed
GroES by regulates ATPase activity
helicaltheconformation (Landryof the GroEL
et al., 1992),sub-the chaperonin
compact,incontaining
GroEL are proteins up to three disulfide
a flexible compact intermediate or molten units, coordinating their action
also interacts with the denatured state of anof all-5 protein
to allow the release
have significant
bonds, and globule state. secondary structure, but bound polypeptide
(Schmidtin and Buchner,productive
a manner 1992). Itfor folding
is not known whether
lack stable tertiary structure and expose hydrophobic
Key words: chaperonin/GroEL/a-lactalbumin/molten (Gray and Fersht, 1991; Bochkareva et al., 1992;
GroEL recognizes primarily contiguous sequence elements Jackson
surfaces. Complex formation
globule/protein requires almost the com-
folding et al., 1993; orMartin et al., 1993a,b;
hydrophobic surfaces Todd typically 1993). by partially
et al.,exposed
plete aLA sequence and is strongly dependent on salts During folding, the polypeptide is maintained in
folded polypeptides. It has been suggested that GroEL a shielded
 GroEL binding
J. Biochem. 124, 319-325 (1998)

The Region of ƒ¿-Lactalbumin Recognized by GroEL

Akio Shimizu,' Takahiro Tanba, Isamu Ogata, Masamichi Ikeguchi, and Shintaro Sugai
Department of Bioengineering, Faculty of Engineering, Soka University, 1-236 Tangi-cho, Hachioji,
Tokyo 192-8577

Received for publication, February 17, 1998

The binding constants between disulfide-intact or various disulfide-reduced bovine ƒ¿-

lactalbumins and an Escherichia coli chaperonin, GroEL, were determined by using the
equilibrium dialysis method. The disulfide-intact and one-disulfide (Cys6-Cys120)-re
duced ƒ¿-lactalbumins were shown not to bind with GroEL both in the presence and absence
of Call. The two-disulfide (Cys6-Cys120 and Cys28-Cyslll)-reduced ƒ¿-lactalbumin, which
has the native-like tertiary structure in its ƒÀ-domain region and an unfolded a-domain in
the presence of Call, showed considerable binding with GroEL. The binding free energy of
the two-disulfide-reduced ƒ¿-lactalbumin in the presence of Call is close to that of the
molten globule state of disulfide-intact ƒ¿-lactalbumin. This result suggests that GroEL
binds to the unfolded a-domain of ƒ¿-lactalbumin regardless of the conformation of the

,ƒÀ-domain. The fully disulfide-reduced and two-disulfide-reduced ƒ¿-lactalbumins were

found to bind more strongly with GroEL in the absence of Ca" than the two-disulfide-
reduced ƒ¿-lactalbumin in the presence of Ca", thus indicating that the unfolding of the
ƒÀ-domain of ƒ¿-lactalbumin leads to stronger interaction with GroEL.

Key words: equilibrium dialysis, GroEL, ƒ¿-lactalbumin, binding constant, binding site.

The Escherichia coli chaperonin GroEL is one of the most

can assume various conformations, to GroEL.
 HSP binding
THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

Roles of Cytosolic Hsp70 and Hsp40 Molecular Chaperones in

Vol. 278, No. 9, Issue of February 28, pp. 7034 –7042, 2003
Printed in U.S.A.

Post-translational Translocation of Presecretory Proteins into the

THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 278, No. 9, Issue of February 28, pp. 7034 –7042, 2003
Printed in U.S.A.

Endoplasmic Reticulum*
Roles of Cytosolic Hsp70 and Hsp40 Molecular Chaperones in
Post-translational Translocation of Presecretory Proteins into the Received for publication, October 15, 2002
Endoplasmic Reticulum* Published, JBC Papers in Press, December 19, 2002, DOI 10.1074/jbc.M210544200

Received for publication, October 15, 2002

Jantra Published,
Ngosuwan, Nancy
JBC Papers in Press,M. Wang,
December Katie
19, 2002, L. Fung, and William J. Chirico‡
DOI 10.1074/jbc.M210544200

From
Jantra Ngosuwan, Nancy M.the Department
Wang, of Anatomy
Katie L. Fung, and J.
and William Cell Biology,
Chirico‡ State University of New York Downstate Medical Center,
Brooklyn, New York 11203
From the Department of Anatomy and Cell Biology, State University of New York Downstate Medical Center,
Brooklyn, New York 11203

Hsp70 molecular chaperones Hsp70 molecular chaperones

and their co-chaperones boundand their
form, co-chaperones
peptide substrates undergo cycles bound
of rapidform,
bindingpeptide substrates undergo cycles of rapid binding
work together in work various together in various
cellular compartments to cellular
and release fromcompartments to and
Hsp70 (3). In the ADP-bound form,release
the inter- from Hsp70 (3). In the ADP-bound form, the inter-
guide the folding of proteins and to aid the translocation actions are slower resulting in higher affinity. Hsp70s stimu-
guide the folding of proteins and to aid the translocation actions are slower resulting in higher affinity. Hsp70s stimu-

Downloaded from http://www.jbc.org/ at SUNG KYUN KWAN UNIVERSITY on March 20, 2017
of proteins across membranes. Hsp70s stimulate protein late protein folding by binding to exposed hydrophobic se-
folding by binding ofexposed
proteins across membranes.
hydrophobic sequences quences Hsp70s(4 – 6)stimulate
and preventing protein late aggregation
their irreversible protein folding by binding to exposed hydrophobic se-
thereby preventing folding
irreversible by aggregation.
binding exposed Hsp40s (7). hydrophobic
The activity of Hsp70s sequences quences
is regulated by Hsp40s and (4 – 6) and preventing their irreversible aggregation
other
stimulate the ATPase activity of Hsp70s and target un- co-chaperones. Hsp40s stimulate the ATPase activity of
thereby preventing irreversible aggregation.
folded proteins to Hsp70s. Genetic and biochemical evi- Hsp70s (8) and can target unfolded proteins Hsp40s (7). toThe activity of Hsp70s is regulated by Hsp40s and other
them (7).
dence supports a role stimulate
for cytosolic the
Hsp70sATPaseand Hsp40sactivity
in of Hsp70s and target un- co-chaperones.
Hsp40s preferentially bind hydrophobic polypeptides (9) and Hsp40s stimulate the ATPase activity of
the post-translational foldedtranslocation
proteins of toprecursor
Hsp70s. pro-Genetic and biochemical evi-
can prevent the aggregation of some unfolded proteins (7,
Hsp70s (8)10).and can target unfolded proteins to them (7).
teins into endoplasmic reticulum and mitochondria. To
gain mechanistic insight, dence we supports
measureda the role for cytosolic
effects of
GeneticHsp70s and Hsp40s
and biochemical in
evidence supports a role for cytoso-
Hsp40s
lic Hsp70s and Hsp40s in the post-translational translocation preferentially bind hydrophobic polypeptides (9) and
Saccharomyces cerevisiae the post-translational
Ssa1p (Hsp70) and Ydj1p translocation of precursor pro-
(Hsp40) on the translocation of histidine-tagged prepro- of precursor proteins into the endoplasmic reticulumcan prevent and mi-the aggregation of some unfolded proteins (7, 10).
teins into endoplasmic reticulum and mitochondria. To
!-factor (pp!F6H) into microsomes. Radiolabeled tochondria (reviewed in Ref. 11). Deshaies et al.Genetic (12) reported
and biochemical evidence supports a role for cytoso-
pp!F6H was affinity gain mechanistic
purified from wheat germ insight,
transla- wethatmeasured the effects
presecretory proteins of
and mitochondrial precursors accu-
Saccharomyces
tion reactions (or Escherichia coli) to removecerevisiae
endoge- Ssa1p
mulated in(Hsp70) and
the yeast cytosol when Ydj1p lic Hsp70s
the concentration of cytosolic and Hsp40s in the post-translational translocation
nous chaperones. We demonstrated that either Ssa1p or Hsp70s was lowered. A temperature-sensitive
(Hsp40) on the translocation of histidine-tagged prepro- of precursor
mutant of theproteins into the endoplasmic reticulum and mi-
Ydj1p stimulates post-translational translocation by yeast cytosolic Hsp70 Ssa1p rapidly accumulated presecretory
-factor (pp
preventing pp!F6H!aggregation. The ! F6H)and/or
binding intohy- microsomes. Radiolabeled
proteins at the nonpermissive
tochondria (reviewed in Ref. 11). Deshaies et al. (12) reported
temperature (13). Chirico et al.
drolysis of ATP by Ssa1p pp!F6H were wasrequired affinity
to maintainpurified
the (14)from
showedwheat germ
that yeast transla-
cytosolic that the
Hsp70s stimulated presecretory
in vitro proteins and mitochondrial precursors accu-
translocation competence of pp!F6H. To clarify the con- translocation of prepro-!-factor (pp!F) into yeast mulated in the yeast cytosol when the concentration of cytosolic
microsomes.
tion reactions (or Escherichia coli) to remove
tributions of membrane-bound and cytosolic Ydj1p, we Complexes containing Hsp70s and presecretory and mitochon- endoge-
compared the efficiency nousofchaperones.
chaperone-dependent We demonstrated thatproteins
trans- drial precursor either Ssa1p
have or Hsp70s
been identified
was lowered. A temperature-sensitive mutant of the
in wheat germ
location into wild-type Ydj1p and Ydj1p-deficient
stimulates microsomes. post-translational translocation
extracts and reticulocyte by Theyeast
lysates (15–17). ability cytosolic
to stimu- Hsp70 Ssa1p rapidly accumulated presecretory
Neither soluble nor membrane-bound Ydj1p was essen- late post-translational translocation is not shared by all
preventing
tial for post-translational proteinpp !F6H aggregation.
translocation. The The binding and/or hy- proteins at the nonpermissive temperature (13). Chirico et al.
Hsp70s or by other stress protein families. Neither Hsp60 (18),
ability of Ssa1p, Ydj1p, drolysis of ATP by
or both chaperones to Ssa1p were
restore the required to maintain the (14) showed
Kar2p (19), nor Hsp90 (18) stimulates translocation. However,that yeast cytosolic Hsp70s stimulated the in vitro
translocation competence of aggregated
translocation pp!F6H was
competence of pp !F6H. To clarify theforcon-
negligible. DnaK can partially substitute translocation
yeast cytosolic Hsp70s in of prepro-!-factor (pp!F) into yeast microsomes.
tributions of membrane-bound vitroand cytosolic Ydj1p, we Complexes containing Hsp70s and presecretory and mitochon-
(19, 20).
compared the efficiency of chaperone-dependent Several laboratories have exploredtrans-the role of DnaJ homologs
Hsp701 molecular chaperones in protein translocation. Caplan et al. (21)drial reportedprecursor
that a proteins have been identified in wheat germ
locationand into
theirwild-type
co-chaperonesand work Ydj1p-deficient
temperature-sensitive microsomes.
mutant of YDJ1 was extracts
defective for and
trans- reticulocyte lysates (15–17). The ability to stimu-
together in a variety of cellular compartments to guide the
Neither soluble nor membrane-bound Ydj1p was essen- late post-translational
folding of proteins and to aid the translocation of proteins location at the nonpermissive temperature. However, Atencio
translocation is not shared by all
across membranes (reviewedtial for post-translational
in Ref. 1). The binding and hy- protein and Yaffe (22)translocation.
and Becker et al. The (13) showed that pp!F is
translocated normally in deletion mutants of Hsp70s
YDJ1. or
Transloca-by other stress protein families. Neither Hsp60 (18),
drolysis of ATP regulate ability of Ssa1p,
the action of Hsp70s Ydj1p,
(2). In theor both
ATP- chaperones to restore the
tion of pp!F was defective in a strain expressing Kar2p
a mutant(19),formnor Hsp90 (18) stimulates translocation. However,
translocation competence ofof aggregated Ydj1p that cannot pp !F6H was
be farnesylated indicating that the lipid-
negligible.
* This work was supported by National Science Foundation Grant
DnaK can partially substitute for yeast cytosolic Hsp70s in
modified version of Ydj1p plays a role in protein translocation
MCB-9905988 and a grant from the American Heart Association (to (21). Hendrick et al. (23) showed that Escherichia vitro (19, 20).
coli DnaJ
W. J. C.). The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby
completely inhibited post-translational translocation Several of pplaboratories
!F have explored the role of DnaJ homologs
 The Journal of Neuroscience, March 15, 2003 • 23(6):2203–2211 • 2203

HSP70 overexpression
Heat Shock Protein 70 Chaperone Overexpression
Ameliorates Phenotypes of the Spinal and Bulbar Muscular
Atrophy Transgenic Mouse Model by Reducing Nuclear-
Localized Mutant Androgen Receptor Protein The Journal of Neuroscience, March 15, 2003 • 23(6):2203–2211 • 2203

Heat Shock Protein 70 Chaperone Overexpression

Hiroaki Adachi,1 Masahisa Katsuno,1 Makoto Minamiyama,1 Chen Sang,1 Gerassimos Pagoulatos,2
Charalampos Angelidis,2 Moriaki Kusakabe,3 Atsushi Yoshiki,4 Yasushi Kobayashi,1 Manabu Doyu,1 and Gen Sobue1
1Department ofAmeliorates Phenotypes
Neurology, Nagoya University of the65 Tsurumai-cho
Graduate School of Medicine, Spinal and Showa-ku,Bulbar Muscular
Nagoya 466-8550, Japan, Department of General 2

Biology, University of Ioannina, School of Medicine, Ioannina GR-45110, Greece, ANB Tsukuba Institute, ALOKA Company, Ltd., 1103 Fukaya,
3

Kasumigaura,Atrophy Transgenic
Niihari, Ibaraki 300-0134, Mouse
Japan, and Experimental
4 Model
Animal by Reducing
Division, Department Nuclear-
of Biological Systems, BioResource Center, The Institute

Localized Mutant Androgen Receptor Protein

of Physical and Chemical Research (RIKEN) Tsukuba Institute 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan

Spinal and bulbar

Hiroakimuscular
Adachi,1 atrophy
Masahisa(SBMA)
Katsuno, is1 an inherited
Makoto motor neuron
Minamiyama, 1 Chendisease caused by the
Sang,1 Gerassimos expansion
Pagoulatos, 2 of the polyglutamine (polyQ)

tract within Charalampos

the androgenAngelidis, Moriaki
receptor2 (AR). Kusakabe,
The Atsushi Yoshiki,
nuclear3 inclusions 4 Yasushi Kobayashi,1 Manabu Doyu,1 and Gen Sobue1
consisting of the mutant AR protein are characteristic and combine with
1Department of Neurology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho Showa-ku, Nagoya 466-8550, Japan, 2Department of General
many components of ubiquitin–proteasome and molecular chaperone pathways, raising the possibility that misfolding and altered
Biology, University of Ioannina, School of Medicine, Ioannina GR-45110, Greece, 3ANB Tsukuba Institute, ALOKA Company, Ltd., 1103 Fukaya,
degradationKasumigaura,
of mutantNiihari,
AR may be300-0134,
Ibaraki involved in and
Japan, the4Experimental
pathogenesis. AnimalWe haveDepartment
Division, reportedofthat the overexpression
Biological Systems, BioResourceof heatThe
Center, shock protein (HSP)
Institute
chaperones ofreduces mutant
Physical and ChemicalAR aggregation
Research and cell
(RIKEN) Tsukuba death
Institute 3-1-1in a neuronal
Koyadai, Tsukuba, cell model
Ibaraki (Kobayashi
305-0074, Japan et al., 2000). To determine whether
increasing the expression level of chaperone improves the phenotype in a mouse model, we cross-bred SBMA transgenic mice with mice
overexpressing the inducible form of human HSP70. We demonstrated that high expression of HSP70 markedly ameliorated the motor
function of Spinal and bulbar
the SBMA model muscular
mice.atrophy (SBMA) is an inherited
In double-transgenic mice,motor
theneuron disease causedmutant
nuclear-localized by the expansion of the polyglutamine
AR protein, particularly(polyQ)
that of the large
tract within the androgen receptor (AR). The nuclear inclusions consisting of the mutant AR protein are characteristic and combine with
complex form, was significantly reduced. Monomeric mutant AR was also reduced in amount by HSP70 overexpression, suggesting the
many components of ubiquitin–proteasome and molecular chaperone pathways, raising the possibility that misfolding and altered
enhanced degradation
degradation ofofmutant
mutant AR.beThese
AR may findings
involved suggest that
in the pathogenesis. WeHSP70 overexpression
have reported ameliorates
that the overexpression SBMA
of heat shockphenotypes
protein (HSP)in mice by
reducing nuclear-localized mutant AR, probably caused by enhanced mutant AR degradation. Our study
chaperones reduces mutant AR aggregation and cell death in a neuronal cell model (Kobayashi et al., 2000). To determine whether may provide the basis for the
developmentincreasing
of an HSP70-related
the expression leveltherapy for SBMA
of chaperone and other
improves polyQ in
the phenotype diseases.
a mouse model, we cross-bred SBMA transgenic mice with mice
overexpressing the inducible form of human HSP70. We demonstrated that high expression of HSP70 markedly ameliorated the motor
Key words: HSP70;
functionchaperone;
of the SBMA polyglutamine; SBMA; transgenic
model mice. In double-transgenic mice, mice; protein degradation
the nuclear-localized mutant AR protein, particularly that of the large
complex form, was significantly reduced. Monomeric mutant AR was also reduced in amount by HSP70 overexpression, suggesting the
enhanced degradation of mutant AR. These findings suggest that HSP70 overexpression ameliorates SBMA phenotypes in mice by
reducing nuclear-localized mutant AR, probably caused by enhanced mutant AR degradation. Our study may provide the basis for the
Introduction
development of an HSP70-related therapy for SBMA and other polyQ nuclei,
diseases.spinal motor neurons, and some visceral organs (Li et al.,
Human HSP70 expression in mouse
2208 • J. Neurosci., March 15, 2003 • 23(6):2203–2211 Adachi et al. • HSP70

Figure 4. HSP70 decreases nuclear-localized mutant AR in double-transgenic mice. Immunohistochemical study of the spinal anterior horn ( A–C) an
AR-97Q/HSP70 double-transgenic mice stained with a monoclonal antibody (1C2) against abnormally expanded polyQ (16 weeks old). AR-97Q/HSP70 (!
for 1C2 in the nucleus (A, D). AR-97Q/HSP70 (tg/!) (B, E), and AR-97Q/HSP70 (tg/tg) (C, F ) mice exhibit low levels of 1C2 staining in the nucleus. G, H, Quant
for 1C2 in the spinal ventral horn ( G) and muscle ( H ). Positively stained nuclei were estimated by counting in the thoracic spinal ventral horn and muscl