Daniel Finley Genetics| Vol 192| 1 October 2012

UBIQUITIN

•Human and yeast ubiquitin share 96%
sequence identity.

Cellular functions of the Ubiquitin-Proteasome
System
D.H. Wolf et al., 2004
Founding member of family of
structurally Conserved proteins.
Cecile M. pickart et al., 2004

Ubiquitinylation, targets proteins to the
Proteosome.




Daniel Finley Genetics| Vol 192| 1 October 2012
The ubiquitin pathway
Yihong Ye et al., 2009, Nature Reviews, Mol. Cell Bio

Ubiquitination
E1 E2
E3
Ubiquitin-activating
enzyme
Ubiquitin-conjugating
enzyme
Ubiquitin ligase
E2 : Ubiquitin Conjugation
Enzymes/ Ubiquitin Carrier Protein


E2
(30kd)
YEAST
Sacchromyces
Cerevisiae



HUMAN
ENCODING
GENES
13 38
TYPES
16-35 35
CONSERVED
DOMAIN
UBC (24.2 kd)
(150-200 a.a)
UBC
(150-200a.a)
Structure of E2
Yihong Ye et al., 2009, Nature Reviews, Mol. Cell Bio

The signature UBC fold shares approximately
35% homology amongst the E2s present in
eukaryotes.
H. T. Marc Timmers et al., 2010

Characterized by the presence of a highly
conserved Ubiquitin-Conjugating (UBC) Domain.

Sjoerd J. L. van Wijk et al., THE FASEB
JOURNAL 2010



Classification of E2
Structurally Functionally
1. Structurally…
Core
catalytic
domain
Class II
Class I
C-
terminal
extension
N-
terminal
extention
Class III
Class IV
N & C-
terminal
extention
H. T. Mark
Timmers
et al., THE FASEB
JOURNAL 2010

2. Functionally…
Monoubiquitination
Polyubiquitination
UBC Viable
a

Biological processes or unique features
Ubc1 +
Essential for the cell growth and
viability.

Ubc2/Rad6 +
DNA repair, N-end rule, H2B
monoubiquitylation.
Ubc3/Cdc34 −
Cell cycle, E2 for SCF ligases
Ubc4 +
Protein quality control outside the nucleus,
degradation of short-lived and
abnormal proteins
Ubc5 +
Comparable to Ubc4 but expression is
elevated in stationary phase
Ubc6 +
ERAD, has transmembrane region, can
synthesize K11-chains in vivo
Ubc7 +
ERAD
Ubc8 +
Regulation of gluconeogenesis
Ubc9
b

E2 for Smt3 (SUMO) conjugation
Ubc10/Pex4 +
Peroxisomal E2 important for peroxisome
biogenesis
Ubc11 +
Cytoplasmic localization
Ubc12
b
+
E2 for Rub1 (Nedd8) conjugation
Ubc13 +
DNA repair, dimerizes with Mms2 for
synthesis of K63 chains
Ubiquitin conjugating enzymes of Saccharomyces cerevisiae
Thomas Sommer et al., Genetics| Vol. 192| 26 October , 2012
Yeast Ubc1 homologue in Homo sapien
(E2-25K)/Hip 2/UBE-2K.

Neurological implications of E2-25K:
Plays a crucial factor in modulating Amyloid β
neurotoxicity in the pathogenesis of Alzheimer’s disease.
Sungmin Song et al., 2003


 Ubiquitin-conjugating enzyme E2-25K increases
aggregate formation and cell death in polyglutamine
Diseases.
Remko de Pril et al., 2007









Loss of UBC1 leads to slow growth,
hypersensitivity to canavanine &
defects in protein degradation.
UBC1 partially complements the
phenotype for ubc4ubc5 mutant cells
W.Seufert, J.P.McGrath and S.Jentsch, 1990


UBC1, UBC4 and UBC5 constitute a subfamily of
ubiquitin-conjugating enzymes required for
growth and viability suggests a vital function for
ubiquitin-dependent protein degradation in
eukaryotic cells.
W.Seufert, J.P.McGrath and S.Jentsch, 1990
UBC4
MSSSKRIAKELSDLERDPPTSCSAGPVGD
DLYHWQASIMGPADSPYAGGVFFLSIHFP
TDYPFKPPKISFTTKIYHPNINANGNICLDI
LKDQWSPALTLSKVLLSICSLLTDANPDDP
LVPEIAHIYKTDRPKYEATAREWTKKYAV

UBC 5
MSSSKRIAKELSDLGRDPPASCSAGPVGD
DLYHWQASIMGPSDSPYAGGVFFLSIHFPT
DYPFKPPKVNFTTKIYHPNINSSGNICLDIL
KDQWSPALTLSKVLLSICSLLTDANPDDPL
VPEIAQIYKTDKAKYEATAKEWTKKYAV

Kate E. Stoll et al., 2011
Closely related in sequence and
complementing in function


Comprise a major part of ubiquitin-
conjugation activity in stressed cells
Studies on UBC4 and UBC5
UBC4
148 residues

alpha/beta protein

four strands forming
an anti-parallel beta-
sheet bounded by four
alpha-helices


Catalytic
domain
Ubiquitin
interaction
site
E3 binding
site
Structure of Ubc4
Peter S. Brzovic et al., 2011
PREVIOUS WORK DONE

• Functional studies of c-UBC1
under various stress condition.
A

• Prediction of c-UBC1 structure
using bioinformatic tool
B
Swapping of yeast Ubc1 linker for mammalian E2-25K
RESULTS
0
20
40
60
80
100
120
4 hours 8 hours 12 hours 16 hours
UBC1
UBC1
induced
c- UBC1
c- UBC1
induced
ubc1
Heat stress (37 C)


%

s
u
r
v
i
v
a
l

Time
0
10
20
30
40
50
60
70
80
without pretreatment with pretreatmemt
wild type
c-UBC1
ubc1
wild type
induced
c-UBC1
induced
Thermotolerance Test














%

s
u
r
v
i
v
a
l

Canavanine Test

ubc1


UBC1



c-UBC1
Control With canavanine
FUNCTIONAL AND
STRUCTURAL
CHARACTERIZATION OF c-UBC1
AND CLONING OF N82S AND
E15G FOR UBC5
PRESENTER
TANVI KHANNA
GUIDE:
DR. C. RATNA PRABHA
MACROMOLECULAR
STRUCTURE AND BIOLOGY LAB
OBJECTIVES
1. Purification of c-UBC1

2.To check formation of Polyubiquitination
chain in c-UBC1 by western blot.
3. Cloning:
•Construction of N82S for UBC5 by site
directed mutagenesis.

•Construction of E15A for UBC5 by site
directed mutagenesis.



OBJECTIVE 1
Purification of c-UBC1
STRATEGY







Harvest the cells after 1mM IPTG induction (overnight)
Pellet down
8K/2min./ 4’C
Wash with lysis Buffer, Vortex, Spin- 3x





DNA Precipitation by polyethylene aniline





40% Ammonium sulphate precipitation
Sonication
Spin 12K/ 15min./4’C
Purified UBC1 is obtained



Spin 12K| 30min.|4’C
85% Ammonium sulphate precipitation
Spin 12K| 30min.|4’c
Discard supernatant, take pellet
Resuspend in 20mM Tris Buffer
Column purification and Dialysis
. OBJECTIVE 2
 WESTERN BLOT
Cut down the nitrocellulose paper according to the gel
Pre- wet the membrane in ethanol for 2min.
Soak them for 10min in Lx Transfer Buffer
Take 2pieces of precut filter paper
Wet and 2 reusable fibre pads in IX Transfer Buffer
Lay one pad on black plastic side of transfer cell and other on
its top, to avoid bubble
BLOTTING PROCEDURE
Place gels on top of filter paper in proper orientations
Add nitrocellulose membranes, bending in middle and
smoothing towards edges
Add last filter paper Wet, again roll from centre of the stack
towards the edges with a damp wedge tool
Place the cell into the cradle to match with black plastic side
Place the Ice Pack into the unit and filled with chill (4’C)
lX Transfer buffer on a small stir bar at the bottom
Transfer take 1hr at 100V/ 350m Amp. Transfer complete
separate the apparatus and filter forceps to handle the blot.
IMMUNODETECTION
Blots immediately rinse in DDH2O(1min) and block in TBS-T plus
blocking agent Skim milk, BSA etc, 1hour, RT
Standard (GADPH) or Primary Ab ( panERK etc), suspended in TBS-T
with blocking agent
Left on blots overnight 4’C with agitation or 2@RT with agitation
Wash membrane in TBS-T 3times, 10-20min. with agitation
Pour off wash buffer and replace with Secondary Ab in TBS-T. Incubate
one hour. Ab diluted (1: 10000)
Wash the membrane TBS-T in 3times 10-20min.
Construction of N82S for UBC5 by
site directed mutagenesis.

Construction of E15A for UBC5 by
site directed mutagenesis.
. OBJECTIVE 3
Mutation in Ubc4 by replacing
amino acid of Ubc5:

Glutamate (glu)
Glycine(gly)
Amino
acid
3 letter 1 letter Residue
location
Side
chain
polarity
Side
chain
acidity
or
basicity
of
neutral
species
Hydropathy
index
Aspargin
e
Asn N 82 Polar Neutral -3.5
Serine Ser S 82 Polar Neutral -0.8
Glutamat
e
Glu E 15 Polar Neutral -3.5
Glycine Gly G 15 Non-
polar
Neutral -0.4
Rationale
To understand if Ubc4 and Ubc5 have
any functions that are distinct from each
other.

If this point mutation can alter the
spatial geometry of the molecule.
Bgl II
Kpn I
pQE9
UBC4
yEP96
PCR
Bgl II Kpn I
Bgl II
Kpn I
Bgl II
Kpn I
yEP96
N82S
82 FR
82 RE
UBC4 FR
UBC4 RE
pQE9
UBC4

PCR
Bgl II
Kpn I
Bgl II
Kpn I
Bgl II Kpn I
yEP96
Kpn I
Bgl II
yEP96
E15G

20 FR
20 RE
UBC4 FR
UBC4 RE


THANK
YOU
The proteasome: a proteolytic nanomachine of
cell regulation and waste disposal


D.H. Wolf Biochimica et Biophysica Acta|(2004)
BACK UP
(UPS)
This system is unique in cellular regulation as—
in contrast to
phosphorylation/dephosphorylation of a
protein—
It allows a complete shutdown of function of a
selected protein molecule due to its irreversible
proteolysis.
A similar pathway has been identified recently in prokaryotes. In a
screen for interacting proteins of the proteosomal ATPase
in Mycobacterium tuberculosum (Mtb), Pearce et al. identified a
6.9-kDa Pup (prokaryotic ubiquitin-like protein) protein. Pup
becomes activated in a manner involving deamidation by Dop,
followed by covalent linkage to lysine residues of acceptor
substrates [such as the proteasomal subunit malonyl co-A acyl
carrier protein (FabD)] and targeting them for proteasomal
degradation. Proteasome-associated factor A (PafA) has been
implicated in the conjugation , since pafA Mtb strains are absent in
Pup-FabD conjugates and the pool of unmodified FabD is
stabilized. It is tempting to speculate about E2-like activities in
prokaryotes resembling eukaryotic E2s. However, no enzymes
bearing the UBC fold have been identified in bacteria . Since PafA
combines E2- and E3-like features in Mycobacteria, their features
have possibly become separated during eukaryotic evolution.
how UBC-fold architecture relates to protein function.
UBC fold can be divided into three shells of amino acids. The first shell consists of the
heart of the enzyme and is formed by the catalytic cysteine and conserved residues
surrounding that, directly involved in UBL conjugation. Residues in the first shell are
solvent accessible to accommodate the activated Ub/UBL. The second shell consists of
the inner core structures, partially covering the first shell. Finally, the third shell, the
actual surface residues, mediates the direct interactions with other proteins, like the
E1 and the E3. We would like to point out, however, that some E2s can be used both
for conjugation of Ub and of ISG15 (24)⇓. Notably, the third shell does not cover the
first shell. The second shell has a dual role. It arranges residues in the third shell for E1
and E3 recognition, and it positions the first shell in optimal position to the third shell
so that the C terminus of the activated Ub/UBL can reach the catalytic center within
the first shell. when changing E2-E3 interaction specificity, catalytic mechanisms of E2s
remain the same, indicating that the first shell is not critically dependent on or linked
to how the third shell is organized.
Sequence comparision of UBC1 &
E2-25K Linker

Wolfgang Seufert and Stefan Jentsch, 1990
•Human and yeast ubiquitin share 96%
sequence identity.
W.Seufert, J.P.McGrath and S.Jentsch, 1990

The catalytic domain of E2 conjugating enzymes is
responsible for the formation of the thiol ester
intermediate, further conjuncting with E3 to fated
substrate.

E2 also have a C termini domain involved in
dimerization of the E2, substrate recognition, and
involvement in ubiquitination chain formation.
Lorick KL, Jensen JP, Weissman AM., 2009
Wolfgang Seufert and Stefan Jentsch, 1990