introduction of ubiquitinylation- e2 enzyme
DESCRIPTION
E2 enzyme act as a carrier protein in ubiquitinylation process.UPS undergo degradation by 26S proteosome.Contain conserved UBC domain, 150 residues..Yeast and Humans show 35% homology...E2 in Yeast( Sacchromyces cerevisiae ) are 16-35 types whiles active 35 in human.Ubc1 homologue in humans is E2- 25K| Hip-2, probing in pathogenesis of Alzheimer's Disease.UBC1, UBC4 and UBC5 show selective degradation of short- lived proteins.E2-25K undergo polyubiquitination without using E3 ligase.TRANSCRIPT
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Daniel Finley Genetics| Vol 192| 1 October 2012
UBIQUITIN
•Human and yeast ubiquitin share 96% sequence identity.
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Cellular functions of the Ubiquitin-Proteasome System
D.H. Wolf et al., 2004
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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
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The ubiquitin pathway
Yihong Ye et al., 2009, Nature Reviews, Mol. Cell Bio
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Ubiquitination
E1 E2
E3
Ubiquitin-activating enzyme
Ubiquitin-conjugating enzyme
Ubiquitin ligase
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E2 : Ubiquitin Conjugation Enzymes/ Ubiquitin Carrier Protein
E2 (30kd)
YEASTSacchromyces Cerevisiae
HUMAN
ENCODING GENES
13 38
TYPES 16-35 35CONSERVED DOMAIN
UBC (24.2 kd) (150-200 a.a)
UBC (150-200a.a)
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Structure of E2
Yihong Ye et al., 2009, Nature Reviews, Mol. Cell Bio
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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
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Classification of E2
Structurally Functionally
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1. Structurally…Core
catalytic domain
Class II
Class I
C-terminalextension
N-terminal extention
Class III
Class IV
N & C-terminal extention
H. T. Mark Timmers et al., THE FASEB JOURNAL 2010
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2. Functionally…
Monoubiquitination
Polyubiquitination
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UBC ViableaBiological 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
Ubc9b − E2 for Smt3 (SUMO) conjugation
Ubc10/Pex4 + Peroxisomal E2 important for peroxisome biogenesis
Ubc11 + Cytoplasmic localization
Ubc12b + 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
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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 increasesaggregate formation and cell death in polyglutamineDiseases. Remko de Pril et al., 2007
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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
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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
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UBC4MSSSKRIAKELSDLERDPPTSCSAGPVGDDLYHWQASIMGPADSPYAGGVFFLSIHFPTDYPFKPPKISFTTKIYHPNINANGNICLDILKDQWSPALTLSKVLLSICSLLTDANPDDPLVPEIAHIYKTDRPKYEATAREWTKKYAV
UBC 5 MSSSKRIAKELSDLGRDPPASCSAGPVGDDLYHWQASIMGPSDSPYAGGVFFLSIHFPTDYPFKPPKVNFTTKIYHPNINSSGNICLDILKDQWSPALTLSKVLLSICSLLTDANPDDPLVPEIAQIYKTDKAKYEATAKEWTKKYAV
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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
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UBC4
148 residues
alpha/beta protein
four strands forming an anti-parallel beta-sheet bounded by four alpha-helices
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Catalytic domain
Ubiquitin interaction site
E3 binding site
Structure of Ubc4
Peter S. Brzovic et al., 2011
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PREVIOUS WORK DONE
A• Functional studies of c-
UBC1 under various stress condition.
B• Prediction of c-UBC1
structure using bioinformatic tool
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Swapping of yeast Ubc1 linker for mammalian E2-25K
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RESULTS
4 hours 8 hours 12 hours 16 hours0
20
40
60
80
100
120
UBC1
UBC1 induced
c- UBC1
c- UBC1 induced
ubc1
Heat stress (37 C)⁰
% s
urv
ival
Time
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without pretreatment with pretreatmemt0
10
20
30
40
50
60
70
80
wild type
c-UBC1
ubc1
wild type induced
c-UBC1 induced
Thermotolerance Test
%
su
rviv
al
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Canavanine Test
ubc1
UBC1
c-UBC1
Control With canavanine
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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
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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.
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OBJECTIVE 1
Purification of c-UBC1
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STRATEGYHarvest the cells after 1mM IPTG induction (overnight)
Pellet down8K/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
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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
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. OBJECTIVE 2
WESTERN BLOT
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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
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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.
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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 agentLeft 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.
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Construction of N82S for UBC5 by site directed mutagenesis.
Construction of E15A for UBC5 by site directed mutagenesis.
. OBJECTIVE 3
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Mutation in Ubc4 by replacing amino acid of Ubc5:
Glutamate (glu) Glycine(gly)
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Amino acid
3 letter 1 letter Residue location
Side chain
polarity
Side chain
acidity or
basicity of
neutral species
Hydropathy index
Aspargine
Asn N 82 Polar Neutral -3.5
Serine Ser S 82 Polar Neutral -0.8
Glutamate
Glu E 15 Polar Neutral -3.5
Glycine Gly G 15 Non-polar
Neutral -0.4
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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.
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Bgl II Kpn I
pQE9 UBC4
yEP96
PCR
Bgl II Kpn I
Li
gatio
n
Bgl II
Kpn I
Bgl II
Kpn IyEP96 N82S
82 FR82 REUBC4 FRUBC4 RE
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pQE9 UBC4
PCR Bgl II
Kpn I
Bgl IIKpn I
Bgl II Kpn I
yEP96
Liga
tion
Kpn I
Bgl II
yEP96 E15G
20 FR20 REUBC4 FRUBC4 RE
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THANK YOU
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The proteasome: a proteolytic nanomachine of cell regulation and waste disposal
D.H. Wolf Biochimica et Biophysica Acta|(2004)
BACK UP
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(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 aselected protein molecule due to its irreversible proteolysis.
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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.
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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.
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Sequence comparision of UBC1 & E2-25K Linker
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Wolfgang Seufert and Stefan Jentsch, 1990
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•Human and yeast ubiquitin share 96% sequence identity.
W.Seufert, J.P.McGrath and S.Jentsch, 1990
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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
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Wolfgang Seufert and Stefan Jentsch, 1990
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