Introduction of Ubiquitinylation- E2 Enzyme

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)

YEASTSacchromyces Cerevisiae

HUMAN

ENCODING GENES

13 38

TYPES 16-35 35CONSERVED 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-terminalextension

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 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

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

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

UBC4MSSSKRIAKELSDLERDPPTSCSAGPVGDDLYHWQASIMGPADSPYAGGVFFLSIHFPTDYPFKPPKISFTTKIYHPNINANGNICLDILKDQWSPALTLSKVLLSICSLLTDANPDDPLVPEIAHIYKTDRPKYEATAREWTKKYAV

UBC 5 MSSSKRIAKELSDLGRDPPASCSAGPVGDDLYHWQASIMGPSDSPYAGGVFFLSIHFPTDYPFKPPKVNFTTKIYHPNINSSGNICLDILKDQWSPALTLSKVLLSICSLLTDANPDDPLVPEIAQIYKTDKAKYEATAKEWTKKYAV

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

A• Functional studies of c-

UBC1 under various stress condition.

B• Prediction of c-UBC1

structure using bioinformatic tool

Swapping of yeast Ubc1 linker for mammalian E2-25K

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

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

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

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

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 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.

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

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

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

Li

gatio

n

Bgl II

Kpn I

Bgl II

Kpn IyEP96 N82S

82 FR82 REUBC4 FRUBC4 RE

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

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 aselected 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