Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.564250
Title: Analysis of binary interactions between OTUB1 and E2 ubiquitin-conjugating enzymes
Author: Zulkifle, Nurulisa
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2012
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Abstract:
Post-translational modification of proteins via ubiquitination is mediated by three enzyme families; E1 activating enzymes, E2 conjugating enzymes and E3 ligases, all of which work in a hierarchical manner to facilitate different forms of protein ubiquitin ranging from mono-ubiquitination to the formation of different forms of ubiquitin chains (Ciechanover et al., 2000). Deubiquitinating enzymes (DUBs) act to remove ubiquitin from modified substrates. Apart from the classic interactions within the E1-E2-E3 enzymatic cascade, an unusual non-hierarchical interaction has been observed between some E2 enzymes and a DUB called OTUB1 (Markson et al., 2009). This observation raises interesting questions concerning the molecular mechanisms and specificity of this unusual E2:DUB partnership. In this study, systematic yeast two-hybrid (Y2H) screens were performed between all human E2 and DUB proteins to analyse the extent of E2:DUB interactions. Putative partnerships between OTUB1 and UBE2D1, UBE2D2, UBE2D3, UBE2D4, UBE2E1, UBE2E2, UBE2E3 and UBE2N were identified. These data correlate well with data from other independent studies, including HTP Y2H screens (Markson et al., 2009) and mass spectrometry (Sowa et al., 2009). An N-terminal truncated form of OTUB1 (ΔNOTUB1) was generated by removing a predicted 39aa N-terminal disordered region (Edelmann et al., 2009). Using this construct in combination with wild type (WT) OTUB1, complementary biophysical studies were performed to investigate the formation of complexes with UBE2D2 and UBE2E1 as these represented the strongest interactions detected in preliminary Y2H studies. Gel filtration chromatography showed convincing complex formation for both ΔNOTUB1:UBE2D2 and ΔNOTUB1:UBE2E1 in 1:1 stoichiometry. The thermodynamic profile of each complex was measured by ITC suggested a stronger affinity between ΔNOTUB1:UBE2D2 (Kd 3.89 µM) than observed for the ΔNOTUB1:UBE2E1 complex (Kd 16.55 µM). The n values for both complexes are 1.16±0.06 sites and 0.92±0.03 sites respectively, confirming that both complexes adopt a 1:1 stoichiometry. Observing the UBE2D2 (1H15N)-HSQC NMR spectral changes that occurred upon addition of unlabelled ΔNOTUB1 allowed the identification of potential residues of contact between the two proteins. From this study, we were able to predict that the 1st α-helix, the L1 loop of the 3rd and 4th β-sheet, the L2 loop connecting the 4th β-strand and the H2 α-helix within UBE2D2 were likely to be the binding surfaces for OTUB1. Point mutants corresponding to predicted contact residues in UBE2D2 were generated and tested in Y2H studies to determine their role in facilitating the formation of both E2:OTUB1 and E2:E3-RING complexes. This data suggests that in some, but not all cases, OTUB1 and E3-RINGs bind competitively to the same interface on E2 proteins. Preliminary immunofluorescence studies show that partner proteins predominantly co-localise in the cytoplasm, except UBE2E1 which is predominantly nuclear. Data from this study allowed us to propose a model of how OTUB1:UBE2D2 complex may forms and functions. Significantly, many of these predictions have now been verified by independent structural studies and subsequent live cell microscopy studies in our lab.
Supervisor: Sanderson, Chris Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.564250  DOI: Not available
Keywords: QP Physiology
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