Philosophiae Doctor - PhD (Biotechnology)

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Browsing by Subject "2-hybrid"

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    Structural characterisation of the interaction between RBBP6 and the multifunctional protein YB-1
    (University of the Western Cape, 2010) Muleya, Victor; Pugh, David J.R.; Dept. of Biotechnology; Faculty of Science
    As a means of further localising the interaction, truncated fragments derived from the C-terminal region of YB-1, were tested for their interaction with the RING finger domain of RBBP6 using three different assays: a directed yeast 2-hybrid assay, co-immunoprecipitation and NMR chemical shift perturbation analysis. Our results suggest that the entire 62 amino acid region at the C-terminal domain of YB-1 may be involved in the interaction with RBBP6. Using chemical shift perturbation analysis, this study provides an indication of where YB-1 binds to the RING finger. This represents the first step towards the design of therapeutics aimed at modulating the interaction between RBBP6 and YB-1 as a means of targeting the oncogenic effects of YB-1. In order to identify E2 enzymes involved in the ubiquitination of YB-1, we examined the efficiencies of selected E2s in an in vitro ubiquitination assay. UbcH5c and UbcH7 were both found to catalyse the ubiquitination of YB-1 in conjuction with RBBP6, whereas Ubc13 was not. Finally, we show using NMR that two single-point mutations of the RING finger-like domain are sufficient to abolish homodimerisation of the domain. These will be used in future studies to investigate the requirement for homodimerisation on the ubiquitination activity of RBBP6.