Structural characterisation of the interaction between RBBP6 and the multifunctional protein YB-l
| dc.contributor.advisor | Pugh, David J. R. | |
| dc.contributor.author | Muleya, Victor | |
| dc.date.accessioned | 2022-03-04T11:16:41Z | |
| dc.date.accessioned | 2024-05-09T07:45:59Z | |
| dc.date.available | 2022-03-04T11:16:41Z | |
| dc.date.available | 2024-05-09T07:45:59Z | |
| dc.date.issued | 2010 | |
| dc.description | >Magister Scientiae - MSc | en_US |
| dc.description.abstract | Retinoblastoma binding protein 6 (RBBP6) is a 250 kDa RING finger-containing protein whose function is known to be mediated through interaction with other proteins. RBBP6 plays a role in the regulation of the tumour suppressor protein p53 and is also thought to be involved in mRNA splicing although its role has yet to be characterised. A recent study utilising a yeast 2-hybrid screen identified the cancer-associated protein known as YB-l as an interacting partner of RBBP6, and showed that RBBP6 ubiquitinates YB-I, leading to its degradation in the proteasome.Human Y-box binding protein 1 (YB-I) is member of the cold-shock domain family of proteins, which regulates a number of growth related genes through both transcriptional and translational mechanisms. YB-l is a cell-survival factor whose expression is increased in proliferating normal and cancer cells. It also protects cells against p53-mediated apoptosis by repressing the p53- promoter and down-regulating endogenous p53. The interaction between RBBP6 and YB-l involves the RING finger-like domain ofRBBP6 and the C-terminal62 amino acids ofYB-l. As a means of further localising the interaction, truncated fragments derived from the C-terminal region of YB-I, 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 ofYB-l may be involved in the interaction with RBBP6. Using chemical shift perturbation analysis, this study provides an indication of where YB-l binds to the RING fmger. This represents the first step towards the design of therapeutics aimed at modulating the interaction between RBBP6 and YB-l as a means of targeting the oncogenic effects ofYB-l. In order to identify E2 enzymes involved in the ubiquitination of YB-I, 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-l in conjuction with RBBP6, whereas Ubc 13 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. www.etd. | en_US |
| dc.identifier.uri | https://hdl.handle.net/10566/13381 | |
| dc.language.iso | en | en_US |
| dc.publisher | University of the Western Cape | en_US |
| dc.rights.holder | University of the Western Cape | en_US |
| dc.subject | RBBP6 | en_US |
| dc.subject | YB-l | en_US |
| dc.subject | Interaction | en_US |
| dc.subject | RING | en_US |
| dc.subject | 15N-HSQC | en_US |
| dc.subject | NMR | en_US |
| dc.subject | Yeast 2-hybrid | en_US |
| dc.subject | Co-immunoprecipitation | en_US |
| dc.subject | Homodimerisation | en_US |
| dc.subject | Ubiquitination | en_US |
| dc.title | Structural characterisation of the interaction between RBBP6 and the multifunctional protein YB-l | en_US |