A wide variety of cellular processes are regulated via the post-translation modification of substrate proteins by either ubiquitin or the small ubiquitin-like modifier, SUMO. In recent years, points of convergence between the once thought distinct and independent SUMO- and ubiquitin-conjugation pathways have been identified. The ubiquitin E3 ligase RNF4 catalyses the addition of ubiquitin modifications to previously SUMO modified proteins. This results in hybrid chains of SUMO and ubiquitin decorating target substrates. Do these SUMO-ubiquitin hybrid chains act as unique signals, distinct from the SUMO and ubiquitin chains that make them? Are there proteins that recognise them as such? The first part of this thesis details a protocol to identify and characterise the affinity of cellular proteins for ‘baits’ consisting of hybrid chains of SUMO and ubiquitin. This affinity chromatography based approach when coupled with high resolution mass spectrometry identified 30 proteins from HEK 293 cellular extracts that were putatively identified as showing affinity specifically for SUMO-ubiquitin hybrid chains. Validation of this approach comes from the identification of the recently postulated SUMO-ubiquitin hybrid chain interacting protein RAP80 amongst the proteins identified as putative SUMO-ubiquitin hybrid chain interacting proteins. SUMO-ubiquitin hybrid chains are then evaluated in an in vivo context. A proximity ligation assay was developed to probe the association between SUMO, ubiquitin and RAP80, suggesting a tight association between the three after DNA damage inducing stimuli. Interesting, the SUMO targeted ubiquitin E3 ligase RNF4 was shown not to be required for the recruitment of RAP80 to sites of DNA damage. The association between RAP80, SUMO, and ubiquitin was probed further by immunoprecipitaion of RAP80 from cell extracts after a SUMO specific proteases treatment. This SUMO specific protease treatment resulted in the loss of high molecular weight ubiquitin conjugates from RAP80 immunoprecipitated material suggesting that some RAP80 associated material is carrying SUMOylation dependent ubiquitin modifications. A SILAC based gel shift assay utilising both ubiquitin and SUMO-specific proteases was then developed to identify proteins that may anchor SUMO-ubiquitin hybrid chains. Although technically challenging, S100A8 was identified as a protein that after the action of ubiquitin and SUMO proteases shifts in a pattern consistent with that expected of a protein that may anchor a SUMO-ubiquitin hybrid chain.
|Date of Award||2015|
|Sponsors||Biotechnology and Biological Sciences Research Council|
|Supervisor||Ronald Hay (Supervisor)|