Interactomics identifies novel regulators of the IRF-1 tumour suppressor protein
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The highly unstructured interferon regulated transcription factor IRF-1 is a tumour suppressor protein that plays vital roles in the antiviral and DNA damage response pathways. To identify interacting factors that regulate IRF-1 function and expand on the available limited information on its interactome, an in vitro screen was developed using peptide-aptamer affinity chromatography coupled with mass spectrometry. Discrete identified which bind to a number of potential transcriptional regulators including NPM1, YB-1 and TRIM28. The screen also proved useful in identifying binding proteins to the C-terminal Mf1 domain, which is vital for IRF-1-mediated growth suppression and Cdk2 repression, and additionally regulates IRF-1 stability. Thus, an LXXLL motif in the MF1 domain was found to be required for the binding of Hsp70 family members and cooperation with Hsp90 to regulated IRF-1 turnover and activity. These conclusions were supproted by the finding the Hsp90 inhibitors suppressed IRF-1-dependent transcription shortly after treatment, whilst at later time points inhibition of Hsp90 led to an Hsp70-dependent depletion of nuclear IRF-1. Conversely, the half-life of IRF-1 was increase by Hsp90 in an ATPase-dependent manner leading to the accumulation of nuclear, but not cytoplasmic, IRF-1. Additionally, a stress specific interaction between IRF-1 and the Hsp70-associated ubiquitin E3 ligase CHIP, that targets Hsp70/Hsp90 clients for proteasomal degradation, was demonstrated. Consequently, decreases in IRF-1 protein levels in cells exposed to heat stress or heavy metal ions were accompanied by the formation of IRF-1:CHIP complexes. Based on observations that CHIP ubiquitination of IRF-1 occurred both in the presence and absence of Hsp70, a model was proposed wherein Hsp70 serves as a factor that recruits CHIP to its substrates and its dissociation from the complex allosterically activates CHIP-dependent substrate ubiquitination. In support of this model, in vitro and biophysical evidence is presented, showing that CHIP in complex with Hsp70 is less flexible and less effective as an E3 ligase that CHIP alone. Thus, in agreement with recent studies, the work done in this thesis highlights the importance of conformational flexibility and of direct binding or 'docking' of CHIP to its substrate(s) in its mechanism of action.