Selective editing of cellular protein degradation by modulating ubiquitin specific protease function

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Manipulation of a cell's proteome by selectively increasing the longevity of beneficial proteins or removing aberrant or undesirable proteins is highly attractive in basic research, therapy and biotechnology applications. This strategy offers advantages over gene silencing and genome editing techniques in that it impacts directly on the protein levels potentially enhancing precision. In eukaryotic cells, the ubiquitin proteasome system regulates selective protein degradation. Therefore, interference with the ubiquitin system offers valuable avenues for modulating cellular protein clearance and in consequence protein levels. Ubiquitin modification of a substrate protein is reversible, whereby ubiquitin specific proteases (USPs), a class of deubiquitinating enzymes, can salvage proteins from destruction by the proteasome by removing the ubiquitin tag. These cysteine proteases are often dysregulated in cancer and also play a role in neurodegenerative disorders and the host's response to infection. The main aim of this project is the identification and evaluation of novel ubiquitin system modulators that interfere with ubiquitin-mediated protein degradation.To this end we will generate engineered fluorescent ubiquitin variants to test ubiquitin specific protease activity in vitro. We will then use the developed assay to identify novel USP inhibitors by screening an in-house compound collection. As a target we will focus on USP15, a multi-functional protease that has been shown to play a regulatory role in transforming growth factor beta (TGF-beta signalling that regulates cell growth, differentiation and apoptosis. In cancer TGF-beta signalling promotes tumour development during the advanced stages of tumorigenesis, but induces cell-cycle arrest and consequently tumour suppression in the early stages. In addition, USP15 suppresses mitophagy, which in turn is related with Parkinson disease and other dysfunctional mitochondria diseases associated with ageing. USP15 also influences the inflammatory response and promotes RIG-I-mediated antiviral signalling. We have recently solved crystal structures of the USP15 N-terminal and catalytic domains. The known crystallisation conditions for the protein will facilitate the determination of structures of USP15-inhibtor complexes to gain insight into the interactions and in combination with binding assays investigate the specificity of identified inhibitors with regards to related USPs. We will then test the top scoring inhibitors in cellular assays focusing on the viability and impact on protein levels of USP15 substrates such as the E3 ubiquitin ligase SMURF2 that targets the TGF beta receptor (T beta R) complex for ubiquitin-mediated degradation. Together, the project will offer skill development in an interdisciplinary setting including protein engineering, fluorescent assays, structural biology and cellular assays and will deliver novel insights into USP activity, structure and inhibition and create tools for the manipulation of protein degradation.