A chemical biology approach for understanding protein prenylation and deubiquitination

Abstract

Chemical proteomics is a powerful and versatile platform for several biological applications, including post-translation modification (PTM) profiling and inhibitor target identification. The approach uses probe analogues of natural substrates or small-molecule inhibitors, coupled to mass spectrometry-based proteomics. Identification of protein PTM or molecule’s target proteins can provide vital information for fundamental biology and disease, drug development and revealing modes of action. This thesis reports development of two novel chemical probes to study farnesylation and ubiquitin carboxy-terminal hydrolase L1 (UCHL1), a deubiquitinating enzyme. Farnesylation, an irreversible lipidation PTM, mediates membrane localisation and protein protein interactions. This PTM is catalysed by farnesyltransferase, reported as a drug target in many cancers, progeria and infections. However, profiling of farnesylated interactomes in living cells is challenging due to a lack of tools. A novel photo-crosslinking farnesyl probe is therefore presented, which closely mimics farnesol to preserve native metabolic incorporation. The probe contains a diazirine for covalent binding with interactomes alongside an alkyne for conjugation with reporters, and was validated in human cells using chemical proteomics. This probe may allow analysis of a previously inaccessible subset of farnesylated interactomes, enhancing understanding of these networks and accelerating drug target validation. UCHL1 is a potential drug target for cancers, neurodegenerative diseases, alongside liver and lung fibrosis. However, bona fide functions and substrates of UCHL1 remain unclear. Novel alkyne-tagged activity-based probes (ABPs) based on UCHL1 covalent inhibitors were therefore used to profile UCHL1 activity and identify target proteins. ABPs in combination with chemical proteomics successfully identified and quantified target proteins in intact cells, establishing an optimised probe which selectively labelled the catalytic cysteine of UCHL1. Further, UCHL1 inhibitors blocked pro-fibrotic effects in idiopathic pulmonary fibrosis models, supporting a potential therapeutic role for UCHL1 inhibition and providing a basis for development of selective UCHL1 inhibitors. This thesis therefore provides chemical proteomics tools that will accelerate drug development and provide fundamental insight into complex biological systems.