Mining the ubiquitin system for catalytic activities through advanced ubiquitin probes

I propose a chemoproteomic platform for the discovery of unchartered eraser activities regulating post-translational modifications within the ubiquitin system. This approach will be enabled by a suite of advanced ubiquitin probes. These reagents recapitulate the complexity of cellular polyubiquitin modifications and are equipped with novel electrophilic handles to report on polyubiquitin chain length- and linkage-dependent activities of deubiquitinating enzymes (DUBs). These enzymes reverse ubiquitin modifications and critically determine the outcome of ubiquitin-mediated signalling, yet how they decode ubiquitin chains is largely unclear. Moreover, activity-based probes with novel designs will enable chemoproteomic experiments to identify ubiquitin-directed serine hydrolases and the elusive eraser enzymes for the cell stress-related ubiquitin-like modifier Urm1. These insights will subsequently reveal molecular mechanisms of chain-length decoding and substrate recognition by facilitating the structural analysis of DUB-probe complexes. Taken together, the proposed work breaks new ground through a rational workflow for comprehensively mining the proteome for unprecedented DUB/Ubl eraser activities. Expected results will have broad implications also for ubiquitin assembly enzymes, pathogen-derived virulence factors and other protein classes. UbiPRO will thus illuminate a fundamentally important and evolutionarily conserved system of molecular information encoding employed by all forms of life.