The development and application of ubiquitin and SUMO-directed photoreactive probes

This PhD thesis focuses on the development and application of ubiquitin (Ub) / Small Ubiquitin-like Modifier (SUMO)-directed photoreactive probes, to identify endogenous interactors of E2-Ub/SUMO conjugates and to study interactions in solution using crosslinking-mass spectrometry (XL-MS). The first chapter describes the design and production of a UbcH5a-based photoreactive probe designed to capture RING E3 ligases. The probe successfully captured a range of RING E3 ligases and the DUB, OTUB1 in vitro. Notably, this revealed an alternative conformation of OTUB1 and UbcH5a-Ub that contradicts existing crystal structures. This finding prompted further investigation into the non-canonical inhibition of ubiquitination mediated by OTUB1. In the second chapter, the objective was to elucidate the mechanisms of degrader-mediated ubiquitination. Thus, stable Ube2R1-Ub conjugates were generated to form a complex with neddylated von Hippel-Lindau Cullin 2 ligase (CRL2VHL) recruited to Bromodomain 2 of Bromodomain-containing protein 4 (Brd4BD2) by the Proteolysis Targeting Chimera (PROTAC), MZ1. The complex was then observed using Cryogenic Electron Microscopy (cryo-EM) by a collaborator. A Ube2R1-Ub-based photoreactive probe was developed and was used with XL-MS to reveal how Rbx1 engages with donor ubiquitin in-solution. To further verify the structure, sites of ubiquitination on the neo-substrate, Brd4BD2 were mapped by MS. This revealed how lysine residues in the degrader-bound neo-substrate are optimally orientated within the neddylated-CRL2VHL complex to facilitate ubiquitination. The third and fourth chapters focus on the development and application of a SUMO2-Ubc9-based photoreactive probe. The probe, in combination with MS-based proteomics identified all verified SUMO E3s and many well-established SUMO substrates. Novel potential SUMO-Ubc9 interactors were enriched by the probe and a subset were explored biochemically. The dataset provides a useful resource for the SUMO field that can be used as a reference for future studies. Overall, the thesis presents innovative methods for studying protein interactions, contributing to a deeper understanding of ubiquitination and SUMOylation mechanisms. The findings offer potential avenues for future research with applications in drug discovery and probe development.