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

Abstract

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.

Date of Award	2025
Original language	English
Awarding Institution	University of Dundee
Supervisor	Ronald Hay (Supervisor)