Mass Spectrometry-Based Proteomics Approaches to Define Enzyme-Substrate Relationships in the Ubiquitin Proteasome System

Abstract

The ubiquitin proteasome system (UPS) is a major regulator of cellular protein abundance, degrading proteins that are tagged with a ubiquitin post-translational modification. Respectively, E3 ubiquitin ligases and deubiquitinating enzymes (DUBs) are the UPS enzymes responsible for the addition and removal of ubiquitin from their substrates. While most small molecule drugs bind and directly modulate protein function, degradation of the entire protein can be achieved by drugs targeting the UPS. DUB inhibitors prevent the removal of ubiquitin from endogenous DUB substrates. Proteolysis-targeting chimeras (PROTACs) and molecular glue degraders redirect E3 ligases to ubiquitinate neo-substrates. Systematic characterization of DUB and E3 ligase substrates has been a challenge in the field and remains crucial to understanding the biological activity of drugs targeting the UPS. Facilitated by the availability of improved and highly selective drugs targeting the UPS, I demonstrate an approach for the systematic meta-analysis of an in-house global expression proteomics database. This work represents the first large-scale analysis of proteomics data for DUB inhibitors, PROTACs, and molecular glue degraders and derives insights on both substrate selectivity and important parameters for DUB inhibitor and PROTAC design. I first demonstrate the identification of endogenous substrates of DUBs using a combination of small molecule DUB inhibitors and mass spectrometry. Using USP7 as a proof-of-concept, I demonstrated that selective USP7 inhibition causes rapid degradation of USP7 substrates, allowing their identification by global expression proteomics. This work optimizes a quantitative proteomics workflow for the small molecule profiling of DUB substrates and systematically nominates substrates of USP7, USP47, USP25, USP28, USP30, UCHL1, and USP1, laying a foundation for the further study of these DUBs. Next, I demonstrate the broad applicability of my chemical proteomics approach by expanding the analysis to molecular glues and PROTACs. The systematic meta-analysis of global expression proteomics allowed for the first large-scale exploration of important PROTAC design principles, including linker length, E3 ligase recruitment, ternary complex formation, as well as recruitment of off-target substrates of the E3 ligase cereblon (CRBN). Hijacking CRBN is a common approach in TPD, but the characterization of its native function and endogenous substrates remains poorly understood. To substantially improve on available chemical and genetic tools to study CRBN biology, I applied insights from my systematic meta-analysis of PROTACs to the development of potent and selective PROTAC degraders of CRBN. In a related effort, I established affinity-purification mass-spectrometry (AP-MS) methods for the identification of drug-induced CRBN neo-substrates. This approach identified a new family of neo-substrates targetable by CRBN-based molecular glue degraders and established a practical approach for the identification of degrader-induced substrates. This body of research has collectively advanced the field’s understanding of endogenous DUB substrates and E3 ligase neo-substrates, uncovered important design principles for development of DUB inhibitors and PROTACs, and improved tools and methods for further substrate identification studies in the UPS.