Mechanistic Dissection of Substrate Recruitment by Ubiquitin Ligases

Abstract

Protein homeostasis is an essential process that needs to be tightly regulated to maintain a delicate balance between protein synthesis and protein degradation. The Ubiquitin Proteasome System (UPS) serves as a fundamental regulatory pathway for controlling protein degradation, of which E3 ligases lend additional specificity in targeting and degrading their substrates. Investigating the variety of mechanisms that ubiquitin ligases rely on allows us to understand how diverse substrates are recruited. Targeted protein degradation (TPD) is an emerging field that utilizes small molecules – either PROTACs or small molecules – to hijack E3 ligases and direct them to neo-substrates for ubiquitylation and subsequent degradation. However, the discovery of new degraders is hindered by the lack of understanding underlying these degraders’ mechanism of action and how they co-opt existing mechanisms in nature. My thesis work includes three projects that leverages a combination of biochemistry, structural biology, chemical biology, and proteomics to dissect the mechanistic details of how ubiquitin ligases interact with their substrates both in endogenous and drug-induced settings. In Chapter 2, I combined biochemistry and structural biology to interrogate DCAF16-based molecular glue degraders and discovered a novel “template-assisted covalent modification” mechanism. In Chapter 3, I contributed to structure-function studies for HUWE1 and characterized various classes of HUWE1 mutants with biochemical assays. In Chapter 4, I developed a novel mass spectrometry-based tool to provide temporal resolution for cellular perturbations, specifically looking at the intertwined network of E3 ligases and DUBs. These findings enhance our existing comprehension of the mechanisms of ubiquitin ligases, potentially advancing the exploration and advancement of degraders.