Probing the molecular choreography: a chemical biology exploration of the Ubiquitin and Ubiquitin-like post-translational machinery

Abstract

This thesis explores the development and application of advanced biochemical tools to study deubiquitinating enzymes (DUBs) and DUB-like proteases, with a focus on uncovering novel enzymatic activities and regulatory mechanisms. It discusses the discovery of cross-reactive DUBs that exhibit diverse and previously uncharacterized biological functions, shedding light on their roles in cellular signaling and host-pathogen interactions. A key part of the work examines E3 ubiquitin ligases, particularly those from the TRIM family, leading to the identification of USP24 as a novel interactor of TRIM25—implicating a potential regulatory axis in innate immune signaling. Additionally, the thesis details the optimization of expression and purification strategies for Sentrin-specific Proteases (SENPs), enabling robust and reproducible preparation of active enzymes. These improvements laid the foundation for high-throughput biochemical screening approaches to investigate SENP substrate specificities and to identify potential SENP inhibitors. Collectively, the thesis provides valuable insights into the enzymology of ubiquitin and ubiquitin-like protein pathways, demonstrating how targeted biochemical methodologies can advance our understanding of post-translational modifications and guide therapeutic discovery.