Structural, Dynamic and Functional Studies of USP14 : A Proteasome-Associated Deubiquitinating Enzyme Regulating Protein Degradation

Abstract: Ubiquitin-Specific Protease 14 (USP14) is a proteasome-associated deubiquitinating enzyme (DUB) involved in regulation of protein degradation and recycling of ubiquitin. USP14 consists of a Ubl domain connected to a USP domain via a flexible linker. The Ubl domain is believed to be responsible for initial association to Rpn1 on the 19S proteasome while the USP domain binds ubiquitin and cleaves the isopeptide bond in ubiquitin chains. Due to its prevalence in various cancers, USP14 has been identified as a promising target for drug development and has been shown to decrease tumor growth and dissemination in zebrafish when inhibited by small molecules.The structure of the USP14 USP domain has been characterized by X-ray crystallography in its free state as well as when bound to ubiquitin or catalytic inhibitors. Additionally, structures of full-length USP14 in complex with the proteasome and ubiquitin have been solved by cryo- EM. However, by these methods, only stable structural elements are visible, limiting the understanding of the dynamic properties of USP14.In this thesis, I have used a wide range of biophysical analysis methods together with nuclear magnetic resonance (NMR) to study the structural, functional, and dynamic properties of USP14. In collaboration with pharmacologists, I have applied biophysical analysis and modelling to the analysis of a library of USP14 binders to elucidate binding affinities and specificities. By combining NMR with small-angle X-ray scattering, I have described the dynamic interaction between the USP14 Ubl domain and its catalytic USP domain and its predisposition toward proteasome binding. Deuteration of USP14 and full NMR analysis of the dynamic properties of the USP domain in the presence and absence of the Ubl domain provided extended understanding of how dynamic networks could be involved in regulating the catalytic activities of USP14. Finally, NMR analysis of USP14 non-Ub-binding mutants jointly with functional analysis has extended our understanding on how USP14 regulates the activities of the proteasome. Taken together, the studies presented here contribute to the investigation of novel therapeutic pathways designed to improve cancer treatment and patient care.