Hazlett, Zachary S., authorYao, Tingting, advisorCohen, Robert, committee memberPeersen, Olve, committee memberDi Pietro, Santiago, committee memberKennan, Alan, committee member2021-01-112022-01-082020https://hdl.handle.net/10217/219567The ubiquitin (Ub) C-terminal hydrolase, Uch37, can be found associated with the 26S proteasome as well as the INO80 chromatin remodeling complex. Bound to the 26S proteasome, it assists in regulating the degradation of Ub modified proteins. The proteasomal subunit Rpn13 binds Uch37, anchors it to the proteasome 19S regulatory particle and enhances the deubiquitinating enzyme's (DUB's) catalytic activity. While the structure of the Uch37/Rpn13 complex bound to a single Ub molecule has been characterized, much still remains unknown regarding the enzyme's substrate specificity, the molecular basis for its substrate specificity, and its function in the regulation of proteasomal degradation. In this thesis we characterize the substrate specificity of Uch37 with and without its proteasomal binding partner Rpn13. By synthesizing poly-Ub chains of various linkage types and topologies and using these Ub chains in in vitro deubiquitination assays, we were able to determine that Uch37/Rpn13 selectively cleaves branched Ub chains. This provides evidence to suggest that Uch37 is the first enzyme with activity specific for branched Ub chains. Branched Ub chains have been identified endogenously and have roles connected to the regulation of nascent misfolded polypeptides, cell cycle control, and the enhancement of proteasomal degradation. The work presented here sets out to characterize the molecular mechanism of branched chain hydrolysis by Uch37 and its binding partner Rpn13, determine the kinetics of this enzymatic reaction, and establish a system for probing the function of "debranching" by Uch37 in proteasomal degradation. The conclusion of our work builds our understanding of the complex system of intracellular signaling by Ub and unveils key elements to the primary system responsible for regulating cellular protein homeostasis.born digitalmasters thesesengCopyright and other restrictions may apply. User is responsible for compliance with all applicable laws. For information about copyright law, please see https://libguides.colostate.edu/copyright.proteasomebranched ubiquitinUch37Text