Biochemical and cell biological studies of the Parkinson's disease-associated ubiquitin hydrolase UCH-L1

Ubiquitin C-terminal hydrolase-L1 (UCH-L1) is a neuronal deubiquitinating enzyme that comprises 1-2% of soluble brain protein, and is associated with several neurodegenerative diseases, most notably Parkinson's disease (PD), and is upregulated in many cancers. Biochemically, UCH-L1 is known to cleave amide and ester conjugates from the C-terminus of ubiquitin, and thus may function in ubiquitin recycling or as a modulator of ubiquitin-dependent pathways. However, neither its in vivo substrate(s), nor its roles in cellular physiology or disease pathogenesis are clearly understood. Despite biological studies which suggest roles for UCH-L1 in critical processes such as ubiquitin homeostasis, apoptosis, and learning and memory, and in spite of the recent solution of its crystal structure, UCH-L1 function and substrates remain relatively enigmatic. This work presents a brief overview of UCH-L1 and its relationship to PD in Chapter 1, followed by several lines of research which aim to improve our understanding of UCH-LI cellular activity and its role in PD. In Chapter 2, we demonstrate that UCH-L1 is monoubiquitinated in cell culture, and that this modification restricts enzyme activity by preventing binding to ubiquitinated targets. We found that ubiquitination sites are located near the active site of the enzyme, and that UCH-L 1 catalyzes its own deubiquitinatinn, thereby regulating the lifetime of this modification in a manner dependent on its catalytic activity level. Chapter 3 addresses the subcellular localization of UCH-L1, and we report that a portion of UCH-L1 is localized to the endoplasmic reticulum in a variety of cell lines commonly used in the study of UCH-L1. Finally, in Chapter 4, we examine biological interactions between UCH-L1 and α-synuclein, another neuronal protein linked to PD, and demonstrate that overexpression of UCH-L1 promotes the monoubiquitination of α-synuclein in cell culture. We also examine the mechanism by which UCH-L1 has been previously shown to increase α-synuclein levels in cells.