Development of Ubiquitin Variants with Selectivity for the Ubiquitin C-Terminal Hydrolase Subfamily of Deubiquitinases

There are over 100 deubiquitinating enzymes (DUBs) that account for seven distinct DUB subfamilies, each having different functions and binding topology to ubiquitin. One of these subfamilies, the ubiquitin C-terminal hydrolases (UCHs), contain four DUBs including the two structurally homologous enzymes UCHL1 and UCHL3. Both UCHL1 and UCHL3 have been described as cancer targets in literature, however development of small molecule probes to study their enzymatic activity in cancerous disease states remains difficult due to active site similarities between not only the UCHs but also all DUBs. This necessitates a novel method of probe development for DUBs. Due to this need, the endogenous substrate, ubiquitin has been utilized to develop macromolecular based probes for DUB enzymes.

UCHL1 and UCHL3 have an intrinsically high binding affinity for ubiquitin, which our lab utilized to our advantage in designing selective mono-ubiquitin variants (UbVs) and UbV-activity based probes containing mutations at amino acid residue sites that perturb binding to other DUBs were designed. Structural data, computational methods, as well as previous literature precedent on UbVs developed for other DUB subfamilies were utilized to derive highly selective mono-UbV for UCHL1 (Chapter 2), a UCHL1/UCHL3 UbV-activity based probe (Chapter 2), as well as a highly selective UbV-activity based probe for UCHL3 (Chapter 3).

Mechanistic studies were completed on Ub:UCHL1 complex formation and provide a novel interpretation of Ub:DUB binding as a whole as the negatively charged catalytic cysteine residue may be providing electrostatic repulsion to the negatively charged C-terminus of Ub post cleavage (Chapter 4). While development of a selective small molecule inhibitor for UCHL1 has proven difficult, a UCHL1 peptide-based inhibitor VAEFMK has yet to be characterized. VAEFMK and analogs display selectivity toward UCHL1 over UCHL3 but require high concentrations to fully inhibit UCHL1 in cells (Chapter 4). Conclusions and future directions to research on the ubiquitin variant projects will be presented in Chapter 5.