Divergent ubiquitin interactions among ubiquitin specific proteases and development of small molecule DUB inhibitors
Date
2016
Authors
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Publisher
University of Delaware
Abstract
Deubiquitinating enzymes (DUBs) are responsible for reversing mono- and poly-ubiquitination of proteins and play essential roles in numerous cellular processes. Close to 100 human DUBs have been identified and are classified into five families, with the ubiquitin-specific protease (USP) family being the largest one (> 50 members). The binding of ubiquitin (Ub) to USP is strikingly different from that observed for the DUBs in the ubiquitin C-terminal hydrolase (UCH) and ovarian tumor domain protease (OTU) families. Unlike UCHs and OTUs that bind ubiquitin largely through its C-terminal peptide, USPs bind Ub more extensively at both the N-terminal region and the body of ubiquitin in addition to the ubiquitin C-terminal peptide. We generated a panel of mutant ubiquitins and used them to probe the ubiquitin’s interaction with a number of USPs. Our results revealed a remarkable divergence of USP-Ub interactions among the USP catalytic domains. Our double mutant cycle analysis targeting the ubiquitin residues located in the N-terminal region, the body, and the C-terminal tail of ubiquitin also demonstrated different crosstalk among the USP-Ub interactions. My work uncovered intriguing divergences in the ubiquitin-binding mode in the USP family DUBs and raised the possibility of targeting the ubiquitin-binding hotspots on USPs for selective inhibition of USPs by small molecule antagonists. To identify new small molecule inhibitors of USPs we employed virtual screening. Utilizing the NCI virtual diversity set we identified two pan-USP inhibitors NSC134149 and NSC127133. Compounds NSC134149 and NSC127133 possess IC50 in the micromolar range. Further testing of analogs of the lead compounds were carried out. No NSC127133 analogs were found to be potent inhibitors, while the NSC134149 analog NSC15590 displayed partial selectivity for USP21. Our studies demonstrated the feasibility of finding inhibitors that target the USP catalytic core using virtual screening and paved the way for future structure activity relationship (SAR) study for more potent and selective USP inhibitors. ☐ Interferon specific gene 15 (ISG15) is an ubiquitin-like (Ubl) that is covalently attached to the ε-amine of a substrate lysine residue as a post-translational modification. ISG15 was discovered as part of the innate immune response. It is conjugated to human and viral proteins to delay viral replication. Currently, the only known human deISGylases are members of the ubiquitin specific protease (USP) family. USP2, USP5, USP13, USP14, USP18 and USP21 have been shown to form complexes with ISG15 probes or turnover ISG15 substrate. Despite being a member of the USP family, USP18 is specific for ISG15 and does not form a complex with an ubiquitin probe. To investigate the deISGylation in humans, we expressed and purified stable truncates of USP18 for biochemical analysis. We found that the USP18 truncates that we generated possessed very low state-state kinetic activity using ISG15-AMC as a substrate. In contrast the truncated USP18 can be efficiently labeled by ISG15 probes. When the putative active cite cysteine (C64) was mutated to alanine, we detected much reduced labeling by the ISG15-Br probe, while a double mutation of C64A/C65A abolished the labeling of USP18 by ISG15-Br.
Description
Keywords
USP18, Ubiquitin, ISG15, Inhibitor, Deubiquitinase, DeISGylase