Divergence in Ubiquitin Interaction and Catalysis among the Ubiquitin-Specific Protease Family Deubiquitinating Enzymes

Deubiquitinating enzymes (DUBs) are responsible for reversing mono- and polyubiquitination 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 (>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. 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 tip, the central body, and the tail of ubiquitin also demonstrated different crosstalk among the USP–Ub interactions. This work uncovered intriguing divergence in the ubiquitin-binding mode in the USP family DUBs and raised the possibility of targeting the ubiquitin-binding hot spots on USPs for selective inhibition of USPs by small molecule antagonists

Chemical Protein Polyubiquitination Reveals the Role of a Noncanonical Polyubiquitin Chain in DNA Damage Tolerance

Polyubiquitination of proteins regulates a variety of cellular processes, including protein degradation, NF-κB pathway activation, apoptosis, and DNA damage tolerance. Methods for generating polyubiquitinated protein with defined ubiquitin chain linkage and length are needed for an in-depth molecular understanding of protein polyubiquitination. However, enzymatic protein polyubiquitination usually generates polyubiquitinated proteins with mixed chain lengths in a low yield. We report herein a new chemical approach for protein polyubiquitination with a defined ubiquitin chain length and linkage under a mild condition that preserves the native fold of the target protein. In DNA damage tolerance, K63-polyubiquitinated proliferating cell nuclear antigen (PCNA) plays an important yet unclear role in regulating the selection of the error-free over error-prone lesion bypass pathways. Using the chemically polyubiquitinated PCNA, we revealed a mechanism of the K63 polyubiquitin chain on PCNA in promoting the error-free lesion bypass by suppressing the DNA translesion synthesis (TLS)

Transient Kinetic Analysis of USP2-Catalyzed Deubiquitination Reveals a Conformational Rearrangement in the K48-Linked Diubiquitin Substrate

Deubiquitination has emerged as an essential regulatory mechanism of a number of cellular processes. An in-depth understanding of deubiquitinating enzyme (DUB) catalysis, particularly the mode of ubiquitin binding and the individual steps in the DUB catalytic turnover, is imperative for exploiting DUBs for therapeutic intervention. In this work, we present a transient kinetic study of USP2 in hydrolyzing a model substrate Ub-AMC and a physiological substrate K48-linked diubiquitin. We conducted stopped-flow fluorescence analyses of the binding of mono- and diubiquitin to an inactive USP2 mutant and unveiled interesting differences in the binding kinetics between the two substrates. While a simple one-step binding of monoubiquitin to USP2 was observed, a biphasic binding was evident for diubiquitin. We further followed the deubiquitination reaction of Ub-AMC and K48-linked IQF-diubiquitin by USP2 using stopped-flow florescence under a single-turnover condition. Global fitting of the reaction traces revealed differences in the microscopic rate constants between Ub-AMC and the physiological diubiquitin substrate. Our binding and single-turnover data support a conformational rearrangement of the diubiquitin substrate in USP2-catalyzed deubiquitination. This finding is significant given the recent finding that the K48-linked diubiquitin is dynamic in its conformation. Our results provide useful insights into the mechanism of how USP recognizes ubiquitin moieties in a chain structure, which is important for understanding USP catalysis and developing inhibitors against USPs

Cell Lysate-Based AlphaLISA Deubiquitinase Assay Platform for Identification of Small Molecule Inhibitors

The deubiquitinases, or DUBs, are associated with various human diseases, including neurological disorders, cancer, and viral infection, making them excellent candidates for pharmacological intervention. Drug discovery campaigns against DUBs require enzymatic deubiquitination assays amenable for high-throughput screening (HTS). Although several DUB substrates and assays have been developed in recent years, they are largely limited to recombinantly purified DUBs. Many DUBs are large multidomain proteins that are difficult to obtain recombinantly in sufficient quantities for HTS. Therefore, an assay that obviates the need of recombinant protein generation and also recapitulates a physiologically relevant environment is highly desirable. Such an assay will open doors for drug discovery against many therapeutically relevant, but currently inaccessible, DUBs. Here, we report a cell lysate DUB assay based on AlphaLISA technology for high throughput screening. This assay platform uses a biotin-tagged ubiquitin probe and a HA-tagged DUB expressed in human cells. The assay was validated and adapted to a 1536-well format, which enabled a screening against UCHL1 as proof of principle using a library of 15 000 compounds. We expect that the new platform can be readily adapted to other DUBs to allow the identification of more potent and selective small molecule inhibitors and chemical probes

Serine Phosphorylation Is Critical for the Activation of Ubiquitin-Specific Protease 1 and Its Interaction with WD40-Repeat Protein UAF1

Deubiquitinating enzymes (DUBs) are important for the normal function of a number of cellular processes, including transcriptional regulation, cell cycle control, and DNA damage response. The enzymatic activity of DUB is regulated by different mechanisms. DUBs in several different families are post-translationally modified by phosphorylation. Large-scale phosphoproteomic studies of human DUBs revealed that a majority of ubiquitin-specific proteases (USPs) are phosphorylated. USP1 is a prototypical DUB that requires a specific interaction with a WD40-repeat protein, UAF1, for its catalytic activity. In this study, we show that Ser313 phosphorylation in USP1 is required for its interaction with UAF1 and for the stimulation of USP1’s activity. In contrast, two other known USP1 serine phosphorylations (Ser42 and Ser67) are dispensable with respect to the activity of the USP1/UAF1 complex. An S313D phosphomimetic mutation in USP1 can substitute for Ser313 phosphorylation in promoting the formation of the USP1/UAF1 complex. We further demonstrated that CDK1 is responsible for Ser313 phosphorylation, and protein phosphatase treatment of USP1 can lead to inactivation of USP1/UAF1. An inserted domain in USP1 (amino acids 235–408) was found to interact with UAF1, and this interaction is mediated by Ser313 phosphorylation. Our findings revealed an intriguing mechanism of regulating USP1 activity that combines phosphorylation of a key serine residue in USP1 and the specific interaction of USP1 with a WD40-repeat protein UAF1. The pSer313-dependent formation of the USP1/UAF1 complex points to a new approach for inhibiting USP1 activity by disrupting the interaction between the UAF1’s WD40-repeat domain and the Ser313-containing phosphopeptide in USP1