Ubiquitination is a crucial posttranslational protein modification involved in a myriad of biological pathways. This modification is reversed by deubiquitinases (DUBs) that deconjugate the single ubiquitin (Ub) moiety or poly-Ub chains from substrates. In the past decade, tremendous efforts have been focused on targeting DUBs for drug discovery. However, most chemical compounds with inhibitory activity for DUBs suffer from mild potency and low selectivity. To overcome these obstacles, we developed a phage display-based protein engineering strategy for generating Ub variant (UbV) inhibitors, which was previously successfully applied to the Ub-specific protease (USP) family of cysteine proteases. In this work, we leveraged the UbV platform to selectively target STAMBP, a member of the JAB1/MPN/MOV34 (JAMM) metalloprotease family of DUB enzymes. We identified two UbVs (UbV^SP.1 and UbV^SP.3) that bind to STAMBP with high affinity but differ in their selectivity for the closely related paralog STAMBPL1. We determined the STAMBPL1-UbV^SP.1 complex structure by X-ray crystallography, revealing hotspots of the JAMM-UbV interaction. Finally, we show that UbV^SP.1 and UbV^SP.3 are potent inhibitors of STAMBP isopeptidase activity, far exceeding the reported small-molecule inhibitor BC-1471. This work demonstrates that UbV technology is suitable to develop molecules as tools to target metalloproteases, which can be used to further understand the cellular function of JAMM family DUBs.

Ubiquitin-specific peptidase 16 (USP16) is a deubiquitinase that plays a role in the regulation of gene expression, cell cycle progression, and various other functions. It was originally identified as the major deubiquitinase for histone H2A and has since been found to deubiquitinate a range of other substrates, including proteins from both the cytoplasm and nucleus. USP16 is phosphorylated when cells enter mitosis and dephosphorylated during the metaphase/anaphase transition. While much of USP16 is localized in the cytoplasm, separating the enzyme from its substrates is considered an important regulatory mechanism. Some of the functions that USP16 has been linked to include DNA damage repair, immune disease, tumorigenesis, protein synthesis, coronary artery health, and male infertility. The strong connection to immune response and the fact that multiple oncogene products are substrates of USP16 suggests that USP16 may be a potential therapeutic target for the treatment of certain human diseases.

Deubiquitinases (DUBs) have been the subject of intense scrutiny in recent years. Many of their diverse enzymatic mechanisms are well characterized in vitro; however, our understanding of these enzymes at the cellular level lags due to the lack of quality tool reagents. DUBs play a role in seemingly every biological process and are central to many human pathologies, thus rendering them very desirable and challenging therapeutic targets. This review aims to provide researchers entering the field of ubiquitination with knowledge of the pharmacological modulators and tool molecules available to study DUBs. A focus is placed on small molecule inhibitors, ubiquitin variants (UbVs), and activity-based probes (ABPs). Leveraging these tools to uncover DUB biology at the cellular level is of particular importance and may lead to significant breakthroughs. Despite significant drug discovery efforts, only approximately 15 chemical probe-quality small molecule inhibitors have been reported, hitting just 6 of about 100 DUB targets. UbV technology is a promising approach to rapidly expand the library of known DUB inhibitors and may be used as a combinatorial platform for structure-guided drug design.

Ubiquitin-specific proteases (USPs) are a family of multi-domain deubiquitinases (DUBs) with variable architectures, some containing regulatory auxiliary domains. Among the USP family, all occurrences of intramolecular regulation presently known are autoactivating. USP8 remains the sole exception as its putative WW-like domain, conserved only in vertebrate orthologs, is autoinhibitory. Here, we present a comprehensive structure-function analysis describing the autoinhibition of USP8 and provide evidence of the physical interaction between the WW-like and catalytic domains. The solution structure of full-length USP8 reveals an extended, monomeric conformation. Coupled with DUB assays, the WW-like domain is confirmed to be the minimal autoinhibitory unit. Strikingly, autoinhibition is only observed with the WW-like domain in cis and depends on the length of the linker tethering it to the catalytic domain. Modeling of the WW:CD complex structure and mutagenesis of interface residues suggests a novel binding site in the S1 pocket. To investigate the interplay between phosphorylation and USP8 autoinhibition, we identify AMP-activated protein kinase as a highly selective modifier of S718 in the 14-3-3 binding motif. We show that 14-3-3γ binding to phosphorylated USP8 potentiates autoinhibition in a WW-like domain-dependent manner by stabilizing an autoinhibited conformation. These findings provide mechanistic details on the autoregulation of USP8 and shed light on its evolutionary significance.

Bacterial non-homologous end joining requires the ligase, LigD and Ku. Ku finds the break site, recruits LigD, and then assists LigD to seal the phosphodiester backbone. Bacterial Ku contains a core domain conserved with eukaryotes but has a unique C-terminus that can be divided into a minimal C-terminal region that is conserved and an extended C-terminal region that varies in sequence and length between species. Here, we examine the role of Mycobacterium tuberculosis Ku C-terminal variants, where we removed either the extended or entire C-terminus to investigate the effects on Ku–DNA binding, rates of Ku-stimulated ligation, and binding affinity of a direct Ku–LigD interaction. We find that the extended C-terminus limits DNA binding and identify key amino acids that contribute to this effect through alanine-scanning mutagenesis. The minimal C-terminus is sufficient to stimulate ligation of double-stranded DNA, but the Ku core domain also contributes to stimulating ligation. We further show that wildtype Ku and the Ku core domain alone directly bind both ligase and polymerase domains of LigD. Our results suggest that Ku-stimulated ligation involves direct interactions between the Ku core domain and the LigD ligase domain, in addition to the extended Ku C-terminus and the LigD polymerase domain.