Ubiquitin-specific protease 11 structure in complex with an engineered substrate mimetic reveals a molecular feature for deubiquitination selectivity.

Ubiquitin-specific proteases (USPs) are crucial for controlling cellular proteostasis and signaling pathways but how deubiquitination is selective remains poorly understood, in particular between paralogues. Here, we developed a fusion tag method by mining the Protein Data Bank and trapped USP11, a key regulator of DNA double-strand break repair, in complex with a novel engineered substrate mimetic. Together, this enabled structure determination of USP11 as a Michaelis-like complex that revealed key S1 and S1' binding site interactions with a substrate. Combined mutational, enzymatic, and binding experiments identified Met77 in linear diubiquitin as a significant residue that leads to substrate discrimination. We identified an aspartate "gatekeeper" residue in the S1' site of USP11 as a contributing feature for discriminating against linear diubiquitin. When mutated to a glycine, the corresponding residue in paralog USP15, USP11 acquired elevated activity toward linear diubiquitin in-gel shift assays, but not controls. The reverse mutation in USP15 confirmed that this position confers paralog-specific differences impacting diubiquitin cleavage rates. The results advance our understanding of the molecular basis for the higher selectivity of USP11 compared to USP15 and may aid targeted inhibitor development. Moreover, the reported carrier-based crystallization strategy may be applicable to other challenging targets.

Structural Insights into the Catalytic Mechanism and Ubiquitin Recognition of USP34.

Ubiquitination, an important posttranslational modification, participates in virtually all aspects of cellular functions and is reversed by deubiquitinating enzymes (DUBs). Ubiquitin-specific protease 34 (USP34) plays an essential role in cancer, neurodegenerative diseases, and osteogenesis. Despite its functional importance, how USP34 recognizes ubiquitin and catalyzes deubiquitination remains structurally uncharacterized. Here, we report the crystal structures of the USP34 catalytic domain in free state and after binding with ubiquitin. In the free state, USP34 adopts an inactive conformation, which contains a misaligned catalytic histidine in the triad. Comparison of USP34 structures before and after ubiquitin binding reveals a structural basis for ubiquitin recognition and elucidates a mechanism by which the catalytic triad is realigned. Transition from an open inactive state to a relatively closed active state is coupled to a process by which the "fingertips" of USP34 intimately grip ubiquitin, and this has not been reported before. Our structural and biochemical analyses provide important insights into the catalytic mechanism and ubiquitin recognition of USP34.

Mitoxantrone stacking does not define the active or inactive state of USP15 catalytic domain.

Ubiquitin specific protease USP15 is a deubiquitinating enzyme reported to regulate several biological and cellular processes, including TGF-ß signaling, regulation of immune response, neuro-inflammation and mRNA splicing. Here we study the USP15 D1D2 catalytic domain and present the crystal structure in its catalytically-competent conformation. We compare this apo-structure to a previous misaligned state in the same crystal lattice. In both structures, mitoxantrone, an FDA approved antineoplastic drug and a weak inhibitor of USP15 is bound, indicating that it is not responsible for inducing a switch in the conformation of active site cysteine in the USP15 D1D2 structure. Instead, mitoxantrone contributes to crystal packing, by forming a stack of 12 mitoxantrone molecules. We believe this reflects how mitoxantrone can be responsible for e.g. nuclear condensate partitioning. We conclude that USP15 can switch between active and inactive states in the absence of ubiquitin, and that this is independent of mitoxantrone binding. These insights can be important for future drug discovery targeting USP15.

Mutation of aspartic acid 199 in USP1 disrupts its deubiquitinating activity and impairs DNA repair.

The deubiquitinating enzyme USP1 contains highly conserved motifs forming its catalytic center. Recently, the COSMIC mutation database identified a mutation in USP1 at Asp-199 in endometrial cancer. Here, we investigated the role of Asp-199 for USP1 function. The mutation of aspartic acid to alanine (D199A) resulted in failure of USP1 to undergo autocleavage and form a complex with ubiquitin, indicating D199A Usp1 is catalytically inactive. The D199A mutation did not affect the interaction with Uaf1. Moreover, D199A Usp1 had defects in deubiquitination of FANCD2 and PCNA and displayed reduced FANCD2 foci formation and DNA repair efficiency. Furthermore, mutation of Asp-199 to glutamic acid resulted in phenotypes similar to the D199A mutation. Collectively, our findings demonstrate the importance of Asp-199 for USP1 activity and suggest the implications of USP1 downregulation in cancer.

The Multifaceted Roles of USP15 in Signal Transduction.

Ubiquitination and deubiquitination are protein post-translational modification processes that have been recognized as crucial mediators of many complex cellular networks, including maintaining ubiquitin homeostasis, controlling protein stability, and regulating several signaling pathways. Therefore, some of the enzymes involved in ubiquitination and deubiquitination, particularly E3 ligases and deubiquitinases, have attracted attention for drug discovery. Here, we review recent findings on USP15, one of the deubiquitinases, which regulates diverse signaling pathways by deubiquitinating vital target proteins. Even though several basic previous studies have uncovered the versatile roles of USP15 in different signaling networks, those have not yet been systematically and specifically reviewed, which can provide important information about possible disease markers and clinical applications. This review will provide a comprehensive overview of our current understanding of the regulatory mechanisms of USP15 on different signaling pathways for which dynamic reverse ubiquitination is a key regulator.

USP39 mediates p21-dependent proliferation and neoplasia of colon cancer cells by regulating the p53/p21/CDC2/cyclin B1 axis.

Ubiquitin-specific protease 39 (USP39) is frequently overexpressed in a variety of cancers, and involved in the regulation of various biological processes, such as cell proliferation, cell cycle progression, apoptosis and pre-messenger RNA splicing. Nevertheless, the biological roles and mechanisms of USP39 in colon cancer remain largely unknown. In this study, we analyzed whether USP39 can be a molecular target for the treatment of colon cancer. Whilst overexpression of USP39 was detected in human colon cancer tissues and cell lines, USP39 knockdown was observed to inhibit the growth and subcutaneous tumor formation of colon cancer cells. Further analysis showed that USP39 knockdown can stabilize p21 by prolonging the half-life of p21 and by upregulating the promoter activity of p21. The RS domain and USP domain of USP39 were found to play an essential role. Additionally, our findings revealed that USP39 plays a regulatory role in the proliferation of colon cancer cells by the p53/p21/CDC2/cyclin B1 axis in a p21-dependent manner. Taken together, this study provided the theoretical basis that may facilitate the development of USP39 as a novel potential target of colon cancer therapy.

Ubiquitin-specific protease 5 was involved in the interferon response to RGNNV in sea perch (Lateolabrax japonicus).

Deubiquitinases are widely involved in the regulation of the virus-triggered type I interferon (IFN) signaling. Here, we found sea perch (Lateolabrax japonicus) ubiquitin-specific protease 5 (LjUSP5) was a negative regulatory factor of the red-spotted grouper nervous necrosis virus (RGNNV)-triggered IFN response. LjUSP5 encoded a polypeptide of 830 amino acids, containing a zinc finger UBP domain (residues 197-270 aa), two ubiquitin-associated domains (residues 593-607 aa; 628-665 aa), and one UBP domain (residues 782-807 aa), and shared the closest genetic relationship with the USP5 of Larimichthys crocea. Quantitative RT-PCR analysis showed that LjUSP5 was ubiquitously expressed and up-regulated significantly in all inspected tissues post RGNNV infection, and its transcripts significantly increased in brain, liver and kidney tissues post RGNNV infection. LjUSP5 was up-regulated in cultured LJB cells after poly I:C and RGNNV treatments. In addition, overexpression of LjUSP5 significantly inhibited the activation of zebrafish IFN 1 promoter and promoted RGNNV replication in vitro. Furthermore, LjUSP5 inhibited the activation of zebrafish IFN 1 promoter induced by key genes of retinoic acid-inducible gene I-like receptors signaling pathway. Our findings provides useful information for further elucidating the mechanism underlying NNV infection.

Deubiquitinase MYSM1 in the Hematopoietic System and beyond: A Current Review.

MYSM1 has emerged as an important regulator of hematopoietic stem cell function, blood cell production, immune response, and other aspects of mammalian physiology. It is a metalloprotease family protein with deubiquitinase catalytic activity, as well as SANT and SWIRM domains. MYSM1 normally localizes to the nucleus, where it can interact with chromatin and regulate gene expression, through deubiquitination of histone H2A and non-catalytic contacts with other transcriptional regulators. A cytosolic form of MYSM1 protein was also recently described and demonstrated to regulate signal transduction pathways of innate immunity, by promoting the deubiquitination of TRAF3, TRAF6, and RIP2. In this work we review the current knowledge on the molecular mechanisms of action of MYSM1 protein in transcriptional regulation, signal transduction, and potentially other cellular processes. The functions of MYSM1 in different cell types and aspects of mammalian physiology are also reviewed, highlighting the key checkpoints in hematopoiesis, immunity, and beyond regulated by MYSM1. Importantly, mutations in MYSM1 in human were recently linked to a rare hereditary disorder characterized by leukopenia, anemia, and other hematopoietic and developmental abnormalities. Our growing knowledge of MYSM1 functions and mechanisms of actions sheds important insights into its role in mammalian physiology and the etiology of the MYSM1-deficiency disorder in human.

TIFAB Regulates USP15-Mediated p53 Signaling during Stressed and Malignant Hematopoiesis.

TRAF-interacting protein with a forkhead-associated domain B (TIFAB) is implicated in myeloid malignancies with deletion of chromosome 5q. Employing a combination of proteomic and genetic approaches, we find that TIFAB regulates ubiquitin-specific peptidase 15 (USP15) ubiquitin hydrolase activity. Expression of TIFAB in hematopoietic stem/progenitor cells (HSPCs) permits USP15 signaling to substrates, including MDM2 and KEAP1, and mitigates p53 expression. Consequently, TIFAB-deficient HSPCs exhibit compromised USP15 signaling and are sensitized to hematopoietic stress by derepression of p53. In MLL-AF9 leukemia, deletion of TIFAB increases p53 signaling and correspondingly decreases leukemic cell function and development of leukemia. Restoring USP15 expression partially rescues the function of TIFAB-deficient MLL-AF9 cells. Conversely, elevated TIFAB represses p53, increases leukemic progenitor function, and correlates with MLL gene expression programs in leukemia patients. Our studies uncover a function of TIFAB as an effector of USP15 activity and rheostat of p53 signaling in stressed and malignant HSPCs.

Phosphorylation of USP15 and USP4 Regulates Localization and Spliceosomal Deubiquitination.

Deubiquitinating enzymes have key roles in diverse cellular processes whose enzymatic activities are regulated by different mechanisms including post-translational modification. Here, we show that USP15 is phosphorylated, and its localization and activity are dependent on the phosphorylation status. Nuclear-cytoplasmic fractionation and mass spectrometric analysis revealed that Thr149 and Thr219 of human USP15, which is conserved among different species, are phosphorylated in the cytoplasm. The phosphorylation status of USP15 at these two positions alters the interaction with its partner protein SART3, consequently leading to its nuclear localization and deubiquitinating activity toward the substrate PRP31. Treatment of cells with purvalanol A, a cyclin-dependent kinase inhibitor, results in nuclear translocation of USP15. USP4, another deubiquitinating enzyme with a high sequence homology and domain structure as USP15, also showed purvalanol A-dependent changes in activity and localization. Collectively, our data suggest that modifications of USP15 and USP4 by phosphorylation are important for the regulation of their localization required for cellular function in the spliceosome.

Mebendazole elicits potent antimyeloma activity by inhibiting the USP5/c-Maf axis.

c-Maf is a critical oncogenic transcription factor that contributes to myelomagenesis. Our previous studies demonstrated that the deubiquitinase USP5 stabilizes c-Maf and promotes myeloma cell proliferation and survival; therefore, the USP5/c-Maf axis could be a potential target for myeloma therapy. As a concept of principle, the present study established a USP5/c-Maf-based luciferase system that was used to screen an FDA-approved drug library. It was found that mebendazole, a typical anthelmintic drug, preferentially induced apoptosis in c-Maf-expressing myeloma cells. Moreover, oral administration of mebendazole delayed the growth of human myeloma xenografts in nude mice but did not show overt toxicity. Further studies showed that the selective antimyeloma activity of mebendazole was associated with the inhibition of the USP5/c-Maf axis. Mebendazole downregulated USP5 expression and disrupted the interaction between USP5 and c-Maf, thus leading to increased levels of c-Maf ubiquitination and subsequent c-Maf degradation. Mebendazole inhibited c-Maf transcriptional activity, as confirmed by both luciferase assays and expression measurements of c-Maf downstream genes. In summary, this study identified mebendazole as a USP5/c-Maf inhibitor that could be developed as a novel antimyeloma agent.

Structural and Functional Characterization of Ubiquitin Variant Inhibitors of USP15.

The multi-domain deubiquitinase USP15 regulates diverse eukaryotic processes and has been implicated in numerous diseases. We developed ubiquitin variants (UbVs) that targeted either the catalytic domain or each of three adaptor domains in USP15, including the N-terminal DUSP domain. We also designed a linear dimer (diUbV), which targeted the DUSP and catalytic domains, and exhibited enhanced specificity and more potent inhibition of catalytic activity than either UbV alone. In cells, the UbVs inhibited the deubiquitination of two USP15 substrates, SMURF2 and TRIM25, and the diUbV inhibited the effects of USP15 on the transforming growth factor ß pathway. Structural analyses revealed that three distinct UbVs bound to the catalytic domain and locked the active site in a closed, inactive conformation, and one UbV formed an unusual strand-swapped dimer and bound two DUSP domains simultaneously. These inhibitors will enable the study of USP15 function in oncology, neurology, immunology, and inflammation.

Catalytic domain of deubiquitinylase USP48 directs interaction with Rel homology domain of nuclear factor kappaB transcription factor RelA.

The activity and close regulation of nuclear factor kappaB (NF-κB) transcription factors is critical for a variety of cellular processes including inflammation, immunity, differentiation and cell survival. Thus, dysregulation of the NF-κB system could lead to serious diseases, e.g. uncoordinated growth of the normal tissue during the development of cancer. Transcriptional activity of the NF-κB factor RelA is regulated by a number of mechanisms which comprise ubiquitinylation by a multimeric ubiquitin ligase containing Elongins B and C, cullin-2 (Cul2) and suppressor of cytokine signaling 1 (SOCS1), but also USP48-dependent deubiquitinylation. Further, USP48 promotes cell survival and antagonizes also other E3 ligase functions which are involved in genome stability and DNA repair. The regulation of RelA by USP48 has been investigated in detail, but the domains of USP48 and RelA for direct interaction are not known. In this study we report that USP48 interacts physically with RelA in the nucleus. Further, we show by overexpression of truncated proteins that the catalytic USP domain of USP48 interacts with the N-terminal region of the Rel homology domain (RHD) of RelA. This study provides first evidence that the USP domain of USP48 is important for the physical association with substrate proteins, and a suitable target for small molecule inhibitors for therapeutic intervention strategies.

Recognition of the Diglycine C-End Degron by CRL2KLHDC2 Ubiquitin Ligase.

Aberrant proteins can be deleterious to cells and are cleared by the ubiquitin-proteasome system. A group of C-end degrons that are recognized by specific cullin-RING ubiquitin E3 ligases (CRLs) has recently been identified in some of these abnormal polypeptides. Here, we report three crystal structures of a CRL2 substrate receptor, KLHDC2, in complex with the diglycine-ending C-end degrons of two early-terminated selenoproteins and the N-terminal proteolytic fragment of USP1. The E3 recognizes the degron peptides in a similarly coiled conformation and cradles their C-terminal diglycine with a deep surface pocket. By hydrogen bonding with multiple backbone carbonyls of the peptides, KLHDC2 further locks in the otherwise degenerate degrons with a compact interface and unexpected high affinities. Our results reveal the structural mechanism by which KLHDC2 recognizes the simplest C-end degron and suggest a functional necessity of the E3 to tightly maintain the low abundance of its select substrates.

The structure of the deubiquitinase USP15 reveals a misaligned catalytic triad and an open ubiquitin-binding channel.

Ubiquitin-specific protease 15 (USP15) regulates important cellular processes, including transforming growth factor ß (TGF-ß) signaling, mitophagy, mRNA processing, and innate immune responses; however, structural information on USP15's catalytic domain is currently unavailable. Here, we determined crystal structures of the USP15 catalytic core domain, revealing a canonical USP fold, including a finger, palm, and thumb region. Unlike for the structure of paralog USP4, the catalytic triad is in an inactive configuration with the catalytic cysteine ∼10 Å apart from the catalytic histidine. This conformation is atypical, and a similar misaligned catalytic triad has so far been observed only for USP7, although USP15 and USP7 are differently regulated. Moreover, we found that the active-site loops are flexible, resulting in a largely open ubiquitin tail-binding channel. Comparison of the USP15 and USP4 structures points to a possible activation mechanism. Sequence differences between these two USPs mainly map to the S1' region likely to confer specificity, whereas the S1 ubiquitin-binding pocket is highly conserved. Isothermal titration calorimetry monoubiquitin- and linear diubiquitin-binding experiments showed significant differences in their thermodynamic profiles, with USP15 displaying a lower affinity for monoubiquitin than USP4. Moreover, we report that USP15 is weakly inhibited by the antineoplastic agent mitoxantrone in vitro A USP15-mitoxantrone complex structure disclosed that the anthracenedione interacts with the S1' binding site. Our results reveal first insights into USP15's catalytic domain structure, conformational changes, differences between paralogs, and small-molecule interactions and establish a framework for cellular probe and inhibitor development.

Active site alanine mutations convert deubiquitinases into high-affinity ubiquitin-binding proteins.

A common strategy for exploring the biological roles of deubiquitinating enzymes (DUBs) in different pathways is to study the effects of replacing the wild-type DUB with a catalytically inactive mutant in cells. We report here that a commonly studied DUB mutation, in which the catalytic cysteine is replaced with alanine, can dramatically increase the affinity of some DUBs for ubiquitin. Overexpression of these tight-binding mutants thus has the potential to sequester cellular pools of monoubiquitin and ubiquitin chains. As a result, cells expressing these mutants may display unpredictable dominant negative physiological effects that are not related to loss of DUB activity. The structure of the SAGA DUB module bound to free ubiquitin reveals the structural basis for the 30-fold higher affinity of Ubp8C146A for ubiquitin. We show that an alternative option, substituting the active site cysteine with arginine, can inactivate DUBs while also decreasing the affinity for ubiquitin.

USP15 inhibits HPV16 E6 degradation and catalytically inactive USP15 has reduced inhibitory activity.

High-risk human papillomaviruses (HPVs) possess transforming activity leading to development of the cancer, including oropharyngeal, anal, penile, vulvar, vaginal, and cervical cancer. The stability of E6 is essential for its complete function as an oncoprotein. Using the yeast two-hybrid system, we identified ubiquitin-specific protease 15 (USP15) as an HPV16 E6-interacting protein. USP15 cleaves polyubiquitin chains of HPV16 E6 and/or ubiquitin precursors. Our results indicate that USP15 could increase the level of HPV16 E6 by inhibiting E6 degradation. USP15 inhibited the degradation of HPV16 E6 in dose-dependent manner. In contrast, catalytically inactive mutants of USP15 had a reduced inhibitory effect on E6 degradation. In particular, USP15 mutants of all three cysteine boxes and the NHL mutant of the KRF box had a drastically reduced inhibitory effect on HPV16 E6 degradation. In addition, HPV16 E6 mRNA was not induced by USP15; therefore, HPV16 E6 appears to be post-translationally regulated. These results suggest that USP15 has the ability to stabilize E6 as a deubiquitinating enzyme, and as an oncoprotein affects biological functions in infected human cells.

938RHRK941 is responsible for Ubiquitin specific protease 48 nuclear translocation which can stabilize NF-κB (p65) in the nucleus.

The transcription factor NF-κB is a key regulator of cellular processes. A mechanism that contributes to timely termination of NF-κB activity is UPS-dependent degradation of p65 in the nucleus or on chromatin. The ubiquitin-specific protease that takes part in this process and its molecular mechanisms are shown in previous study, but which structural feature of USP48 was responsible for these effects is unknown. Here, we show that maybe the stability of NF-κB is controlled by proteasome-mediated degradation and ubiquitin-specific protease 48 (USP48), also known as synaptic ubiquitin-specific protease (synUSP) or USP31, can enhance NF-κB stability through proteasome-dependent regulation in the nucleus. USP48 contains a carboxyl-terminal nuclear localizing signal, 938RHRK941, which is responsible for its nuclear translocation. Our results demonstrate a more detailed mechanism for this member of the USP gene family in cellular processes.

USP2a Supports Metastasis by Tuning TGF-ß Signaling.

TGF-ß has been demonstrated to promote tumor metastasis, and the regulatory mechanisms are poorly understood. Here, we report the role of USP2a in promoting metastasis by facilitating TGF-ß-triggered signaling. USP2a interacts with TGFBR1 and TGFBR2 upon TGF-ß stimulation and removes K33-linked polyubiquitin chains from Lys502 of TGFBR1, promoting the recruitment of SMAD2/3. Simultaneously, TGFBR2 phosphorylates Ser207/Ser225 of USP2a, leading to the disassociation of SMAD2/3 from TGFBR1. The phosphorylation of USP2a and SMAD2 is positively correlated in human tumor biopsies, and USP2a is hyper-phosphorylated in lung adenocarcinomas with lymph node invasion. Depletion or pharmacologic inhibition of USP2a dampens TGF-ß-triggered signaling and metastasis. Our findings have characterized an essential role of USP2a as a potential target for treatment of metastatic cancers.

K48-linked polyubiquitination of dengue virus NS1 protein inhibits its interaction with the viral partner NS4B.

Dengue virus (DENV) is a member of the Flaviviridae family, which is transmitted to mammalian species through arthropods, and causes dengue fever or severe dengue fever in humans. The DENV genome encodes for multiple nonstructural (NS) proteins including NS1. NS1 plays an essential role in replication by interacting with other viral proteins including NS4B, however how these interactions are regulated during virus infection is not known. By using bioinformatics, mass spectrometry analysis, and co-immunoprecipitation assays, here we show that DENV-NS1 is ubiquitinated on multiples lysine residues during DENV infection, including K189, a lysine residue previously shown to be important for efficient DENV replication. Data from in vitro and cell culture experiments indicate that dengue NS1 undergoes modification with K48-linked polyubiquitin chains, which usually target proteins to the proteasome for degradation. Furthermore, ubiquitinated NS1 was detected in lysates as well as in supernatants of human and mosquito infected cells. Ubiquitin deconjugation of NS1 using the deubiquitinase OTU resulted in increased interaction with the viral protein NS4B suggesting that ubiquitinated NS1 has reduced affinity for NS4B. In support of these data, a K189R mutation on NS1, which abrogates ubiquitination on amino acid residue 189 of NS1, also increased NS1-NS4B interactions. Our work describes a new mechanism of regulation of NS1-NS4B interactions and suggests that ubiquitination of NS1 may affect DENV replication.