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.

Synthesis and structure-activity relationships of USP48 deubiquitinylase inhibitors.

Ubiquitin-specific proteases represent a family of enzymes that catalyze the cleavage of ubiquitin from specific substrate proteins to regulate their activity. USP48 is a rarely studied USP, which has recently been linked to inflammatory signaling via regulation of the transcription factor nuclear factor kappa B. Nonetheless, a crystal structure of USP48 has not yet been resolved and potent inhibitors are not known. We screened a set of 14 commercially available USP inhibitors for their activity against USP48 and identified the USP2 inhibitor "ML364" as a candidate for further optimization. Using a ligand-based approach, we derived and synthesized a series of ML364 analogs. The IC50 concentrations of the new compounds to inhibit USP48 were determined in a deubiquitinylase activity assay by measuring the fluorescence intensity using tetra-ubiquitin rhodamine110 as substrate. A compound containing a carboxylic acid functionalization (17e) inhibited USP48 activity toward tetra-ubiquitin rhodamine110 with an IC50 of 12.6 µM. Further structure-based refinements are required to improve the inhibition activity and specificity.

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.

A Panel of Engineered Ubiquitin Variants Targeting the Family of Domains Found in Ubiquitin Specific Proteases (DUSPs).

Domains found in ubiquitin specific proteases (DUSPs) occur in seven members of the ubiquitin specific protease (USP) family. DUSPs are defined by a distinct structural fold but their functions remain largely unknown, although studies with USP4 suggest that its DUSP enhances deubiquitination activity. We used phage-displayed libraries of ubiquitin variants (UbVs) to derive protein-based tools to target DUSP family members with high affinity and specificity. We designed a UbV library based on insights from the structure of a previously identified UbV bound to the DUSP of USP15. The new library yielded 33 unique UbVs that bound to DUSPs from five different USPs (USP4, USP11, USP15, USP20 and USP33). For each USP, we were able to identify at least one DUSP that bound with high affinity and absolute specificity relative to the other DUSPs. We showed that UbVs targeting the DUSPs of USP15, USP11 and USP20 inhibited the catalytic activity of the enzyme, despite the fact that the DUSP is located outside of the catalytic domain. These findings provide an alternative means of inhibiting USP activity by targeting DUSPs, and this mechanism could be potentially extended other DUSP-containing USPs.

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.

Protein Loop Conformational Free Energy Changes via an Alchemical Path without Reaction Coordinates.

We introduce a method called restrain-free energy perturbation-release 2.0 (R-FEP-R 2.0) to estimate conformational free energy changes of protein loops via an alchemical path. R-FEP-R 2.0 is a generalization of the method called restrain-free energy perturbation-release (R-FEP-R) that can only estimate conformational free energy changes of protein side chains but not loops. The reorganization of protein loops is a central feature of many biological processes. Unlike other advanced sampling algorithms such as umbrella sampling and metadynamics, R-FEP-R and R-FEP-R 2.0 do not require predetermined collective coordinates and transition pathways that connect the two endpoint conformational states. The R-FEP-R 2.0 method was applied to estimate the conformational free energy change of a ß-turn flip in the protein ubiquitin. The result obtained by R-FEP-R 2.0 agrees with the benchmarks very well. We also comment on problems commonly encountered when applying umbrella sampling to calculate protein conformational free energy changes.

Self-stabilizing regulation of deubiquitinating enzymes in an enzymatic activity-dependent manner.

Deubiquitinating enzymes (DUBs) play important roles in many physiological and pathological processes by modulating the ubiquitination of their substrates. DUBs undergo post-translational modifications including ubiquitination. However, whether DUBs can reverse their own ubiquitination and regulate their own protein stability requires further investigation. To answer this question, we screened an expression library of DUBs and their enzymatic activity mutants and found that some DUBs regulated their own protein stability in an enzymatic activity- and homomeric interaction-dependent manner. Taking Ubiquitin-specific-processing protease 29 (USP29) as an example, we found that USP29 deubiquitinates itself and protects itself from proteasomal degradation. We also revealed that the N-terminal region of USP29 is critical for its protein stability. Taken together, our work demonstrates that at least some DUBs regulate their own ubiquitination and protein stability. Our findings provide novel molecular insight into the diverse regulation of DUBs.

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.

USP13 interacts with cohesin and regulates its ubiquitination in human cells.

Cohesin is a multiprotein ring complex that regulates 3D genome organization, sister chromatid cohesion, gene expression, and DNA repair. Cohesin is known to be ubiquitinated, although the mechanism, regulation, and effects of cohesin ubiquitination remain poorly defined. We previously used gene editing to introduce a dual epitope tag into the endogenous allele of each of 11 known components of cohesin in human HCT116 cells. Here we report that mass spectrometry analysis of dual-affinity purifications identified the USP13 deubiquitinase as a novel cohesin-interacting protein. Subsequent immunoprecipitation/Western blots confirmed the endogenous interaction in HCT116, 293T, HeLa, and RPE-hTERT cells; demonstrated that the interaction occurs specifically in the soluble nuclear fraction (not in the chromatin); requires the ubiquitin-binding domains (UBA1/2) of USP13; and occurs preferentially during DNA replication. Reciprocal dual-affinity purification of endogenous USP13 followed by mass spectrometry demonstrated that cohesin is its primary interactor in the nucleus. Ectopic expression and CRISPR knockout of USP13 showed that USP13 is paradoxically required for both deubiquitination and ubiquitination of cohesin subunits in human cells. USP13 was dispensable for sister chromatid cohesion in HCT116 and HeLa cells, whereas it was required for the dissociation of cohesin from chromatin as cells transit through mitosis. Together these results identify USP13 as a new cohesin-interacting protein that regulates the ubiquitination of cohesin and its cell cycle regulated interaction with chromatin.

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.

Restriction of Cellular Plasticity of Differentiated Cells Mediated by Chromatin Modifiers, Transcription Factors and Protein Kinases.

Ectopic expression of master regulatory transcription factors can reprogram the identity of specific cell types. The effectiveness of such induced cellular reprogramming is generally greatly reduced if the cellular substrates are fully differentiated cells. For example, in the nematode C. elegans, the ectopic expression of a neuronal identity-inducing transcription factor, CHE-1, can effectively induce CHE-1 target genes in immature cells but not in fully mature non-neuronal cells. To understand the molecular basis of this progressive restriction of cellular plasticity, we screened for C. elegans mutants in which ectopically expressed CHE-1 is able to induce neuronal effector genes in epidermal cells. We identified a ubiquitin hydrolase, usp-48, that restricts cellular plasticity with a notable cellular specificity. Even though we find usp-48 to be very broadly expressed in all tissue types, usp-48 null mutants specifically make epidermal cells susceptible to CHE-1-mediated activation of neuronal target genes. We screened for additional genes that allow epidermal cells to be at least partially reprogrammed by ectopic che-1 expression and identified many additional proteins that restrict cellular plasticity of epidermal cells, including a chromatin-related factor (H3K79 methyltransferase, DOT-1.1), a transcription factor (nuclear hormone receptor NHR-48), two MAPK-type protein kinases (SEK-1 and PMK-1), a nuclear localized O-GlcNAc transferase (OGT-1) and a member of large family of nuclear proteins related to the Rb-associated LIN-8 chromatin factor. These findings provide novel insights into the control of cellular plasticity.

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.

Examination of the Deubiquitylation Site Selectivity of USP51 by Using Chemically Synthesized Ubiquitylated Histones.

Histone ubiquitylation and deubiquitylation processes and the mechanisms of their regulation are closely relevant to the field of epigenetics. Recently, the deubiquitylating enzyme USP51 was reported to selectively cleave ubiquitylation on histone H2A at K13 or K15 (i.e., H2AK13Ub and H2AK15Ub), but not at K119 (i.e., H2AK119Ub), in nucleosomes in vivo. To elucidate the mechanism for the selectivity of USP51, we constructed structurally well-defined in vitro protein systems with a ubiquitin modification at precise sites. A total chemical protein synthesis procedure was developed, wherein hydrazide-based native chemical ligation was used to efficiently generate five ubiquitylated histones (H2AK13Ub, H2AK15Ub, H2AK119Ub, H2BK34Ub, and H2BK120Ub). These synthetic ubiquitylated histones were assembled into nucleosomes and subjected to in vitro USP51 deubiquitylation assays. Surprisingly, USP51 did not show preference between H2AK13/15Ub and H2AK119Ub, in contrast to previous in vivo observations. Accordingly, an understanding of the selectivity of USP51 may require consideration of other factors, such as alternative pre-existing histone modifications, competitive reader proteins, or different nucleosome quality among the in vivo extraction nucleosome and the in vitro reconstitution one. Further experiments established that USP51 in vitro could deubiquitylate a nucleosome carrying H2BK120Ub, but not H2BK34Ub. Molecular dynamics simulations suggested that USP51-catalyzed hydrolysis of ubiquitylated nucleosomes was affected by steric hindrance of the isopeptide bond.