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The ubiquitin-proteasome pathway is responsible for the turnover of different cellular proteins, such as transport proteins, presentation of antigens to the immune system, control of the cell cycle, and activities that promote cancer. The enzymes which remove ubiquitin, deubiquitylating enzymes (DUBs), play a critical role in central and peripheral immune tolerance to prevent the development of autoimmune diseases and thus present a potential therapeutic target for the treatment of autoimmune diseases. DUBs function by removing ubiquitin(s) from target protein and block ubiquitin chain elongation. The addition and removal of ubiquitin molecules have a significant impact on immune responses. DUBs and E3 ligases both specifically cleave target protein and modulate protein activity and expression. The balance between ubiquitylation and deubiquitylation modulates protein levels and also protein interactions. Dysregulation of the ubiquitin-proteasome pathway results in the development of various autoimmune diseases such as inflammatory bowel diseases (IBD), psoriasis, multiple sclerosis (MS), systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). This review summarizes the current understanding of ubiquitination in autoimmune diseases and focuses on various DUBs responsible for the progression of autoimmune diseases.

Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is a dual lipid and protein phosphatase. Multiple mechanisms contributing to the regulation of PTEN levels have been identified thus far, including post-translational modifications, epigenetic mechanisms, and transcriptional mechanisms. In the present study, we identified Ubiquitin specific peptidase 22 (USP22) as a novel deubiquitination-modifying enzyme of PTEN. Furthermore, by inducing deubiquitination and inhibiting the degradation of PTEN, USP22 could induce Cyclin dependent kinase inhibitor 1A (CDKN1A, also symbolled as p21) expression in pancreatic cancer. Besides, MDM2 proto-oncogene (MDM2) inhibitor enhanced the anti-pancreatic cancer effects of USP22 overexpression. In addition to its regulation of MDM2-Tumor protein p53 (p53) signaling, we found that PTEN could induce p21 expression by interacting with Ankyrin repeat and KH domain containing 1 (ANKHD1) and inhibiting ANKHD1 binding to the p21 promoter. Taken together, our results indicate that ANKHD1 and MDM2 might be novel therapeutic targets in pancreatic cancer.

Castration-resistant prostate cancer (CRPC) is a highly malignant type of advanced cancer resistant to androgen deprivation therapy. One of the important mechanisms for the development of CRPC is the persistent imbalanced regulation of AR and AR splice variants (AR/AR-Vs). In this study, we reported KDM4A-AS1, a recently discovered lncRNA, as a tumor promoter that was significantly increased in CRPC cell lines and cancer tissues. Depletion of KDM4A-AS1 significantly reduced cell viability, proliferation, migration in vitro, and tumor growth in vivo. We found that by binding to the NTD domain, KDM4A-AS1 enhances the stability of USP14-AR/AR-Vs complex, and promoted AR/AR-Vs deubiquitination to protect it from MDM2-mediated ubiquitin-proteasome degradation. Moreover, KDM4A-AS1 was found to enhance CRPC drug resistance to enzalutamide by repressing AR/AR-Vs degradation; antisense oligonucleotide drugs targeting KDM4A-AS1 significantly reduced the growth of tumors with enzalutamide resistance. Taken together, our results indicated that KDM4A-AS1 played an important role in the progression of CRPC and enzalutamide resistance by regulating AR/AR-Vs deubiquitination; targeting KDM4A-AS1 has broad clinical application potential.

Aurora A kinase is a cell cycle regulator that is dysregulated in several different malignancies. Nevertheless, its regulatory mechanisms are still not fully understood. Here, we report that ubiquitin specific peptidase 3 (USP3) promotes proliferation and metastasis of esophageal squamous cell carcinoma (ESCC) cells by mediating deubiquitination of Aurora A. Analysis of human clinical samples indicated that USP3 and Aurora A are highly expressed in ESCC. Cellular experiments confirmed that high expression of USP3 and Aurora A in ESCC cells promoted malignant cell proliferation and invasion. In this mechanism, USP3 leads to suppression of Aurora A ubiquitination, resulting less proteasome degradation. We constructed the deubiquitinated mimetic K143R of Aurora A and found that K143R significantly promoted the proliferation and invasion of ESCC cells and was not regulated by the deubiquitination of USP3. Moreover, Aurora A K143R potentiated the kinase activity of Aurora A in ESCC cells. Thus, our findings demonstrate that the tumorigenic feature of ESCC is in part mediated by USP3-facilitated deubiquitination of Aurora A.

Mounting studies have substantiated that abrogating autophagy contributes to cardiac hypertrophy (CH). Sirtuin 1 (SIRT1) has been reported to support autophagy and inhibit CH. However, the upstream regulation mechanism behind the regulation of SIRT1 level in CH remains unclear. Circular RNAs (circRNAs) are vital modulators in diverse human diseases including CH. This study intended to investigate the regulatory mechanism of circRNA on SIRT1 expression in CH. CH model was established by angiotensin II (Ang II) fusion or transverse aortic constriction (TAC) surgery and Ang II treatment on hiPSC-CMs and H9c2 cells in vitro. Our results showed that circ-SIRT1 (hsa_circ_0093884) expression was downregulated in Ang II-treated hiPSC-CMs, and confirmed that its conserved mouse homolog circ-Sirt1 (mmu_circ_0002354) was expressed at low levels in Ang II-treated H9c2 cells and TAC-induced mice model. Functionally, circ-SIRT1/circ-Sirt1 attenuated Ang II-induced CH and induced autophagy in hiPSC-CMs and H9c2 cardiomyocytes. Mechanistically, circ-SIRT1 could upregulate its host gene SIRT1 at the post-transcriptional level by sponging miR-3681-3p/miR-5195-3p and stabilized SIRT1 protein at the post-translational level by recruiting USP22 to induce deubiquitination on SIRT1 protein. Further, SIRT1 knockdown could rescue the effect of circ-SIRT1 upregulation on Ang II-induced CH and autophagy in vitro and in vivo. In conclusion, we first uncovered that circ-SIRT1 restrains CH via activating SIRT1 to promote autophagy, indicating circ-SIRT1 as a promising target to alleviate CH.

As an important post-translational modification, ubiquitination mediates ∼80% of protein degradation in eukaryotes. The degree of protein ubiquitination is tightly determined by the delicate balance between specific ubiquitin ligase (E3)-mediated ubiquitination and deubiquitinase-mediated deubiquitination. In 2017, we developed UbiBrowser 1.0, which is an integrated database for predicted human proteome-wide E3–substrate interactions. Here, to meet the urgent requirement of proteome-wide E3/deubiquitinase–substrate interactions (ESIs/DSIs) in multiple organisms, we updated UbiBrowser to version 2.0 (http://ubibrowser.ncpsb.org.cn). Using an improved protocol, we collected 4068/967 known ESIs/DSIs by manual curation, and we predicted about 2.2 million highly confident ESIs/DSIs in 39 organisms, with >210-fold increase in total data volume. In addition, we made several new features in the updated version: (i) it allows exploring proteins’ upstream E3 ligases and deubiquitinases simultaneously; (ii) it has significantly increased species coverage; (iii) it presents a uniform confidence scoring system to rank predicted ESIs/DSIs. To facilitate the usage of UbiBrowser 2.0, we also redesigned the web interface for exploring these known and predicted ESIs/DSIs, and added functions of ‘Browse’, ‘Download’ and ‘Application Programming Interface’. We believe that UbiBrowser 2.0, as a discovery tool, will contribute to the study of protein ubiquitination and the development of drug targets for complex diseases.

Ubiquitination and deubiquitination of signaling molecules are critical regulatory mechanisms in various biological contexts such as inflammatory signaling and the DNA damage response. Thus, finely tuned regulation of protein ubiquitination is essential for maintaining cellular homeostasis. Here, we showed that the RING finger protein RNF126 interacts with TRAF3 and promotes its K63-linked polyubiquitination, which is a crucial step in the TRAF3-dependent antiviral response. We found that RNF126 also interacts with OTUB1, a deubiquitinating enzyme that negatively regulates K63-linked ubiquitination of TRAF3. RNF126 promotes ubiquitination of OTUB1, leading to reduced deubiquitinating activity toward TRAF3. Moreover, RNF126 promotes ubiquitination of OTUB1 on cysteine 91, which is reportedly required for its catalytic activity. Taken together, our results suggest that RNF126 positively regulates the antiviral response by directly promoting K63-linked polyubiquitination of TRAF3 and by reducing OTUB1 activity.

Here, we describe a novel interaction between the RNA helicase DDX3 and the deubiquitinase ubiquitin-specific peptidase 9 X-linked (USP9X) in human cells. Domain mapping studies reveal that the C-terminal region of DDX3 interacted with the N terminus of USP9X. USP9X was predominantly localized in the cytoplasm where the interaction between DDX3 and USP9X occurred. USP9X was not visibly enriched in cytoplasmic stress granules (SGs) under oxidative stress conditions, whereas overexpression of GFP-DDX3 induced SG formation and recruited USP9X to SGs in HeLa cells. Luciferase reporter assays showed that depletion of USP9X had no significant effect on DDX3-mediated translation. Given that DDX3 is not ubiquitinated upon ubiquitin overexpression, it is unlikely that DDX3 serves as a substrate of USP9X. Importantly, we found that ubiquitinated MCL1 was accumulated upon depletion of USP9X and/or DDX3 in MG132-treated cells, suggesting that USP9X and DDX3 play a role in regulating MCL1 protein stability and anti-apoptotic function. This study indicates that DDX3 exerts anti-apoptotic effects probably by coordinating with USP9X in promoting MCL1 deubiquitination.

The ubiquitination and deubiquitination of proteins govern signal transduction in every aspect of physiology and pathology, especially in cancer, inflammation, and autoimmune diseases. Rapid progress has been made in obtaining an in-depth understanding of the ubiquitination system since its first discovery during the 1970s. Manipulation of ubiquitination by small molecules is considered a novel therapeutic avenue. In this review, we summarize key applications of small molecules targeting ubiquitination enzymes and currently available technologies applied to the discovery of small molecules that control ubiquitination.

Innate immune sensing of cytosolic DNA via absent in melanoma 2 (AIM2) is a key mechanism leading to inflammatory responses. As aberrant immune responses by dysregulated AIM2 are associated with autoinflammatory diseases, activation of the AIM2 inflammasome should be tightly controlled. In this study, we discovered that ubiquitination and deubiquitination of AIM2 are critical events that regulate AIM2 inflammasome activation. In resting human macrophage cells, AIM2 is constitutively ubiquitinated and undergoes proteasomal degradation to avoid autoinflammation. Upon DNA stimulation, USP21 binds to AIM2 and deubiquitinates it, thereby increasing its protein stability. In addition to the role of USP21 in regulating AIM2 turnover, we uncovered that USP21-mediated deubiquitination of AIM2 is required for the assembly of the AIM2 inflammasome. Depletion of USP21 does not affect the DNA-binding ability of AIM2 but inhibits the formation of the AIM2–ASC complex. Our findings establish that fine-tuning of AIM2 by the ubiquitin system is important for regulating AIM2 inflammasome activation.

Protein Disulfide Isomerase Family A Member 6 (PDIA6) is an endoplasmic reticulum protein that is capable of catalyzing protein folding and disulfide bond formation. Abnormally elevated expression of PDIA6 has been reported to predict poor outcomes in various cancers. Herein, gain-of- and loss-of-function experiments were performed to investigate how PDIA6 participated in the carcinogenesis of pancreatic cancer (PC). By analyzing the protein expression of PDIA6 in 28 paired PC and para carcinoma specimens, we first found that PDIA6 expression was higher in PC samples. Both the overall survival and disease-free survival rates of PC patients with higher PDIA6 expression were poorer than those with lower PDIA6 (n = 178). Furthermore, knockdown of PDIA6 impaired the malignancies of PC cells — suppressed cell proliferation, invasion, migration, cisplatin resistance, and xenografted tumor growth. PDIA6-silenced PC cells were more sensitive to cytotoxic natural killer (NK) cells. Overexpression of PDIA6 had opposite effects on PC cells. Interestingly, COP9 signalosome subunit 5 (CSN5), a regulator of E3 ubiquitin ligases known to promote deubiquitination of its downstream targets, was demonstrated to interact with PDIA6, and its expression was increased in PC cells overexpressing PDIA6. Additionally, PDIA6 overexpression promoted deubiquitination of β-catenin and PD-L1 and subsequently upregulated their expression in PC cells. These alterations were partly reversed by CSN5 shRNA. Collectively, the above results demonstrate that PDIA6 contributes to PC progression, which may be associated with CSN5-regulated deubiquitination of β-catenin and PD-L1. Our findings suggest PDIA6 as a potential target for the treatment of PC., Graphical Abstract Image, graphical abstract

Dysregulation of deubiquitination has been reported to contribute to carcinogenesis. However, the function and mechanism of deubiquitinating enzyme 26S proteasome non‐ATPase regulatory subunit 14 (PSMD14) in the progression of ovarian cancer (OV), the deadliest gynecological cancer, still remains to be characterized. The present study demonstrated that PSMD14 was overexpressed in OV tissues and its higher levels correlated with a higher International Federation of Gynecology and Obstetrics (FIGO) stage in OV patients. A high level of PSMD14 expression was related to poor survival in OV patients. Knockdown and overexpression experiments elucidated that PSMD14 stimulated OV cell proliferation, invasion, and migration in vitro. Repression of PSMD14 suppressed OV tumor growth in vivo. PSMD14 inhibitor O‐phenanthroline (OPA) effectively attenuated malignant behaviors of OV cells in vitro and OV tumor growth in vivo. Mechanistically, we uncovered that PSMD14 was involved in post‐translational regulation of pyruvate kinase M2 isoform (PKM2). PSMD14 decreased K63‐linked ubiquitination on PKM2, downregulated the ratio of PKM2 tetramers to dimers and monomers, and subsequently diminished pyruvate kinase activity and induced nuclear translocation of PKM2, contributing to aerobic glycolysis in OV cells. Collectively, our findings highlight the potential roles of PSMD14 as a biomarker and therapeutic candidate for OV., PSMD14 decreases K63‐linked ubiquitination on PKM2 and downregulates the ratio of PKM2 tetramers to dimers and monomers. Subsequently, this diminishes pyruvate kinase activity and induces nuclear translocation of PKM2 as well as transcription of downstream genes, contributing to aerobic glycolysis in ovarian cancer cells and progression of ovarian cancer.

Purpose: To determine the effect of Bu-Shen-Zhu-Yun Decoction (BSZY-D) on the kisspeptin through JAK2/STAT5 signaling pathway in hyperprolactinemia (HPRL) infertility. Method: SD rats were treated with BSZY-D for cerebrospinal fluid (CSF) extraction. GT1-7 cells were subjected to different treatments. The phosphorylation levels of JAK2 and STAT5, and the expressions of PRLR and kisspeptin of GT1-7 cells in different groups were detected by western blot, RT-qPCR and immunofluorescence. The expressions of CSN5 and GATA1 and other molecular features were checked by western blot, RT-PCR, co-immunoprecipitation and renilla luciferase activity. Results: The phosphorylation levels of JAK2 and STAT5, and the expressions of PRLR and kisspeptin in the HPRL group were significantly decreased, and these changes could be reversed after BSZY-D treatment. In addition, the presence of PRLR deubiquitination was detected in the HPRL group, which could be reversed by shRNA-CSN5, suggesting that BSZY-D played a role through targeting CSN5. The binding level of GATA1 and CSN5 promoter in the HPRL group was significantly decreased, but elevated in the HPRL (BSZY-D/CSF) group (P < 0.05). Conclusion: BSZY-D improved the transcription activity of GATA1 and increased the binding of GATA1 and CSN5. BSZY-D was involved in the deubiquitination of PRLR, which contributes to alleviating the symptoms of HPRL infertility.

New neurons are generated throughout life in distinct regions of the mammalian brain due to the proliferation and differentiation of neural stem cells (NSCs). Ubiquitin, a post-translational modification of cellular proteins, is an important factor in regulating neurogenesis. Deubiquitination is a biochemical process that mediates the removal of ubiquitin moieties from ubiquitin-conjugated substrates. Recent studies have provided growing evidence that deubiquitylases (DUBs) which reverse ubiquitylation process play critical roles in NSCs maintenance, differentiation and maturation. This review mainly focused on the relationship of DUBs and NSCs, and further summarized recent advances in our understanding of DUBs on regulating NSCs biological behaviors.

Recently, many studies have shown that deubiquitination modification of proteins is of great significance in major physiological processes such as cell proliferation, apoptosis, and differentiation. The ubiquitin-specific peptidase (USP) family is one of the most numerous and structurally diverse of the deubiquitinates known to date. USP22, an important member of the USP family, has been found to be closely associated with tumor cell cycle regulation, stemness maintenance, invasion and metastasis, chemoresistance, and immune regulation. We focus on recent advances regarding USP22's function in cancer and discuss the prospect of USP22 in this review.

Fanconi anemia (FA) is a rare genetic disorder caused by mutations in any of the currently 22 known FA genes. The products of these genes, along with other FA-associated proteins, participate in a biochemical pathway, known as the FA pathway. This pathway is responsible for the repair of DNA interstrand cross-links (ICL) and the maintenance of genomic stability in response to replication stress. At the center of the pathway is the monoubiquitination of two FA proteins, FANCD2 and FANCI, on two specific lysine residues. This is achieved by the combined action of the UBE2T ubiquitin-conjugating enzyme and a large multicomponent E3 ligase, known as the FA-core complex. This E2-E3 pair specifically targets the FANCI-FANCD2 heterodimer (ID2 complex) for ubiquitination on DNA. Deubiquitination of both FANCD2 and FANCI, which is also critical for ICL repair, is achieved by the USP1-UAF1 complex. Recent work suggests that FANCD2 ubiquitination transforms the ID2 complex into a sliding DNA clamp. Further, ID2 ubiquitination on FANCI does not alter the closed ID2 conformation observed upon FANCD2 ubiquitination and the associated ID2Ub complex with high DNA affinity. However, the resulting dimonoubiquitinated complex is highly resistant to USP1-UAF1 deubiquitination. This review will provide an update on recent work focusing on how specificity in FANCD2 ubiquitination and deubiquitination is achieved. Recent findings shedding light to the mechanisms, molecular functions, and biological roles of FANCI/FANCD2 ubiquitination and deubiquitination will be also discussed. ENZYMES: UBA1 (6.2.1.45), UBE2T (2.3.2.23), FANCL (2.3.2.27), USP1 (3.4.19.12).

Ovarian tumor domain-containing 6B (OTUD6B) belongs to the OTU deubiquitylating enzyme family. In this study, we report that zebrafish otud6b is induced upon viral infection, and overexpression of otud6b suppresses cellular antiviral response. Disruption of otud6b in zebrafish increases the survival rate upon spring viremia of carp virus and grass carp reovirus exposure. Further assays indicate that otud6b interacts with irf3 and irf7 and diminishes traf6-mediated K63-linked polyubiquitination of irf3 and irf7. In addition, the OTU domain is required for otud6b to repress IFN-1 activation and K63-linked polyubiquitination of irf3 and irf7. Moreover, otud6b also attenuates tbk1 to bind to irf3 and irf7, resulting in the impairment of irf3 and irf7 phosphorylation. This study provides, to our knowledge, novel insights into otud6b function and sheds new lights on the regulation of irf3 and irf7 by deubiquitination in IFN-1 signaling.

NLRP3 inflammasome plays an important role in innate immune system through recognizing pathogenic microorganisms and danger-associated molecules. Deubiquitination of NLRP3 has been shown to be essential for its activation, yet the functions of Ubc13, the K63-linked specific ubiquitin-conjugating enzyme E2, in NLRP3 inflammasome activation are not known. In this study, we found that in mouse macrophages, Ubc13 knockdown or knockout dramatically impaired NLRP3 inflammasome activation. Catalytic activity is required for Ubc13 to control NLRP3 activation, and Ubc13 pharmacological inhibitor significantly attenuates NLRP3 inflammasome activation. Mechanistically, Ubc13 associates with NLRP3 and promotes its K63-linked polyubiquitination. Through mass spectrum and biochemical analysis, we identified lysine 565 and lysine 687 as theK63-linked polyubiquitination sites of NLRP3. Collectively, our data suggest that Ubc13 potentiates NLRP3 inflammasome activation via promoting site-specific K63-linked ubiquitination of NLRP3. Our study sheds light on mechanisms of NLRP3 inflammasome activation and identifies that targeting Ubc13 could be an effective therapeutic strategy for treating aberrant NLRP3 inflammasome activation–induced pathogenesis.

Interleukin-1β (IL-1β) is activated by inflammasome-associated caspase-1 in rare autoinflammatory conditions and in a variety of other inflammatory diseases. Therefore, IL-1β activity must be fine-tuned to enable anti-microbial responses whilst limiting collateral damage. Here, we show that precursor IL-1β is rapidly turned over by the proteasome and this correlates with its decoration by K11-linked, K63-linked and K48-linked ubiquitin chains. The ubiquitylation of IL-1β is not just a degradation signal triggered by inflammasome priming and activating stimuli, but also limits IL-1β cleavage by caspase-1. IL-1β K133 is modified by ubiquitin and forms a salt bridge with IL-1β D129. Loss of IL-1β K133 ubiquitylation, or disruption of the K133:D129 electrostatic interaction, stabilizes IL-1β. Accordingly, Il1bK133R/K133R mice have increased levels of precursor IL-1β upon inflammasome priming and increased production of bioactive IL-1β, both in vitro and in response to LPS injection. These findings identify mechanisms that can limit IL-1β activity and safeguard against damaging inflammation.

CARD9 is an essential adaptor protein in antifungal innate immunity mediated by C-type lectin receptors. The activity of CARD9 is critically regulated by ubiquitination; however, the deubiquitinases involved in CARD9 regulation remain incompletely understood. In this study, we identified ovarian tumor deubiquitinase 1 (OTUD1) as an essential regulator of CARD9. OTUD1 directly interacted with CARD9 and cleaved polyubiquitin chains from CARD9, leading to the activation of the canonical NF-κB and MAPK pathway. OTUD1 deficiency impaired CARD9-mediated signaling and inhibited the proinflammatory cytokine production following fungal stimulation. Importantly, Otud1–/– mice were more susceptible to fungal infection than wild-type mice in vivo. Collectively, our results identify OTUD1 as an essential regulatory component for the CARD9 signaling pathway and antifungal innate immunity through deubiquitinating CARD9.

Background NOD-like receptors affect multiple stages of cancer progression in many malignancies. NACHT, LRR, and PYD domain-containing protein 7 (NLRP7) is a member of the NOD-like receptor family, although its role in tumorigenesis remains unclear. By analyzing clinical samples, we found that NLRP7 protein levels were upregulated in colorectal cancer (CRC). We proposed the hypothesis that a high level of NLRP7 in CRC may promote tumor progression. Here, we further investigated the role of NLRP7 in CRC and the underlying mechanism. Methods NLRP7 expression in human CRC and adjacent non-tumorous tissues was examined by quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, and immunohistochemistry. The effect of NLRP7 in CRC progression was investigated in vitro and in vivo. Proteins interacting with NLRP7 were identified by immunoprecipitation and mass spectrometry analysis while immunofluorescence staining revealed the cellular location of the proteins. Cellular ubiquitination and protein stability assays were applied to demonstrate the ubiquitination effect on NLRP7. Cloning and mutagenesis were used to identify a lysine acceptor site that mediates NLRP7 ubiquitination. Cytokines/chemokines affected by NLRP7 were identified by RNA sequencing, qRT-PCR, and enzyme-linked immunosorbent assay. Macrophage phenotypes were determined using qRT-PCR, flow cytometry, and immunohistochemistry. Results NLRP7 protein levels, but not mRNA levels, were upregulated in CRC, and increased NLRP7 protein expression was associated with poor survival. NLRP7 promoted tumor cell proliferation and metastasis in vivo and in vitro and interacted with ubiquitin-specific protease 10, which catalyzed its deubiquitination in CRC cells. NLRP7 stability and protein levels in CRC cells were modulated by ubiquitination and deubiquitination, and NLRP7 was involved in the ubiquitin-specific protease 10 promotion of tumor progression and metastasis in CRC. K379 was an important lysine acceptor site that mediates NLRP7 ubiquitination in CRC cells. In CRC, NLRP7 promoted the polarization of pro-tumor M2-like macrophages by inducing the secretion of C-C motif chemokine ligand 2. Furthermore, NLRP7 promoted NF-κB nuclear translocation and activation of C-C motif chemokine ligand 2 transcription. Conclusions We showed that NLRP7 promotes CRC progression and revealed an as-yet-unidentified mechanism by which NLRP7 induces the polarization of pro-tumor M2-like macrophages. These results suggest that NLRP7 could serve as a biomarker and novel therapeutic target for the treatment of CRC.

The oncogenic transcription factor c-Maf has been proposed as an ideal therapeutic target for multiple myeloma (MM), but how to achieve it is still elusive. In the present study, we found the Otub1/c-Maf axis could be a potential target. Otub1, an OTU family deubiquitinase, was found to interact with c-Maf by mass spectrometry. Otub1 abrogates c-Maf K48-linked polyubiquitination, thus preventing its degradation and enhancing its transcriptional activity. Specifically, this deubiquitinating activity depends on its Lys71 and the N terminus but is independent of UBE2O, a known E2 of c-Maf. Otub1 promotes MM cell survival and MM tumor growth. In contrast, silence of Otub1 leads to c-Maf degradation and c-Maf-expressing MM cell apoptosis. Therefore, the Otub1/c-Maf axis could be a therapeutic target of MM. In order to explore this concept, we performed a c-Maf recognition element–driven luciferase-based screen against US Food and Drug Administration–approved drugs and natural products, from which the generic cardiac glycoside lanatoside C (LanC) is found to prevent c-Maf deubiquitination and induces its degradation by disrupting the interaction of Otub1 and c-Maf. Consequently, LanC inhibits c-Maf transcriptional activity, induces c-Maf-expressing MM cell apoptosis, and suppresses MM growth and prolongs overall survival of model mice, but without apparent toxicity. Therefore, the present study identifies Otub1 as a novel deubiquitinase of c-Maf and establishes that the Otub1/c-Maf axis is a potential therapeutic target for MM.

A growing amount of evidence suggests that ubiquitination and deubiquitination of programmed death 1 (PD-1)/programmed death-ligand 1 (PD-L1) play crucial roles in the regulation of PD-1 and PD-L1 protein stabilization and dynamics. PD-1/PD-L1 is a major coinhibitory checkpoint pathway that modulates immune escape in cancer patients, and its engagement and inhibition has significantly reshaped the landscape of tumor clearance. The abnormal ubiquitination and deubiquitination of PD-1/PD-L1 influence PD-1/PD-L1-mediated immunosuppression. In this review, we describe the ubiquitination- and deubiquitination-mediated modulation of PD-1/PD-L1 signaling through a variety of E3 ligases and deubiquitinating enzymes (DUBs). Moreover, we briefly expound on the anticancer potential of some agents that target related E3 ligases, which further modulate the ubiquitination of PD-1/PD-L1 in cancers. Therefore, this review reveals the development of a highly promising therapeutic approach for cancer immunotherapy by targeting PD-1/PD-L1 ubiquitination.

The transcription factor nuclear factor (erythroid-derived 2)-like 2 (NRF2) plays a key role in cancer progression and is tightly regulated by the proteasome pathway. E3 ligases that mediate NRF2 ubiquitination have been widely reported, but the mechanism of NRF2 deubiquitination remains largely unclear. Here, we identified ubiquitin-specific-processing protease 11 (USP11) in NRF2 complexes and confirmed an interaction between these two proteins. We further found that USP11 deubiquitinates NRF2; this modification stabilizes NRF2. Functionally, USP11 depletion contributes to the suppression of cell proliferation and induction of ferroptotic cell death due to ROS-mediated stress, which can be largely abrogated by overexpression of NRF2. Finally, immunohistochemical staining of USP11 and NRF2 was performed using a lung tissue microarray, which revealed that USP11 is highly expressed in patients with NSCLC and positively correlated with NRF2 expression. Together, USP11 stabilizes NRF2 and is thus an important player in cell proliferation and ferroptosis.

Radioresistance is regarded as the main barrier to effective radiotherapy in lung cancer. However, the underlying mechanisms of radioresistance remain elusive. Here, we show that lysine-specific demethylase 4C (KDM4C) is overexpressed and correlated with poor prognosis in lung cancer patients. We provide evidence that genetical or pharmacological inhibition of KDM4C impairs tumorigenesis and radioresistance in lung cancer in vitro and in vivo. Moreover, we uncover that KDM4C upregulates TGF-β2 expression by directly reducing H3K9me3 level at the TGF-β2 promoter and then activates Smad/ATM/Chk2 signaling to confer radioresistance in lung cancer. Using tandem affinity purification technology, we further identify deubiquitinase USP9X as a critical binding partner that deubiquitinates and stabilizes KDM4C. More importantly, depletion of USP9X impairs TGF-β2/Smad signaling and radioresistance by destabilizing KDM4C in lung cancer cells. Thus, our findings demonstrate that USP9X-mediated KDM4C deubiquitination activates TGF-β2/Smad signaling to promote radioresistance, suggesting that targeting KDM4C may be a promising radiosensitization strategy in the treatment of lung cancer.

The ubiquitin system regulates a wide variety of cellular functions. Not surprisingly, dysregulation of the ubiquitin system is associated with various disorders. Therefore, drugs that can modulate the functions of the ubiquitin system have been actively developed to treat these disorders. Chemical knockdown of pathogenic proteins using the ubiquitin-proteasome system is also a promising approach. The ubiquitin system regulates the assemble and disassemble of primary cilia through balanced control over the ubiquitination and deubiquitination of ciliary proteins. Primary cilia are antenna-like structures present in many vertebrate cells that sense and transduce extracellular cues to control cellular processes such as proliferation and differentiation. Impairment of primary cilia is associated with many diseases, including cancer and ciliopathy, a group of multisystem developmental disorders. In this review, we focus on the role of the ubiquitin system on cilia-related disorders and discuss the possibility of the ubiquitin system as therapeutic targets for these diseases through regulation of primary cilia formation.

The glucagon receptor (GCGR) activated by the peptide hormone glucagon is a seven-transmembrane G protein-coupled receptor (GPCR) that regulates blood glucose levels. Ubiquitination influences trafficking and signaling of many GPCRs, but its characterization for the GCGR is lacking. Using endocytic colocalization and ubiquitination assays, we have identified a correlation between the ubiquitination profile and recycling of the GCGR. Our experiments revealed that GCGRs are constitutively ubiquitinated at the cell surface. Glucagon stimulation not only promoted GCGR endocytic trafficking through Rab5a early endosomes and Rab4a recycling endosomes, but also induced rapid deubiquitination of GCGRs. Inhibiting GCGR internalization or disrupting endocytic trafficking prevented agonist-induced deubiquitination of the GCGR. Furthermore, a Rab4a dominant negative (DN) that blocks trafficking at recycling endosomes enabled GCGR deubiquitination, whereas a Rab5a DN that blocks trafficking at early endosomes eliminated agonist-induced GCGR deubiquitination. By down-regulating candidate deubiquitinases that are either linked with GPCR trafficking or localized on endosomes, we identified signal-transducing adaptor molecule-binding protein (STAMBP) and ubiquitin-specific protease 33 (USP33) as cognate deubiquitinases for the GCGR. Our data suggest that USP33 constitutively deubiquitinates the GCGR, whereas both STAMBP and USP33 deubiquitinate agonist-activated GCGRs at early endosomes. A mutant GCGR with all five intracellular lysines altered to arginines remains deubiquitinated and shows augmented trafficking to Rab4a recycling endosomes compared with the WT, thus affirming the role of deubiquitination in GCGR recycling. We conclude that the GCGRs are rapidly deubiquitinated after agonist-activation to facilitate Rab4a-dependent recycling and that USP33 and STAMBP activities are critical for the endocytic recycling of the GCGR.

The process of ubiquitination and deubiquitination is widely present in the human body's protein reactions and plays versatile roles in multiple diseases. Deubiquitinating enzymes (DUBs) are significant regulators of this process, which cleave the ubiquitin (Ub) moiety from various substrates and maintain protein stability. Lung adenocarcinoma (LUAD) is the most common type of non-small cell lung cancer (NSCLC) and remains refractory to treatment. To elucidate the mechanism of LUAD and advance new therapeutic targets, we review the latest research progress on DUBs in LUAD. We summarize the biological capabilities of these DUBs and further highlight those DUBs that may serve as anticancer target candidates for precision treatment. We also discuss deubiquitinase inhibitors, which are expected to play a role in targeted LUAD therapy.

The 26S proteasome is responsible for regulated proteolysis in eukaryotic cells. Its substrates are diverse in structure, function, sequence length, and amino acid composition, and are targeted to the proteasome by post-translational modification with ubiquitin. Ubiquitination occurs through a complex enzymatic cascade and can also signal for other cellular events, unrelated to proteasome-catalyzed degradation. Like other post-translational protein modifications, ubiquitination is reversible, with ubiquitin chain hydrolysis catalyzed by the action of deubiquitinating enzymes (DUBs), ~90 of which exist in humans and allow for temporal events as well as dynamic ubiquitin-chain remodeling. DUBs have been known for decades to be an integral part of the proteasome, as deubiquitination is coupled to substrate unfolding and translocation into the internal degradation chamber. Moreover, the proteasome also binds several ubiquitinating enzymes as well as shuttle factors that recruit ubiquitinated substrates. The role of this intricate machinery and how ubiquitinated substrates interact with proteasomes remains an area of active investigation. Here, we review what has been learned about the mechanisms used by the proteasome to bind ubiquitinated substrates, substrate shuttle factors, ubiquitination machinery, and DUBs. We also discuss many open questions that require further study or the development of innovative approaches to be answered. Finally, we address the promise of expanded therapeutic targeting that could benefit from such new discoveries.

Programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) blocking therapy has become a major pillar of cancer immunotherapy. Compared with antibodies targeting, small-molecule checkpoint inhibitors which have favorable pharmacokinetics are urgently needed. Here we identified berberine (BBR), a proven anti-inflammation drug, as a negative regulator of PD-L1 from a set of traditional Chinese medicine (TCM) chemical monomers. BBR enhanced the sensitivity of tumour cells to co-cultured T-cells by decreasing the level of PD-L1 in cancer cells. In addition, BBR exerted its antitumor effect in Lewis tumor xenograft mice through enhancing tumor-infiltrating T-cell immunity and attenuating the activation of immunosuppressive myeloid-derived suppressor cells (MDSCs) and regulatory T-cells (Tregs). BBR triggered PD-L1 degradation through ubiquitin (Ub)/proteasome-dependent pathway. Remarkably, BBR selectively bound to the glutamic acid 76 of constitutive photomorphogenic-9 signalosome 5 (CSN5) and inhibited PD-1/PD-L1 axis through its deubiquitination activity, resulting in ubiquitination and degradation of PD-L1. Our data reveals a previously unrecognized antitumor mechanism of BBR, suggesting BBR is small-molecule immune checkpoint inhibitor for cancer treatment.

PTEN protein levels are regulated through ubiquitination. Here, we identify BAP1 as a deubiquitinase that physically binds to and deubiquitinates PTEN, thereby inhibiting PTEN degradation. Stabilized PTEN suppresses prostate cancer progression by inhibiting Akt phosphorylation., Deubiquitinase BAP1 is an important tumor suppressor in several malignancies, but its functions and critical substrates in prostate cancer (PCa) remain unclear. Here, we report that the mRNA and protein expression levels of BAP1 are downregulated in clinical PCa specimens. BAP1 can physically bind to and deubiquitinate PTEN, which inhibits the ubiquitination‐mediated degradation of PTEN and thus stabilizes PTEN protein. Ectopically expressed BAP1 in PCa cells increases PTEN protein level and subsequently inhibits the AKT signaling pathway, thus suppressing PCa progression. Conversely, knockdown of BAP1 in PCa cells leads to the decrease in PTEN protein level and the activation of the Akt signaling pathway, therefore promoting malignant transformation and cancer metastasis. However, these can be reversed by the re‐expression of PTEN. More importantly, we found that BAP1 protein level positively correlates with PTEN in a substantial fraction of human cancers. These findings demonstrate that BAP1 is an important deubiquitinase of PTEN for its stability and the BAP1‐PTEN signaling axis plays a crucial role in tumor suppression.

Impaired protein stability or trafficking underlies diverse ion channelopathies and represents an unexploited unifying principle for developing common treatments for otherwise dissimilar diseases. Ubiquitination limits ion channel surface density, but targeting this pathway for the purposes of basic study or therapy is challenging because of its prevalent role in proteostasis. We developed engineered deubiquitinases (enDUBs) that enable selective ubiquitin chain removal from target proteins to rescue the functional expression of disparate mutant ion channels that underlie long QT syndrome (LQT) and cystic fibrosis (CF). In an LQT type 1 (LQT1) cardiomyocyte model, enDUB treatment restored delayed rectifier potassium currents and normalized action potential duration. CF-targeted enDUBs synergistically rescued common (ΔF508) and pharmacotherapy-resistant (N1303K) CF mutations when combined with the US Food and Drug Administation (FDA)-approved drugs Orkambi (lumacaftor/ivacaftor) and Trikafta (elexacaftor/tezacaftor/ivacaftor and ivacaftor). Altogether, targeted deubiquitination via enDUBs provides a powerful protein stabilization method that not only corrects diverse diseases caused by impaired ion channel trafficking, but also introduces a new tool for deconstructing the ubiquitin code in situ.

NOD-like receptor protein 3 (NLRP3) detects microbial infections or endogenous danger signals and activates the NLRP3 inflammasome, which has important functions in host defense and contributes to the pathogenesis of inflammatory diseases, and thereby needs to be tightly controlled. Deubiquitination of NLRP3 is considered a key step in NLRP3 inflammasome activation. However, the mechanisms by which deubiquitination controls NLRP3 inflammasome activation are unclear. Here, we show that the UAF1/USP1 deubiquitinase complex selectively removes K48-linked polyubiquitination of NLRP3 and suppresses its ubiquitination-mediated degradation, enhancing cellular NLRP3 levels, which are indispensable for subsequent NLRP3 inflammasome assembly and activation. In addition, the UAF1/USP12 and UAF1/USP46 complexes promote NF-κB activation, enhance the transcription of NLRP3 and proinflammatory cytokines (including pro-IL-1β, TNF, and IL-6) by inhibiting ubiquitination-mediated degradation of p65. Consequently, Uaf1 deficiency attenuates NLRP3 inflammasome activation and IL-1β secretion both in vitro and in vivo. Our study reveals that the UAF1 deubiquitinase complexes enhance NLRP3 and pro-IL-1β expression by targeting NLRP3 and p65 and licensing NLRP3 inflammasome activation., NLRP3 inflammasome activation is regulated by various signaling pathways to ensure inflammation does not go unchecked. Here the authors show how deubiquitination avoids this regulation to activate the NLRP3 inflammasome through the function of UAF1/USP deubiquitinase complexes.

Na(+)/H(+) exchanger NHE3 of human or primates differs from NHE3 of other animals by having a PY motif, which mediates interaction with the E3 ubiquitin (Ub) ligase Nedd4–2. Ub-conjugation of human NHE3 by Nedd4–2 regulates endocytosis of NHE3 but does not affect its cellular expression. Because Ub-conjugation is a reversible process, the aim of this study is to identify deubiquitinating enzyme (DUB) regulating the post-endosomal fate of human NHE3. Using an activity-based chemical screening, we identified ubiquitin specific protease-7 (USP7) and USP10 that bind NHE3. The roles of DUBs in regulation of NHE3 were studied by determining the effects of silencing of USP7 and USP10. Knockdown of USP7 or USP10 resulted in increased NHE3 ubiquitination and decreased NHE3 expression at the surface membrane and cellular level. The endocytic retrieval of NHE3 was promoted by depletion of USP7 or USP10, with increased association of NHE3 with Rab5a and Rab7. Inhibition of USP7 and USP10 by chemical inhibitors or knockdown had an additive effect on NHE3. In addition, NHE3 half-life was reduced accounting for decreased NHE3 protein abundance. NHE3 is inhibited by protein kinase A. Activation of PKA by forskolin decreased the binding of USP7 and USP10 to NHE3, while increasing ubiquitination of NHE3. Knockdown of USP10 had an additive effect on PKA-dependent inhibition of NHE3. These findings demonstrate that USP7 and USP10 are DUBs that regulate NHE3 ubiquitination and expression, and reveal a new mechanism of NHE3 inhibition involving DUBs.

Deubiquitination, the inverse process of ubiquitination, is catalyzed by deubiquitinases (DUBs) that remove ubiquitin from target proteins and subsequently prevent their degradation by proteasomes. Previously, deubiquitination has been found to be involved in hepatocellular carcinoma (HCC) progression. As yet, however, little is known about the exact role of deubiquitination in the development and/or progression of this type of cancer. HCC tissues and tissue microarrays were used to detect expression of the DUB ubiquitin-specific protease 2a (USP2a). The critical role of USP2a in HCC development and progression was assessed in both in vitro cell and in vivo animal models. LC-MS/MS analyses were performed to identify potential targets of USP2a in HCC cells, after which regulation of target protein stability and ubiquitin status by USP2a were investigated. We found that USP2a was significantly upregulated in HCC tissues, and that a high expression was positively associated with a poor prognosis. Subsequently, we found that USP2a silencing resulted in inhibition of HCC cell proliferation, migration and invasion, whereas exogenous USP2a overexpression resulted in the opposite effects, both in vitro and in vivo. Mechanistically, LC-MS/MS analysis revealed that RAB1A, a key regulator of the ER and Golgi vesicular transport system, serves as a potential target of USP2a in HCC cells. In addition, we found that USP2a can deubiquitinate and stabilize RAB1A and prevent its degradation, and that this process is required for inducing HCC progression by USP2a. Our data indicate that USP2a can promote HCC progression via deubiquitination and stabilization of RAB1A. This observation indicates that DUB targeting may serve as a novel approach to improve the treatment of HCC.

13 páginas, 5 figuras, 1 tabla. Contiene información suplementaria en: Supplementary information accompanies this paper at https://doi.org/10.1186/s13072-020-00367-3, Background: Histone H2B deubiquitination is performed by numerous deubiquitinases in eukaryotic cells including Ubp8, the catalytic subunit of the tetrameric deubiquitination module (DUBm: Ubp8; Sus1; Sgf11; Sgf73) of the Spt-Ada-Gcn5 acetyltransferase (SAGA). Ubp8 is linked to the rest of SAGA through Sgf73 and is activated by the adaptors Sus1 and Sgf11. It is unknown if DUBm/Ubp8 might also work in a SAGA-independent manner. Results: Here we report that a tetrameric DUBm is assembled independently of the SAGA-CORE components SPT7, ADA1 and SPT20. In the absence of SPT7, i.e., independent of the SAGA complex, Ubp8 and Sus1 are poorly recruited to SAGA-dependent genes and to chromatin. Notably, cells lacking Spt7 or Ada1, but not Spt20, show lower levels of nuclear Ubp8 than wild-type cells, suggesting a possible role for SAGA-CORE subunits in Ubp8 localization. Last, deletion of SPT7 leads to defects in Ubp8 deubiquitinase activity in in vivo and in vitro assays. Conclusions: Collectively, our studies show that the DUBm tetrameric structure can form without a complete intact SAGA-CORE complex and that it includes full-length Sgf73. However, subunits of this SAGA-CORE influence DUBm association with chromatin, its localization and its activity., This study was supported by funds to SR-N from the Spanish MINECO, MICIIN (BFU2014-57636, BFU2015-71978, PGC2018-099872-B-I00) and the Generalitat Valenciana (PROM/2012/061, ACOMP2014/061 and PROMETEO 2016/093). This work was supported by FEDER 2014–2020 and the Ministerio de Economia y Competitividad (MINECO) of Spain. V.G-M was supported by the FPU program from the Ministerio de Educación y Ciencia (AP2009-0917); C.C-N by the Generailtat Valenciana PROMETEO/2016/093; P.O-C by the FPI program from MINECO (BES2012-058587); and M.M-E by the GVA (Val I+D: ACIF/2015/025). The M.I-V lab was co-funded by European Regional Development Funds (ERDF) and the Horizon 2020 Framework Programme of the European Union under the grant agreement 688945 (Euro-BioImaging Prep Phase II).

Metabolic reprogramming, including enhanced biosynthesis of macromolecules, altered energy metabolism, and maintenance of redox homeostasis, is considered a hallmark of cancer, sustaining cancer cell growth. Multiple signaling pathways, transcription factors and metabolic enzymes participate in the modulation of cancer metabolism and thus, metabolic reprogramming is a highly complex process. Recent studies have observed that ubiquitination and deubiquitination are involved in the regulation of metabolic reprogramming in cancer cells. As one of the most important type of post-translational modifications, ubiquitination is a multistep enzymatic process, involved in diverse cellular biological activities. Dysregulation of ubiquitination and deubiquitination contributes to various disease, including cancer. Here, we discuss the role of ubiquitination and deubiquitination in the regulation of cancer metabolism, which is aimed at highlighting the importance of this post-translational modification in metabolic reprogramming and supporting the development of new therapeutic approaches for cancer treatment.

Sentrin/small ubiquitin-like modifier (SUMO) is protein modification pathway that regulates multiple biological processes, including cell division, DNA replication/repair, signal transduction, and cellular metabolism. In this review, we will focus on recent advances in the mechanisms of disease pathogenesis, such as cancer, diabetes, seizure, and heart failure, which have been linked to the SUMO pathway. SUMO is conjugated to lysine residues in target proteins through an isopeptide linkage catalyzed by SUMO-specific activating (E1), conjugating (E2), and ligating (E3) enzymes. In steady state, the quantity of SUMO-modified substrates is usually a small fraction of unmodified substrates due to the deconjugation activity of the family Sentrin/SUMO-specific proteases (SENPs). In contrast to the complexity of the ubiquitination/deubiquitination machinery, the biochemistry of SUMOylation and de-SUMOylation is relatively modest. Specificity of the SUMO pathway is achieved through redox regulation, acetylation, phosphorylation, or other posttranslational protein modification of the SUMOylation and de-SUMOylation enzymes. There are three major SUMOs. SUMO-1 usually modifies a substrate as a monomer; however, SUMO-2/3 can form poly-SUMO chains. The monomeric SUMO-1 or poly-SUMO chains can interact with other proteins through SUMO-interactive motif (SIM). Thus SUMO modification provides a platform to enhance protein-protein interaction. The consequence of SUMOylation includes changes in cellular localization, protein activity, or protein stability. Furthermore, SUMO may join force with ubiquitin to degrade proteins through SUMO-targeted ubiquitin ligases (STUbL). After 20 yr of research, SUMO has been shown to play critical roles in most, if not all, biological pathways. Thus the SUMO enzymes could be targets for drug development to treat human diseases.

Circadian clocks are internal timing systems that enable organisms to adjust their behavioral and physiological rhythms to the daily changes of their environment. These clocks generate self-sustained oscillations at the cellular, tissue, and behavioral level. The rhythm-generating mechanism is based on a gene expression network with a delayed negative feedback loop that causes the transcripts to oscillate with a period of approximately 24 hr. This oscillatory nature of the proteins involved in this network necessitates that they are intrinsically unstable, with a short half-life. Hence, post-translational modifications (PTMs) are important to precisely time the presence, absence, and interactions of these proteins at appropriate times of the day. Ubiquitination and deubiquitination are counter-balancing PTMs which play a key role in this regulatory process. In this review, we take a comprehensive look at the roles played by the processes of ubiquitination and deubiquitination in the clock machinery of the most commonly studied eukaryotic models of the circadian clock: plants, fungi, fruit flies, and mammals. We present the effects exerted by ubiquitinating and deubiquitinating enzymes on the stability, but also the activity, localization, and interactions of clock proteins. Overall, these PTMs have key roles in regulating not only the pace of the circadian clocks but also their response to external cues and their control of cellular functions.

Purpose: Ubiquitin specific peptidase 5 (USP5) has been reported to promote the progression of several malignant tumors. It may affect cancer development via modulating cell cycle and colony formation. In pancreatic cancer, the biological function of USP5, especially in migration and invasion remains unclear. Methods: USP5 protein expression levels in primary pancreatic cancer and lymph node metastasis tissues were detected using immunohistochemistry (IHC). χ2 test, Kaplan-Meier analysis, univariate and multivariate analyses were used to evaluate the relationship between USP5 expression and clinicopathological feature. RT-qPCR were carried out to quantitate the mRNA expression levels of USP5 in pancreatic cancer cell lines. CCK8 and Colony formation assay were performed to prove how USP5 works in proliferation. Evaluation of tumor metastasis was made by Transwell and wound healing assay. EMT and STAT3 signaling related markers were detected by western blot. Results: (1) USP5 protein expression levels were related to tumor differentiation, CEA and CA19-9 level. (2) Univariate and multivariate analyses showed that high USP5 expression is an unfavorable prognostic factor for pancreatic cancer. Kaplan-Meier analysis directly indicated that patients with high USP5 expression had shorter overall survival. (3) Increased USP5 expression is related to pancreatic cancer in both proliferation and metastasis. (4) USP5 was proved to mediate STAT3 signaling in pancreatic cancer cells. Conclusions: The results suggest that USP5 is highly expressed and might have clinical significance for pancreatic cancer patients. High USP5 expression promotes both progression and metastasis by activating STAT3 signaling. Thus, USP5 might be a potential target in pancreatic cancer treatment.

Ubiquitin (Ub) is a highly conserved protein that is covalently attached to substrate proteins as a post-translational modification to regulate signaling pathways such as proteasomal degradation and cell cycle/transcriptional regulation in the eukaryotic cellular environment. Ub signaling is regulated by the homeostasis of substrate protein ubiquitination/deubiquitination by E3 ligases and deubiquitinating enzymes (DUBs) in healthy eukaryotic systems. One such DUB, ubiquitin C-terminal hydrolase L1 (UCHL1), is endogenously expressed in the central nervous system under normal physiological conditions, but overexpression and/or mutation has been linked to various cancers and neurodegenerative diseases. The lack of UCHL1 probing strategies suggests development of a selective Ub variant (UbV) for probing UCHL1's role in these disease states would be beneficial. We describe a computational design approach to investigate UbVs that lend selectivity, both binding and inhibition, to UCHL1 over the close structural homologue UCHL3 and members of other DUB families. A number of UbVs, mainly those containing Thr9 mutations, displayed appreciable binding and inhibition selectivity for UCHL1 over UCHL3, compared to wild-type Ub in in vitro assays. By appending reactive electrophiles to the C-terminus of the UbVs, we created the first activity-based probe (ABP) with demonstrated reaction selectivity for UCH family DUBs over other families in cell lysates. Further kinetic analysis of covalent inhibition by the UbV-ABP with UCHL1 and UCHL3 offers insight into the future design of UCHL1 selective UbV-ABP. These studies serve as a proof of concept of the viability of the in silico design of ubiquitin variants for UCH family DUBs as a step toward the development of macromolecular UCHL1 inhibitors.

Background Overexpression of eukaryotic translation initiation factor 3H (EIF3H) predicts cancer progression and poor prognosis, but the mechanism underlying EIF3H as an oncogene remains unclear in esophageal squamous cell carcinoma (ESCC). Methods TCGA database and the immunohistochemistry (IHC) staining of ESCC samples were used and determined the upregulation of EIF3H in ESCC. CCK8 assay, colony formation assay and transwell assay were performed to examine the ability of cell proliferation and mobility in KYSE150 and KYSE510 cell lines with EIF3H overexpression or knockdown. Xenograft and tail-vein lung metastatic mouse models of KYSE150 cells with or without EIF3H knockdown were also used to confirm the function of EIF3H on tumor growth and metastasis in vivo. A potential substrate of EIF3H was screened by co-immunoprecipitation assay (co-IP) combined with mass spectrometry in HEK293T cells. Their interaction and co-localization were confirmed using reciprocal co-IP and immunofluorescence staining assay. The function of EIF3H on Snail ubiquitination and stability was demonstrated by the cycloheximide (CHX) pulse-chase assay and ubiquitination assay. The correlation of EIF3H and Snail in clinical ESCC samples was verified by IHC. Results We found that EIF3H is significantly upregulated in esophageal cancer and ectopic expression of EIF3H in ESCC cell lines promotes cell proliferation, colony formation, migration and invasion. Conversely, genetic inhibition of EIF3H represses ESCC tumor growth and metastasis in vitro and in vivo. Moreover, we identified EIF3H as a novel deubiquitinating enzyme of Snail. We demonstrated that EIF3H interacts with and stabilizes Snail through deubiquitination. Therefore, EIF3H could promote Snail-mediated EMT process in ESCC. In clinical ESCC samples, there is also a positive correlation between EIF3H and Snail expression. Conclusions Our study reveals a critical EIF3H-Snail signaling axis in tumor aggressiveness in ESCC and provides EIF3H as a promising biomarker for ESCC treatment.

Ubiquitination is one of the major post-translational modifications and entails conjugation of ubiquitin molecules to target proteins. To make free ubiquitin molecules available for conjugation, in cells ubiquitin is not only synthesized de novo, but is also provided by cleaving off existing conjugated ubiquitin molecules, so-called deubiquitination reaction. Therefore, intracellular ubiquitin molecules are thought to be recycled, but the recycling frequency remains elusive. The main reason for the lack of such mechanistic details is that the original and recycled ubiquitin molecules are indistinguishable in their chemical and physical properties. To tackle this issue, here we applied 18O-labeling to trace how ubiquitin is recycled in a simultaneous ubiquitination/deubiquitination reaction (ubiquitin cycle reaction). Because deubiquitination is a hydrolysis reaction, the two 16O atoms of the C-terminal carboxy group of a ubiquitin molecule can be exchanged with 18O atoms by deubiquitination in 18O-labeled aqueous solution. By using quantitative mass spectrometry, we detected 18O atom incorporation into the C-terminal carboxy group of ubiquitin in the course of a deubiquitination reaction, in addition, we were able to quantify the 18O-incorporation in a ubiquitin cycle reaction. Unexpectedly, kinetic analysis suggested that ubiquitination reactivity was accelerated in the presence of a deubiquitinating enzyme. Collectively, we have established a quantitative method to trace ubiquitin cycle reactions by analyzing deubiquitination-associated 18O-incorporation into ubiquitin.

Ubiquitin specific peptidase 28 (USP28) is closely associated to the occurrence and development of various malignancies, and thus has been validated as a promising therapeutic target for cancer therapy. To date, only few USP28 inhibitors with moderate inhibitory activity have been reported, highly potent and selective USP28 inhibitors with new chemotypes remain to be discovered for pathologically investigating the roles of deubiquitinase. In this current study, we reported the synthesis and biological evaluation of new [1,2,3]triazolo[4,5-d]pyrimidine derivatives as potent USP28 inhibitors. Especially, compound 19 potently inhibited USP28 (IC50 = 1.10 ± 0.02 μmol/L, Kd = 40 nmol/L), showing selectivity over USP7 and LSD1 (IC50 > 100 μmol/L). Compound 19 was cellularly engaged to USP28 in gastric cancer cells. Compound 19 reversibly bound to USP28 and directly affected its protein levels, thus inhibiting the proliferation, cell cycle at S phase, and epithelial-mesenchymal transition (EMT) progression in gastric cancer cell lines. Docking studies were performed to rationalize the potency of compound 19. Collectively, compound 19 could serve as a new tool compound for the development of new USP28 inhibitors for exploring the roles of deubiquitinase in cancers., Graphical abstract A new series of [1,2,3]triazolo[4,5-d]pyrimidine derivatives were identified to inhibit USP28. Compound 19 potently inhibited USP28 with the IC50 and Kd values of 1.1 μmol/L and 40 nmol/L, respectively.Image 1

Rationale: A number of guanine nucleotide exchange factors (GEFs) including epithelial cell transforming factor ECT2 are believed to drive carcinogenesis through activating distinct oncogenic GTPases. Yet, whether GEF-independent activity of ECT2 also plays a role in tumorigenesis remains unclear. Methods: Immunohistochemical (IHC) staining, colony formation and xenograft assays were used to examine the role of ECT2 in breast carcinogenesis. Co-immunoprecipitation, immunofluorescent stainings, in vivo deubiquitination and in vitro deubiquitination experiments were performed to examine the physical and functional interaction between ECT2 and ubiquitin-specific protease USP7. High-throughput RNA sequencing, quantitative reverse transcription-PCR and Western blotting were employed to investigate the biological significance of the interplay between ECT2 and USP7. Results: We report that ECT2 plays a tumor-promoting role in breast cancer, and GEF activity-deficient ECT2 is able to alleviate ECT2 depletion associated growth defects in breast cancer cells. Mechanistically, we demonstrated that ECT2 physically interacts with ubiquitin-specific protease USP7 and functionally facilitates USP7 intermolecular self-association, -deubiquitination and -stabilization in a GEF activity-independent manner. USP7 in turn, deubiquitinates and stabilizes ECT2, resulting in a feedforward regulatory circuit that ultimately sustains the expression of oncogenic protein MDM2. Conclusion: Our study uncovers a GEF-independent role of ECT2 in promoting survival of breast cancer cells, provides a molecular insight for the reciprocal regulation of ECT2 and USP7, and supports the pursuit of ECT2/USP7 as potential targets for breast cancer intervention.

Chlamydia trachomatis is the cause of several diseases such as sexually transmitted urogenital disease and ocular trachoma. The pathogen contains a small genome yet, upon infection, expresses two enzymes with deubiquitinating activity, termed ChlaDUB1 and ChlaDUB2, presumed to have redundant deubiquitinase (DUB) function because of the similarity of the primary structure of their catalytic domain. Previous studies have led to structural characterization of the enzymatic properties of ChlaDUB1; however, ChlaDUB2 has yet to be investigated thoroughly. In this study, we investigated the deubiquitinase properties of ChlaDUB2 and compared them to those of ChlaDUB1. This revealed a distinct difference in hydrolytic activity with regard to di- and polyubiquitin chains while showing similar ability to cleave a monoubiquitin-based substrate, ubiquitin aminomethylcoumarin (Ub-AMC). ChlaDUB2 was unable to cleave a diubiquitin substrate efficiently, whereas ChlaDUB1 could rapidly hydrolyze this substrate like a prototypical prokaryotic DUB, SdeA. With polyubiquitinated green fluorescent protein substrate (GFP-Ubn), whereas ChlaDUB1 efficiently disassembled the polyubiquitin chains into the monoubiquitin product, the deubiquitination activity of ChlaDUB2, while showing depletion of the substrate, did not produce appreciable levels of the monoubiquitin product. We report the structures of a catalytic construct of ChlaDUB2 and its complex with ubiquitin propargyl amide. These structures revealed differences in residues involved in substrate recognition between the two Chlamydia DUBs. On the basis of the structures, we conclude that the distal ubiquitin binding is equivalent between the two DUBs, consistent with the Ub-AMC activity result. Therefore, the difference in activity with longer ubiquitinated substrates may be due to the differential recognition of these substrates involving additional ubiquitin binding sites.

Ubiquitin-specific protease 14 (USP14) is one of the three proteasome-associated deubiquitinating enzymes and implicated in the progression of various cancers. However, the role of USP14 in ovarian cancer remains unknown. By using an unbiased qRT-PCR screen, here we show that USP14 is considerably increased in cisplatin-resistant ovarian cancer cells. Overexpression of USP14 confers resistance to cisplatin-sensitive ovarian cancer cells. Genetic or pharmacological inhibition of USP14 is able to reverse cisplatin-resistance of ovarian cancer cells, which was accompanied by decreased protein expression of BCL6. Besides, BCL6 protein level was also increased in cisplatin-resistant ovarian cancer cells and silencing of BCL6 in these cells restored their sensitivity to cisplatin. At the molecular lever, we found that USP14 interacted with BCL6 and prevented it from proteasomal-dependent degradation. Thus, our results provide a rationale to target USP14-BCL6 axis in ovarian cancer that may be therapeutically beneficial.

Breast cancer is one of the most common malignancies worldwide, while the luminal types (ERα positive) accounts for two third of all breast cancer cases. Although ERα positive breast cancer could be effective controlled by endocrine therapy, most of the patients will develop endocrine resistance, which becomes a headache clinical issue for breast cancer field. Endocrine resistance could be caused by multiple pathway disorders, the dys-regulation of ERα signaling might be a critical factor, which makes it urgent and important to reveal the potential molecular mechanism of ERα signaling. In our current study, we identified a new deubiquitination enzyme USP1 through screening the whole DUB (Deubiquitinases) siRNA library. The expression of USP1 is elevated in human breast cancer compared with normal mammary tissues. Importantly, USP1 expression levels are specially correlated with poor survival in ERα positive patients. USP1 depletion inhibited breast cancer cell progression and ERα signaling activity. Immuno-precipitation assays indicate that USP1 associates with ERα and promotes its stability possibly via inhibiting ERα K48-linked poly-ubiquitination. In conclusion, our data implicate a non-genomic mechanism by USP1 via stabilizing ERα protein controls ERα target gene expression linked to breast cancer progression.

The ubiquitin system, known as a common feature in eukaryotes, participates in multiple cellular processes, such as signal transduction, cell-cycle progression, receptor trafficking and endocytosis, and even the immune response. In lung cancer, evidence has revealed that aberrant events in ubiquitin-mediated processes can cause a variety of pathological outcomes including tumorigenesis and metastasis. Likewise, ubiquitination on the core components contributing to the activity of cell signaling controls bio-signal turnover and cell final destination. Given this, inhibitors targeting the ubiquitin system have been developed for lung cancer therapies and have shown great prospects for clinical application. However, the exact biological effects and physiological role of the drugs used in lung cancer therapies are still not clearly elucidated, which might seriously impede the progress of treatment. In this work, we summarize current research advances in cell signal regulation processes mediated through the ubiquitin system during the development of lung cancer, with the hope of improving the therapeutic effects by means of aiming at efficient targets.