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Cholangiocarcinoma (CCA) is a hepatobiliary carcinoma with uncontrolled cell proliferation, poor prognosis, and high mortality. The ovarian tumor structural domain (OTU) containing protein 6B (OTUD6B) belongs to the OTU deubiquitin family and is vital in tumor development. However, its expression and biological function in CCA remain unknown. The expression of OTUD6B in CCA was analyzed using TIMER2.0, UALCAN, and GEO databases. MTT, clonal formation assay, immunofluorescence staining, immunohistochemistry staining, and flow cytometry examined the regulation of OTUD6B on cell proliferation, cycle, and apoptosis. The effects of OTUD6B on tumor volume and weight were assessed using the xenograft tumor model. The activities of PTK2 and STAT3 were detected by western blot and CO‐IP. The biological database identified that OTUD6B was upregulated in CCA. In CCA cells, OTUD6B knockdown reduced CCA cell proliferation and promoted apoptosis. Cell cycle analysis indicated that the cycle stopped at the G0/G1 phase after OTU6B downregulation. Furthermore, OTUD6B knockdown resulted in a decrease in tumor volume and weight in xenograft tumor models. Mechanistically, OTUD6B is involved in the deubiquitination of PTK2. PTK2 further affected the phosphorylation of STAT3 thereby regulating the CCA process. Our study demonstrates that OTUD6B knockdown participates in the ubiquitination of PTK2 and phosphorylation of STAT3 to alleviate the process of CCA. These results suggest that OTUD6B may be a potential new strategy for CCA treatment. [ABSTRACT FROM AUTHOR]

Meiosis is a form of cell division that is essential to sexually reproducing organisms and is therefore highly regulated. Each event of meiosis must occur at the correct developmental stage to ensure that chromosomes are segregated properly during both meiotic divisions. One unique meiosis-specific structure that is tightly regulated in terms of timing of assembly and disassembly is the synaptonemal complex (SC). While the mechanism(s) for assembly and disassembly of the SC are poorly understood in Drosophila melanogaster, posttranslational modifications, including ubiquitination and phosphorylation, are known to play a role. Here, we identify a role for the deubiquitinase Usp7 in the maintenance of the SC in early prophase and show that its function in SC maintenance is independent of the meiotic recombination process. Using two usp7 shRNA constructs that result in different knockdown levels, we have shown that the presence of SC through early/mid-pachytene is critical for normal levels and placement of crossovers. [ABSTRACT FROM AUTHOR]

Many bacterial pathogens have evolved effective strategies to interfere with the ubiquitination network to evade clearance by the innate immune system. Here, we report that OTUB1, one of the most abundant deubiquitinases (DUBs) in mammalian cells, is subjected to both canonical and noncanonical ubiquitination during Legionella pneumophila infection. The effectors SidC and SdcA catalyze OTUB1 ubiquitination at multiple lysine residues, resulting in its association with a Legionella-containing vacuole. Lysine ubiquitination by SidC and SdcA promotes interactions between OTUB1 and DEPTOR, an inhibitor of the MTORC1 pathway, thus suppressing MTORC1 signaling. The inhibition of MTORC1 leads to suppression of host protein synthesis and promotion of host macroautophagy/autophagy during L. pneumophila infection. In addition, members of the SidE family effectors (SidEs) induce phosphoribosyl (PR)-linked ubiquitination of OTUB1 at Ser16 and Ser18 and block its DUB activity. The levels of the lysine and serine ubiquitination of OTUB1 are further regulated by effectors that function to antagonize the activities of SidC, SdcA and SidEs, including Lem27, DupA, DupB, SidJ and SdjA. Our study reveals an effectors-mediated complicated mechanism in regulating the activity of a host DUB. Abbreviations: BafA1: bafilomycin A1; BMDMs: bone marrow-derived macrophages; DUB: deubiquitinase; Dot/Icm: defective for organelle trafficking/intracellular multiplication; DEPTOR: DEP domain containing MTOR interacting protein; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; L. pneumophila: Legionella pneumophila; LCV: Legionella-containing vacuole; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; MTORC1: mechanistic target of rapamycin kinase complex 1; OTUB1: OTU deubiquitinase, ubiquitin aldehyde binding 1; PR-Ub: phosphoribosyl (PR)-linked ubiquitin; PTM: posttranslational modification; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SidEs: SidE family effectors; Ub: ubiquitin. [ABSTRACT FROM AUTHOR]

Neuroblastoma (NB) is a pediatric malignancy originating from neural crest cells of the sympathetic nervous system that accounts for 15% of all pediatric cancer deaths. Despite advances in treatment, high-risk NB remains difficult to cure, highlighting the need for novel therapeutic approaches. Ubiquitin-specific protease 7 (USP7) is a deubiquitinase that plays a critical role in tumor suppression and DNA repair, and USP7 overexpression has been associated with tumor aggressiveness in a variety of tumors, including NB. Therefore, USP7 is a potential therapeutic target for NB. The tumor suppressor p53 is a known target of USP7, and therefore reactivation of the p53 pathway may be an effective therapeutic strategy for NB treatment. We hypothesized that inhibition of USP7 would be effective against NB tumor growth. Using a novel USP7 inhibitor, Almac4, we have demonstrated significant antitumor activity, with significant decreases in both cell proliferation and cell viability in TP53 wild-type NB cell lines. USP7 inhibition in NB cells activated the p53 pathway via USP7 and MDM2 degradation, leading to reduced p53 ubiquitination and increased p53 expression in all sensitive NB cells. In addition, USP7 inhibition led to decreased N-myc protein levels in both MYCN-amplified and -nonamplified NB cell lines, but no correlation was observed between MYCN amplification and treatment response. USP7 inhibition induced apoptosis in all TP53 wild-type NB cell lines. USP7 inhibition also induced EZH2 ubiquitination and degradation. Lastly, the combination of USP7 and MDM2 inhibition showed enhanced efficacy. Our data suggests that USP7 inhibition may be a promising therapeutic strategy for children with high-risk and relapsed NB.

The host ESCRT-machinery repairs damaged endolysosomal membranes. If damage persists, selective macroautophagy/autophagy clears the damaged compartment. Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that damages the phagosomal membrane and targets ESCRT-mediated repair as part of its virulence program. The E3 ubiquitin ligases PRKN and SMURF1 promote autophagic capture of damaged, Mtb-containing phagosomes. Because ubiquitination is a reversible process, we anticipated that host deubiquitinases (DUBs) would also be involved. Here, we screened all predicted mouse DUBs for their role in ubiquitin targeting and control of intracellular Mtb. We show that USP8 (ubiquitin specific peptidase 8) colocalizes with intracellular Mtb, recognizes phagosomal membrane damage, and is required for ESCRT-dependent membrane repair. Furthermore, we show that USP8 regulates the NFE2L2/NRF2-dependent antioxidant signature. Taken together, our study demonstrates a central role of USP8 in promoting Mtb intracellular growth by promoting phagosomal membrane repair, limiting ubiquitin-driven selective autophagy, and reducing oxidative stress.Abbreviation: BMDMs: bone marrow-derived macrophages; CFUs: colony-forming units; DUB: deubiquitinase; ESCRT: endosomal sorting complexes required for transport; LLOMe: L-leucyl-L-leucine methyl ester; MFI: mean fluorescence intensity; MOI: multiplicity of infection; Mtb: Mycobacterium tuberculosis; NFE2L2/NRF2: nuclear factor, erythroid derived 2, like 2; PMA: phorbol 12-myristate 13-acetate; ROS: reactive oxygen species; USP8: ubiquitin specific peptidase 8 [ABSTRACT FROM AUTHOR]

Background: Metastasis accounts for the majority of cancer-related deaths. Actin dynamics and actin-based cell migration and invasion are important factors in cancer metastasis. Metastasis is characterized by actin polymerization and depolymerization, which are precisely regulated by molecular changes involving a plethora of actin regulators, including actin-binding proteins (ABPs) and signalling pathways, that enable cancer cell dissemination from the primary tumour. Research on deubiquitinating enzymes (DUBs) has revealed their vital roles in actin dynamics and actin-based migration and invasion during cancer metastasis. Conclusion: Here, we review how DUBs drive tumour metastasis by participating in actin rearrangement and actin-based migration and invasion. We summarize the well-characterized and essential actin cytoskeleton signalling molecules related to DUBs, including Rho GTPases, Src kinases, and ABPs such as cofilin and cortactin. Other DUBs that modulate actin-based migration signalling pathways are also discussed. Finally, we discuss and address therapeutic opportunities and ongoing challenges related to DUBs with respect to actin dynamics. [ABSTRACT FROM AUTHOR]

The extensive cellular signalling events controlled by posttranslational ubiquitination are tightly regulated through the action of specialized proteases termed deubiquitinases. Among them, the OTU family of deubiquitinases can play very specialized roles in the regulation of discrete subtypes of ubiquitin signals that control specific cellular functions. To exert control over host cellular functions, some pathogenic bacteria have usurped the OTU deubiquitinase fold as a secreted virulence factor that interferes with ubiquitination inside infected cells. Herein, we provide a review of the function of bacterial OTU deubiquitinases during infection, the structural basis for their deubiquitinase activities and the bioinformatic approaches leading to their identification. Understanding bacterial OTU deubiquitinases holds the potential for discoveries not only in bacterial pathogenesis but in eukaryotic biology as well. [ABSTRACT FROM AUTHOR]

Ubiquitination, one of the most common posttranslational modifications of proteins, has a significant impact on their functions, such as stability, activity, and cellular localization. Disorders in the ubiquitination and deubiquitination processes are associated with various oncological and neurodegenerative diseases. The complexity of ubiquitin signaling, specifically monoubiquitination and polyubiquitination with different lengths and types of ubiquitin– ubiquitin linkages determines their versatility and ability to regulate hundreds of different cellular processes. Advanced biochemical, mass spectrometric, and computational studies are required for in-depth understanding of the mechanisms of assembly and disassembly, as well as detection of ubiquitin chains and their signal transmission. Recent scientific achievements make it possible to identify protein ubiquitination and the structure of ubiquitin chains, but there are a lot of issues in this area to be clarified. The present review provides a detailed analysis of the current understanding of the architectonics of ubiquitin chains. [ABSTRACT FROM AUTHOR]

p53 is a tumor suppressor gene activated in response to cellular stressors that inhibits cell cycle progression and induces pro-apoptotic signaling. The protein level of p53 is well balanced by the action of several E3 ligases and deubiquitinating enzymes (DUBs). Several DUBs have been reported to negatively regulate and promote p53 degradation in tumors. In this study, we identified USP19 as a negative regulator of p53 protein level. We demonstrate a direct interaction between USP19 and p53 by pull down assay. The overexpression of USP19 promoted ubiquitination of p53 and reduced its protein half-life. We also demonstrate that CRISPR/Cas9-mediated knockout of USP19 in cervical cancer cells elevates p53 protein levels, resulting in reduced colony formation, cell migration, and cell invasion. Overall, our results indicate that USP19 negatively regulates p53 protein levels in cervical cancer progression. [ABSTRACT FROM AUTHOR]

Background: Tumorigenesis and progression of prostate cancer (PCa) are indispensably dependent on androgen receptor (AR). Antiandrogen treatment is the principal preference for patients with advanced PCa. However, the molecular characteristics of PCa with antiandrogen intervention have not yet been fully uncovered. Methods: We first performed proteome analysis with 32 PCa tumor samples and 10 adjacent tissues using data-independent acquisition (DIA)- parallel accumulation serial fragmentation (PASEF) proteomics. Then label-free quantification (LFQ) mass spectrometry was employed to analyze protein profiles in LNCaP and PC3 cells. Results: M-type creatine kinase CKM and cartilage oligomeric matrix protein COMP were demonstrated to have the potential to be diagnostic biomarkers for PCa at both mRNA and protein levels. Several E3 ubiquitin ligases and deubiquitinating enzymes (DUBs) were significantly altered in PCa and PCa cells under enzalutamide treatment, and these proteins might reprogram proteostasis at protein levels in PCa. Finally, we discovered 127 significantly varied proteins in PCa samples with antiandrogen therapy and further uncovered 4 proteins in LNCaP cells upon enzalutamide treatment. Conclusions: Our research reveals new potential diagnostic biomarkers for prostate cancer and might help resensitize resistance to antiandrogen therapy. [ABSTRACT FROM AUTHOR]

The regulation of PD‐L1 is the key question, which largely determines the outcome of the immune checkpoint inhibitors (ICIs) based therapy. However, besides the transcription level, the protein stability of PD‐L1 is closely correlated with its function and has drawn increasing attention. In this study, EZH2 inhibition enhances PD‐L1 expression and protein stability, and the deubiquitinase ubiquitin‐specific peptidase 22 (USP22) is identified as a key mediator in this process. EZH2 inhibition transcriptionally upregulates USP22 expression, and upregulated USP22 further stabilizes PD‐L1. Importantly, a combination of EZH2 inhibitors with anti‐PD‐1 immune checkpoint blockade therapy improves the tumor microenvironment, enhances sensitivity to immunotherapy, and exerts synergistic anticancer effects. In addition, knocking down USP22 can potentially enhance the therapeutic efficacy of EZH2 inhibitors on colon cancer. These findings unveil the novel role of EZH2 inhibitors in tumor immune evasion by upregulating PD‐L1, and this drawback can be compensated by combining ICI immunotherapy. Therefore, these findings provide valuable insights into the EZH2‐USP22‐PD‐L1 regulatory axis, shedding light on the optimization of combining both immune checkpoint blockade and EZH2 inhibitor‐based epigenetic therapies to achieve more efficacies and accuracy in cancer treatment. [ABSTRACT FROM AUTHOR]

Atherosclerosis is a chronic artery disease that causes various types of cardiovascular dysfunction. Vascular smooth muscle cells (VSMCs), the main components of atherosclerotic plaque, switch from contractile to synthetic phenotypes during atherogenesis. Ubiquitylation is crucial in regulating VSMC phenotypes in atherosclerosis, and it can be reversely regulated by deubiquitinases. However, the specific effects of deubiquitinases on atherosclerosis have not been thoroughly elucidated. In this study, RNAi screening in human aortic smooth muscle cells was performed to explore the effects of OTU family deubiquitinases, which revealed that silencing OTUB1 inhibited PDGF-BB-stimulated VSMC phenotype switch. Further in vivo studies using Apoe−/− mice revealed that knockdown of OTUB1 in VSMCs alleviated atherosclerosis plaque burden in the advanced stage and led to a stable plaque phenotype. Moreover, VSMC proliferation and migration upon PDGF-BB stimulation could be inhibited by silencing OTUB1 in vitro. Unbiased RNA-sequencing data indicated that knocking down OTUB1 influenced VSMC differentiation, adhesion, and proliferation. Mass spectrometry of ubiquitinated protein confirmed that proteins related to cell growth and migration were differentially ubiquitylated. Mechanistically, we found that OTUB1 recognized the K707 residue ubiquitylation of PDGFRβ with its catalytic triad, thereby reducing the K48-linked ubiquitylation of PDGFRβ. Inhibiting OTUB1 in VSMCs could promote PDGFRβ degradation via the ubiquitin–proteasome pathway, so it was beneficial in preventing VSMCs' phenotype switch. These findings revealed that knocking down OTUB1 ameliorated VSMCs' phenotype switch and atherosclerosis progression, indicating that OTUB1 could be a valuable translational therapeutic target in the future. [ABSTRACT FROM AUTHOR]

OTULIN deficiency is a complex disease characterized by a wide range of clinical manifestations, including skin rash, joint welling, lipodystrophy to pulmonary abscess, and sepsis shock. This disease is mechanistically linked to mutations in the OTULIN gene, resulting in an immune disorder that compromises the body's ability to effectively combat pathogens and foreign stimuli. The OTULIN gene is responsible for encoding a deubiquitinating enzyme crucial for hydrolyzing Met1-poly Ub chains, and its dysfunction leads to dysregulated immune responses. Patients with OTULIN deficiency often exhibit an increase in monocytes, including neutrophils and macrophages, along with inflammatory clinical features. The onset of symptoms typically occurs at an early age. However, individuals with OTULIN haploinsufficiency are particularly susceptible to life-threatening staphylococcal infections. Currently, the most effective treatment for patients with OTULIN biallelic mutations involves the use of TNF-blocking agents, which target the dysregulated immune response. In conclusion, OTULIN deficiency presents a complex clinical picture with diverse manifestations, attributed to mutations in the OTULIN gene. Understanding the underlying mechanisms is crucial for developing targeted therapeutic interventions to address this challenging condition. Further research into the pathophysiology of OTULIN deficiency is essential for improving clinical management and outcomes for affected individuals. [ABSTRACT FROM AUTHOR]

TEAD transcription factors play a central role in the Hippo signaling pathway. In this study, we focused on transcriptional enhancer factor TEF‐3 (TEAD4), exploring its regulation by the deubiquitinase OTU domain‐containing protein 6A (OTUD6A). We identified OTUD6A as a TEAD4‐interacting deubiquitinase, positively influencing TEAD‐driven transcription without altering TEAD4 stability. Structural analyses revealed specific interaction domains: the N‐terminal domain of OTUD6A and the YAP‐binding domain of TEAD4. Functional assays demonstrated the positive impact of OTUD6A on the transcription of YAP–TEAD target genes. Despite no impact on TEAD4 nuclear localization, OTUD6A selectively modulated nuclear interactions, enhancing YAP–TEAD4 complex formation while suppressing VGLL4 (transcription cofactor vestigial‐like protein 4)–TEAD4 interaction. Critically, OTUD6A facilitated YAP–TEAD4 complex binding to target gene promoters. Our study unveils the regulatory landscape of OTUD6A on TEAD4, providing insights into diseases regulated by YAP–TEAD complexes. [ABSTRACT FROM AUTHOR]

Cactin, a highly conserved protein, plays a crucial role in various physiological processes in eukaryotes, including innate immunity. Recently, the function of Cactin in the innate immunity of Drosophila has been explored, revealing that Cactin regulates a non-canonical signaling pathway associated with the Toll and Imd pathways via the Cactin-Deaf1 axis. In addition, Cactin exhibits specific antiviral activity against the Drosophila C virus (DCV) in Drosophila, with an unknown mechanism. During DCV infection, it has been confirmed that the protein level and antiviral activity of Cactin are regulated by ubiquitination. However, the precise ubiquitination and deubiquitination mechanisms of Cactin in Drosophila remain unexplored. In this study, we identified ubiquitin-specific protease 14 (Usp14) as a major deubiquitinase for Cactin through comprehensive deubiquitinase screening. Our results demonstrate that Usp14 interacts with the C_Cactus domain of Cactin via its USP domain. Usp14 efficiently removes K48- and K63-linked polyubiquitin chains from Cactin, thereby preventing its degradation through the ubiquitin-proteasome pathway. Usp14 significantly inhibits DCV replication in Drosophila cells by stabilizing Cactin. Moreover, Usp14-deficient fruit flies exhibit increased susceptibility to DCV infection compared to wild-type flies. Collectively, our findings reveal the regulation of ubiquitination and antiviral activity of Cactin by the deubiquitinase Usp14, providing valuable insights into the modulation of Cactin-mediated antiviral activity in Drosophila. [ABSTRACT FROM AUTHOR]

Ubiquitin-specific protease 18 (USP18) is a protein recognized for its dual enzymatic and non-enzymatic nature. It is involved in many physiological processes like the cell cycle and cell signaling. It also suppresses heart muscle remodeling upon an increase in the afterload. The role of USP18 in kidney pathology remains unknown. The objective of the study was to assess the relationship between serum and urine USP18 levels, the factors contributing to cardiovascular risk, and the markers of kidney disease activity at different stages of chronic kidney disease (CKD). One hundred participants, aged between 24 and 85 years (mean 53.1 ± 17.1 years), were included. Five groups (n = 20 each) were recruited according to their renal status (healthy individuals, patients with proteinuric glomerulonephritis, patients with non-proteinuric CKD, patients who were treated with hemodialysis, and kidney transplant recipients). The measurements of serum and urine USP18 levels were performed using ELISA. The median serum USP18 level was the highest in healthy participants (1143.0 pg/mL) and kidney transplant recipients (856.6 pg/mL), whereas, in individuals with different forms of CKD, it fitted within the range of 402.1–471.9 pg/mL. Urinary USP18 reached the highest level in the group of CKD patients not yet on dialysis (303.3 pg/mL). Only in this group did it correlate with serum creatinine and urea concentrations. Our results suggest the inhibition of cardioprotective USP18 signaling when kidney function is impaired. Moreover, an increased level of urinary USP18 may indicate chronic tubular damage. [ABSTRACT FROM AUTHOR]

The ubiquitin proteasome system maintains protein homeostasis by regulating the breakdown of misfolded proteins, thereby preventing misfolded protein aggregates. The efficient elimination is vital for preventing damage to the cell by misfolded proteins, known as proteotoxic stress. Proteotoxic stress can lead to the collapse of protein homeostasis and can alter the function of the ubiquitin proteasome system. Conversely, impairment of the ubiquitin proteasome system can also cause proteotoxic stress and disrupt protein homeostasis. This review examines two impacts of proteotoxic stress, 1) disruptions to ubiquitin homeostasis (ubiquitin stress) and 2) disruptions to proteasome homeostasis (proteasome stress). Here, we provide a mechanistic description of the relationship between proteotoxic stress and the ubiquitin proteasome system. This relationship is illustrated by findings from several protein misfolding diseases, mainly neurodegenerative diseases, as well as from basic biology discoveries from yeast to mammals. In addition, we explore the importance of the ubiquitin proteasome system in endoplasmic reticulum quality control, and how proteotoxic stress at this organelle is alleviated. Finally, we highlight how cells utilize the ubiquitin proteasome system to adapt to proteotoxic stress and how the ubiquitin proteasome system can be genetically and pharmacologically manipulated to maintain protein homeostasis. [ABSTRACT FROM AUTHOR]

Objective To observe the effect of deubiquitinase Abraxas brother 1 (ABRO1) on the release of interleu‐ kin (IL)-1β from monocyte macrophage J774A. 1 in mice induced by listeriolysin O (LLO), and to explore the relevant mechanisms. Methods J774A. 1 cells were cultured and infected with wild-type Listeria monocytogenes (LM) strains with LLO (wild-type group), hly gene deficient (Δhly) LM strains with LLO knockout (gene deficient group), and Δhly strains supplemented with hly gene LM strains (replenishment group), respectively. Western blotting was used to detect ABRO1 protein, and ELISA was used to detect IL-1β in the supernatant of cell culture medium. We divided J774A. 1 cells into NI group (uninfected LM strain), WT group (infected WT LM strain), and Δhly group (infected Δhly LM strain), and cells in each group were transfected with NCsiRNA and ABRO1siRNA, respectively. The IL-1β in the super‐ natant of the cell culture medium was detected by ELISA, and Western blotting was used to detect inflammasome-related molecules Caspase-1, p20, and IL-1β, and p17. Results Over the infection time, the expression of ABRO1 and IL-1β levels gradually increased in the wild-type group and the replenishment group, reaching their highest levels at 120 minutes of infection, and both were higher than those in the gene deficient group (all P<0. 05). There were no significant changes in ABRO1 expression or IL-1β levels at different time points in the gene deficient group. The level of IL-1β and expression of p20 and p17 in the supernatant of cells transfected with ABRO1 siRNA in the WT group were lower than those in cells transfected with NC siRNA （all P<0. 05）. The level of IL-1β and expression of p20 and p17 in the supernatant of cells transfected with NC siRNA in the WT group were higher than those in the NI group （all P<0. 05）. The level of IL-1β and expression of p20 and p17 in the supernatant of cells transfected with NC siRNA in the Δhly group were lower than those in the WT group （all P<0. 05）. Conclusions Under the induction of LLO， the expression of ABRO1 increased in J774A. 1 cells. After down-regulating ABRO1 expression， the release of IL-1β in LLO-induced J774A. 1 cells decreased； LLO may promote LLO-mediated IL-1β release by activating inflammasomes. [ABSTRACT FROM AUTHOR]

Abstract The regulation of PD‐L1 is the key question, which largely determines the outcome of the immune checkpoint inhibitors (ICIs) based therapy. However, besides the transcription level, the protein stability of PD‐L1 is closely correlated with its function and has drawn increasing attention. In this study, EZH2 inhibition enhances PD‐L1 expression and protein stability, and the deubiquitinase ubiquitin‐specific peptidase 22 (USP22) is identified as a key mediator in this process. EZH2 inhibition transcriptionally upregulates USP22 expression, and upregulated USP22 further stabilizes PD‐L1. Importantly, a combination of EZH2 inhibitors with anti‐PD‐1 immune checkpoint blockade therapy improves the tumor microenvironment, enhances sensitivity to immunotherapy, and exerts synergistic anticancer effects. In addition, knocking down USP22 can potentially enhance the therapeutic efficacy of EZH2 inhibitors on colon cancer. These findings unveil the novel role of EZH2 inhibitors in tumor immune evasion by upregulating PD‐L1, and this drawback can be compensated by combining ICI immunotherapy. Therefore, these findings provide valuable insights into the EZH2‐USP22‐PD‐L1 regulatory axis, shedding light on the optimization of combining both immune checkpoint blockade and EZH2 inhibitor‐based epigenetic therapies to achieve more efficacies and accuracy in cancer treatment.

Abstract Background USP54, a ubiquitin-specific protease in the deubiquitinase (DUB) family, facilitates the malignant progression of several types of cancer. However, the role of USP54 in prostate cancer (PCa), especially castration-resistant prostate cancer (CRPC), remains unknown. Methods We established the CRPC LNCaP-AI cell line from the hormone-sensitive prostate cancer (HSPC) LNCaP cell line. RNA-Seq was utilized to explore DUB expression levels in LNCaP and LNCaP-AI. USP54 was knocked down, and its effects on cell growth were evaluated in vitro and in vivo. Bioinformatics analyses were conducted to explore signaling pathways affected by USP54 in PCa. Quantitative polymerase chain reaction was used to confirm key signaling pathways involved. Results USP54 was the most strongly upregulated DUB in LNCaP-AI cells compared with LNCaP cells. USP54 levels were higher in PCa than in normal tissues. USP54 silencing suppressed the proliferation of PCa cell lines, both in vitro and in vivo. USP54 expression was positively correlated with the androgen receptor (AR) signaling level in PCa samples, and USP54 knockdown inhibited AR signaling in PCa cells. Conclusions USP54 was upregulated during HSPC progression to CRPC. USP54 depletion suppressed CRPC cell proliferation both in vitro and in vivo. USP54 may facilitate PCa progression by regulating AR signaling.

Abstract Toxoplasma gondii is among the most important parasites worldwide. The apicoplast is a unique organelle shared by all Apicomplexan protozoa. Increasing lines of evidence suggest that the apicoplast possesses its own ubiquitination system. Deubiquitination is a crucial step executed by deubiquitinase (DUB) during protein ubiquitination. While multiple components of ubiquitination have been identified in T. gondii, the deubiquitinases involved remain unknown. The aim of the current study was to delineate the localization of TgOTU7 and elucidate its functions. TgOTU7 was specifically localized at the apicoplast, and its expression was largely regulated during the cell cycle. Additionally, TgOTU7 efficiently breaks down ubiquitin chains, exhibits linkage-nonspecific deubiquitinating activity and is critical for the lytic cycle and apicoplast biogenesis, similar to the transcription of the apicoplast genome and the nuclear genes encoding apicoplast-targeted proteins. Taken together, the results indicate that the newly described deubiquitinase TgOTU7 specifically localizes to the apicoplast and affects the cell growth and apicoplast homeostasis of T. gondii.

Background: USP54, a ubiquitin-specific protease in the deubiquitinase (DUB) family, facilitates the malignant progression of several types of cancer. However, the role of USP54 in prostate cancer (PCa), especially castration-resistant prostate cancer (CRPC), remains unknown. Methods: We established the CRPC LNCaP-AI cell line from the hormone-sensitive prostate cancer (HSPC) LNCaP cell line. RNA-Seq was utilized to explore DUB expression levels in LNCaP and LNCaP-AI. USP54 was knocked down, and its effects on cell growth were evaluated in vitro and in vivo. Bioinformatics analyses were conducted to explore signaling pathways affected by USP54 in PCa. Quantitative polymerase chain reaction was used to confirm key signaling pathways involved. Results: USP54 was the most strongly upregulated DUB in LNCaP-AI cells compared with LNCaP cells. USP54 levels were higher in PCa than in normal tissues. USP54 silencing suppressed the proliferation of PCa cell lines, both in vitro and in vivo. USP54 expression was positively correlated with the androgen receptor (AR) signaling level in PCa samples, and USP54 knockdown inhibited AR signaling in PCa cells. Conclusions: USP54 was upregulated during HSPC progression to CRPC. USP54 depletion suppressed CRPC cell proliferation both in vitro and in vivo. USP54 may facilitate PCa progression by regulating AR signaling. [ABSTRACT FROM AUTHOR]

Ubiquitination, a vital post-translational modification in plants, plays a significant role in regulating protein activity, localization, and stability. This process occurs through a complex enzyme cascade that involves E1, E2, and E3 enzymes, leading to the covalent attachment of ubiquitin molecules to substrate proteins. Conversely, deubiquitinating enzymes (DUBs) work in opposition to this process by removing ubiquitin moieties. Despite extensive research on ubiquitination in plants, our understanding of the function of DUBs is still emerging. UBP12 and UBP13, two plant DUBs, have received much attention recently and are shown to play pivotal roles in hormone signaling, light perception, photoperiod responses, leaf development, senescence, and epigenetic transcriptional regulation. This review summarizes current knowledge of these two enzymes, highlighting the central role of deubiquitination in regulating the abundance and activity of critical regulators such as receptor kinases and transcription factors during phytohormone and developmental signaling. [ABSTRACT FROM AUTHOR]

Kidney fibrosis is a prominent pathological feature of hypertensive kidney diseases (HKD). Recent studies have highlighted the role of ubiquitinating/deubiquitinating protein modification in kidney pathophysiology. Ovarian tumor domain-containing protein 6 A (OTUD6A) is a deubiquitinating enzyme involved in tumor progression. However, its role in kidney pathophysiology remains elusive. We aimed to investigate the role and underlying mechanism of OTUD6A during kidney fibrosis in HKD. The results revealed higher OTUD6A expression in kidney tissues of nephropathy patients and mice with chronic angiotensin II (Ang II) administration than that from the control ones. OTUD6A was mainly located in tubular epithelial cells. Moreover, OTUD6A deficiency significantly protected mice against Ang II-induced kidney dysfunction and fibrosis. Also, knocking OTUD6A down suppressed Ang II-induced fibrosis in cultured tubular epithelial cells, whereas overexpression of OTUD6A enhanced fibrogenic responses. Mechanistically, OTUD6A bounded to signal transducer and activator of transcription 3 (STAT3) and removed K63-linked-ubiquitin chains to promote STAT3 phosphorylation at tyrosine 705 position and nuclear translocation, which then induced profibrotic gene transcription in epithelial cells. These studies identified STAT3 as a direct substrate of OTUD6A and highlighted the pivotal role of OTUD6A in Ang II-induced kidney injury, indicating OTUD6A as a potential therapeutic target for HKD. NEW & NOTEWORTHY Ovarian tumor domain-containing protein 6 A (OTUD6A) knockout mice are protected against angiotensin II-induced kidney dysfunction and fibrosis. OTUD6A promotes pathological kidney remodeling and dysfunction by deubiquitinating signal transducer and activator of transcription 3 (STAT3). OTUD6A binds to and removes K63-linked-ubiquitin chains of STAT3 to promote its phosphorylation and activation, and subsequently enhances kidney fibrosis. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii is among the most important parasites worldwide. The apicoplast is a unique organelle shared by all Apicomplexan protozoa. Increasing lines of evidence suggest that the apicoplast possesses its own ubiquitination system. Deubiquitination is a crucial step executed by deubiquitinase (DUB) during protein ubiquitination. While multiple components of ubiquitination have been identified in T. gondii, the deubiquitinases involved remain unknown. The aim of the current study was to delineate the localization of TgOTU7 and elucidate its functions. TgOTU7 was specifically localized at the apicoplast, and its expression was largely regulated during the cell cycle. Additionally, TgOTU7 efficiently breaks down ubiquitin chains, exhibits linkage-nonspecific deubiquitinating activity and is critical for the lytic cycle and apicoplast biogenesis, similar to the transcription of the apicoplast genome and the nuclear genes encoding apicoplast-targeted proteins. Taken together, the results indicate that the newly described deubiquitinase TgOTU7 specifically localizes to the apicoplast and affects the cell growth and apicoplast homeostasis of T. gondii. [ABSTRACT FROM AUTHOR]

Here, we describe an adult female with severe fasciitis and skin necrosis who carried a private, predicted deleterious missense mutation in OTULIN in heterozygosity. OTULIN is a cellular regulator of deubiquitination that has been shown to play a key role in intrinsic immunity against staphylococcal α-toxin. The patient was treated with broad-spectrum antibiotics, and multiple surgical explorations were conducted without clinical response. Since autoinflammation was the predominant clinical feature, TNF inhibition was started with a good clinical response. We show that excessive inflammation in OTULIN haploinsufficiency can be effectively treated by TNF inhibition. [ABSTRACT FROM AUTHOR]

Ubiquitin-specific protease7 (USP7) regulates the stability of the p53 tumor suppressor protein and several other proteins critical for tumor cell survival. Aberrant expression of USP7 facilitates human malignancies by altering the activity of proto-oncogenes/proteins, and tumor suppressor genes. Therefore, USP7 is a validated anti-cancer drug target. In this study, a drug repurposing approach was used to identify new hits against the USP7 enzyme. It is one of the most strategic approaches to find new uses for drugs in a cost- and time-effective way. Nuclear Magnetic Resonance-based screening of 172 drugs identified 11 compounds that bind to the catalytic domain of USP7 with dissociation constant (Kd) values in the range of 0.6–1.49 mM. These 11 compounds could thermally destabilize the USP7 enzyme by decreasing its melting temperature up to 9 °C. Molecular docking and simulation studies provided structural insights into the ligand-protein complexes, suggesting that these compounds bind to the putative substrate binding pocket of USP7, and interact with its catalytically important residues. Among the identified 11 hits, compound 6 (oxybutynin), 7 (ketotifen), 10 (pantoprazole sodium), and 11 (escitalopram) also showed anti-cancer activity with an effect on the expression of proto-oncogenes and tumor-suppressor gene at mRNA level in HCT116 cells. The compounds identified in this study can serve as potential leads for further studies.

Abstract Deubiquitinases (DUBs) play important roles in various human cancers and targeting DUBs is considered as a novel anticancer therapeutic strategy. Overexpression of ubiquitin specific protease 7 and 22 (USP7 and USP22) are associated with malignancy, therapy resistance, and poor prognosis in many cancers. Although both DUBs are involved in the regulation of similar genes and signaling pathways, such as histone H2B monoubiquitination (H2Bub1), c-Myc, FOXP3, and p53, the interdependence of USP22 and USP7 expression has never been described. In the study, we found that targeting USP7 via either siRNA-mediated knockdown or pharmaceutical inhibitors dramatically upregulates USP22 in cancer cells. Mechanistically, the elevated USP22 occurs through a transcriptional pathway, possibly due to desuppression of the transcriptional activity of SP1 via promoting its degradation upon USP7 inhibition. Importantly, increased USP22 expression leads to significant activation of downstream signal pathways including H2Bub1 and c-Myc, which may potentially enhance cancer malignancy and counteract the anticancer efficacy of USP7 inhibition. Importantly, targeting USP7 further suppresses the in vitro proliferation of USP22-knockout (USP22-Ko) A549 and H1299 lung cancer cells and induces a stronger activation of p53 tumor suppressor signaling pathway. In addition, USP22-Ko cancer cells are more sensitive to a combination of cisplatin and USP7 inhibitor. USP7 inhibitor treatment further suppresses in vivo angiogenesis and tumor growth and induced more apoptosis in USP22-Ko cancer xenografts. Taken together, our findings demonstrate that USP7 inhibition can dramatically upregulate USP22 in cancer cells; and targeting USP7 and USP22 may represent a more effective approach for targeted cancer therapy, which warrants further study. Video Abstract

Abstract Background USP51 is a deubiquitinase (DUB), that is involved in diverse cellular processes. Accumulating evidence has demonstrated that USP51 contributes to cancer development. However, its impact on non-small cell lung carcinoma (NSCLC) cell malignancy is largely unknown. Methods In this study, we performed bioinformatics analysis on a dataset from The Cancer Genome Atlas to determine the association between USP51 and cell stemness marker expression in NSCLC patients. RT‒qPCR, Western blotting, and flow cytometry were performed to examine the effects of USP51 depletion on stemness marker expression. Colony formation and tumor sphere formation assays were used to assess the stemness of NSCLC cells. A cycloheximide chase time-course assay and a polyubiquitination assay were carried out to analyze the effects of USP51 on the TWIST1 protein level. TWIST1 was overexpressed in USP51 knockdown NSCLC cells to determine whether TWIST1 is required. The effect of USP51 on the in vivo growth of NSCLC cells was tested through subcutaneous injections in mice. Results We found that USP51 deubiquitinates TWIST1, which is significantly upregulated in the tissues of patients with NSCLC and is closely associated with poor prognosis. USP51 expression was positively correlated with the expression of stemness marker CD44, SOX2, NANOG, and OCT4 in NSCLC patients. USP51 depletion attenuated mRNA, protein, and cell surface expression of stemness markers and the stemness of NSCLC cells. Ectopic USP51 expression potentiated the stability of the TWIST1 protein by attenuating its polyubiquitination. In addition, TWIST1 re-expression in NSCLC cells reversed the inhibitory effect of USP51 knockdown on cell stemness. Furthermore, the in vivo results confirmed the suppressive effect of USP51 depletion on NSCLC cell growth. Conclusions Our results show that USP51 maintains the stemness of NSCLC cells by deubiquitinating TWIST1. Knocking it down reduces both cell stemness and growth of NSCLC cells.

Proteases that cleave ubiquitin or ubiquitin-like proteins (UBLs) are critical players in maintaining the homeostasis of the organism. Concordantly, their dysregulation has been directly linked to various diseases, including cancer, neurodegeneration, developmental aberrations, cardiac disorders and inflammation. Given their potential as novel therapeutic targets, it is essential to fully understand their mechanisms of action. Traditionally, observed effects resulting from deficiencies in deubiquitinases (DUBs) and UBL proteases have often been attributed to the misregulation of substrate modification by ubiquitin or UBLs. Therefore, much research has focused on understanding the catalytic activities of these proteins. However, this view has overlooked the possibility that DUBs and UBL proteases might also have significant non-catalytic functions, which are more prevalent than previously believed and urgently require further investigation. Moreover, multiple examples have shown that either selective loss of only the protease activity or complete absence of these proteins can have different functional and physiological consequences. Furthermore, DUBs and UBL proteases have been shown to often contain domains or binding motifs that not only modulate their catalytic activity but can also mediate entirely different functions. This review aims to shed light on the non-catalytic, moonlighting functions of DUBs and UBL proteases, which extend beyond the hydrolysis of ubiquitin and UBL chains and are just beginning to emerge.

Many cellular processes are regulated by ubiquitin-mediated proteasomal degradation. Pathogens can regulate eukaryotic proteolysis through the delivery of proteins with de-ubiquitinating (DUB) activities. The obligate intracellular pathogen Chlamydia trachomatis secretes Cdu1 (ChlaDUB1), a dual deubiquitinase and Lys-acetyltransferase, that promotes Golgi remodeling and survival of infected host cells presumably by regulating the ubiquitination of host and bacterial proteins. Here, we determined that Cdu1’s acetylase but not its DUB activity is important to protect Cdu1 from ubiquitin-mediated degradation. We further identified three C. trachomatis proteins on the pathogen-containing vacuole (InaC, IpaM, and CTL0480) that required Cdu1‘s acetylase activity for protection from degradation and determined that Cdu1 and these Cdu1-protected proteins are required for optimal egress of Chlamydia from host cells. These findings highlight a non-canonical mechanism of pathogen-mediated protection of virulence factors from degradation after their delivery into host cells and the coordinated regulation of secreted effector proteins.

Protein ubiquitination plays a pivotal role in protein homeostasis. Ubiquitination may regulate the stability, activity, protein–protein interaction, and localization of a protein. Ubiquitination is subject to regulation by two groups of counteracting enzymes, the E3 ubiquitin ligases and deubiquitinases. Consistently, deubiquitinases are involved in essentially all biological processes. OTUB1, an OTU-family deubiquitinase, is a critical regulator of development, cancer, DNA damage response, and immune response. OTUB1 antagonizes the ubiquitination of a wide-spectrum of proteins through at least two different mechanisms. Besides direct deubiquitination, OTUB1 can also inhibit ubiquitination by non-canonically blocking ubiquitin transfer from certain ubiquitin-conjugases (E2). In this review, we start with a general background of protein ubiquitination and deubiquitination. Next, we introduce the basic characteristics of OTUB1 and then elaborate on the updated biological functions of OTUB1. Afterwards, we discuss potential mechanisms underlying the versatility and specificity of OTUB1 functions. In the end, we discuss the perspective that OTUB1 can be a potential therapeutic target for cancer.

ABSTRACTThe ubiquitin-proteasome system (UPS) and autophagy are highly conserved processes that maintain cellular health through the clearance of misfolded/aberrant proteins and damaged organelles. Ubiquitination is a crucial protein modification to regulate entry in these two pathways. However, the function of deubiquitinases (DUBs) in the UPS and autophagy remains largely unclear. The Leon/USP5 deubiquitinase is essential for maintaining ubiquitin homeostasis and proteasome function. In our recent study, we found that Leon/USP5 depletion resulted in the induction of autophagosome formation and an enhancement of the autophagic flux. Additionally, a genetic analysis in Drosophila revealed that Leon overexpression suppressed Atg1-induced cell death. We further showed that Leon/USP5 interacts with the autophagy initiator Atg1/ULK1, regulating its levels and thus modulating autophagosome formation. These findings suggest that Leon/USP5 plays a dual role in regulation of UPS and autophagy.Abbreviations: Atg1: autophagy-related 1; Atg7: autophagy-related 7; DUB: deubiquitinase; ED: enzyme dead; PTM: post-translational modification; SQSTM1: sequestosome 1; ULK1: unc-51 like autophagy activating kinase 1; UPS: ubiquitin-proteasome system; USP5: ubiquitin-specific proteinase 5; WT: wild-type.

Deubiquitinases (DUBs) are a family of >100 proteases that hydrolyze isopeptide bonds linking ubiquitin to protein substrates, often leading to reduced substrate degradation through the ubiquitin proteasome system. Deregulation of DUB activity has been implicated in many diseases, including cancer, neurodegeneration and auto‐inflammation, and several have been recognized as attractive targets for therapeutic intervention. Ubiquitin‐derived covalent activity‐based probes (ABPs) provide a powerful tool for DUB activity profiling, but their large recognition element impedes cellular permeability and presents an unmet need for small molecule ABPs which can account for regulation of DUB activity in intact cells or organisms. Here, through comprehensive chemoproteomic warhead profiling, we identify cyanopyrrolidine (CNPy) probe IMP‐2373 (12) as a small molecule pan‐DUB ABP to monitor DUB activity in physiologically relevant live cells. Through proteomics and targeted assays, we demonstrate that IMP‐2373 quantitatively engages more than 35 DUBs across a range of non‐toxic concentrations in diverse cell lines. We further demonstrate its application to quantification of changes in intracellular DUB activity during pharmacological inhibition and during MYC deregulation in a model of B cell lymphoma. IMP‐2373 thus offers a complementary tool to ubiquitin ABPs to monitor dynamic DUB activity in the context of disease‐relevant phenotypes. [ABSTRACT FROM AUTHOR]

Deubiquitinases (DUBs) play important roles in various human cancers and targeting DUBs is considered as a novel anticancer therapeutic strategy. Overexpression of ubiquitin specific protease 7 and 22 (USP7 and USP22) are associated with malignancy, therapy resistance, and poor prognosis in many cancers. Although both DUBs are involved in the regulation of similar genes and signaling pathways, such as histone H2B monoubiquitination (H2Bub1), c-Myc, FOXP3, and p53, the interdependence of USP22 and USP7 expression has never been described. In the study, we found that targeting USP7 via either siRNA-mediated knockdown or pharmaceutical inhibitors dramatically upregulates USP22 in cancer cells. Mechanistically, the elevated USP22 occurs through a transcriptional pathway, possibly due to desuppression of the transcriptional activity of SP1 via promoting its degradation upon USP7 inhibition. Importantly, increased USP22 expression leads to significant activation of downstream signal pathways including H2Bub1 and c-Myc, which may potentially enhance cancer malignancy and counteract the anticancer efficacy of USP7 inhibition. Importantly, targeting USP7 further suppresses the in vitro proliferation of USP22-knockout (USP22-Ko) A549 and H1299 lung cancer cells and induces a stronger activation of p53 tumor suppressor signaling pathway. In addition, USP22-Ko cancer cells are more sensitive to a combination of cisplatin and USP7 inhibitor. USP7 inhibitor treatment further suppresses in vivo angiogenesis and tumor growth and induced more apoptosis in USP22-Ko cancer xenografts. Taken together, our findings demonstrate that USP7 inhibition can dramatically upregulate USP22 in cancer cells; and targeting USP7 and USP22 may represent a more effective approach for targeted cancer therapy, which warrants further study. 51f9aZhqcv_kcNHk2RbtxA Video Abstract [ABSTRACT FROM AUTHOR]

SQSTM1/p62 (sequestosome 1) is a multifunctional protein that serves as a receptor for selective autophagy and scaffold. In selective autophagy, p62 functions as a bridge between polyubiquitinated proteins and autophagosomes. Further, p62 acts as a signaling hub for many cellular pathways including mTORC1, NF-κB, and Keap1-Nrf2. Post-translational modifications of p62, such as ubiquitination and phosphorylation, are known to determine its binding partners and regulate their intracellular functions. However, the mechanism of p62 deubiquitination remains unclear. In this study, we found that ubiquitin-specific protease 13 (USP13), a member of the USP family, directly binds p62 and removes ubiquitin at Lys7 (K7) of the PB1 domain. USP13-mediated p62 deubiquitination enhances p62 protein stability and facilitates p62 oligomerization, resulting in increased autophagy and degradation of Keap1, which is a negative regulator of the antioxidant response that promotes Nrf2 activation. Thus, USP13 can be considered a therapeutic target as a deubiquitination enzyme of p62 in autophagy-related diseases. [Display omitted] • USP13 is a deubiquitination enzyme of p62/SQSTM1. • USP13 enhances p62 stability by removing K48 and K63 ubiquitin at residue p62 K7. • p62-mediated autophagy is promoted by USP13. • USP13 induces p62-Keap1 binding, leading to free Nrf2 enter the nucleus for antioxidant gene expression. [ABSTRACT FROM AUTHOR]

Reversible protein ubiquitination represents an essential determinator of cellular homeostasis, and the ubiquitin-specific enzymes, particularly deubiquitinases (DUBs), are emerging as promising targets for drug development. DUBs are composed of seven different subfamilies, out of which ubiquitin-specific proteases (USPs) are the largest family with 56 members. One of the well-characterized USPs is USP1, which contributes to several cellular biological processes including DNA damage response, immune regulation, cell proliferation, apoptosis, and migration. USP1 levels and activity are regulated by multiple mechanisms, including transcription regulation, phosphorylation, autocleavage, and proteasomal degradation, ensuring that the cellular function of USP1 is performed in a suitably modulated spatio-temporal manner. Moreover, USP1 with deregulated expression and activity are found in several human cancers, indicating that targeting USP1 is a feasible therapeutic approach in anti-cancer treatment. In this review, we highlight the essential role of USP1 in cancer development and the regulatory landscape of USP1 activity, which might provide novel insights into cancer treatment. [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV) infection is a major public health problem worldwide, causing nearly one million deaths annually. OTUD5 is a deubiquitinase associated with cancer development and innate immunity response. However, the regulatory mechanisms of OTUD5 underlying HBV replication need to be deeply elucidated. In the present investigation, we found that HBV induced significant up-regulation of OTUD5 protein in HBV-infected cells. Further study showed that OTUD5 interacted with HBV core/precore, removing their K48-linked ubiquitination chains and protecting their stability. Meanwhile, overexpression of OTUD5 could inhibit the MAPK pathway and then increase the expression of HNF4ɑ, and ERK1/2 signaling was required for OTUD5-mediated activation of HNF4α, promoting HBV replication. Together, these data indicate that OTUD5 could deubiquitinate HBV core protein degradation by its deubiquitinase function and promote HBV activity by up-regulating HNF4α expression via inhibition of the ERK1/2 pathway. These results might present a novel therapeutic strategy against HBV infection. [ABSTRACT FROM AUTHOR]

Simple Summary: Triple-negative breast cancer (TNBC) has a high recurrence rate and metastasis rate, and the prognosis for patients is poor. Identifying novel treatment avenues for TNBC is crucial for the treatment and prognosis of TNBC patients. Deubiquitinase deficiency is associated with the occurrence and progression of several diseases, including cancer. As a member of deubiquitinase, ubiquitin-specific peptidase 53 (USP53) is essential for the development of tumors such as hepatocellular carcinoma and renal clear cell carcinoma. However, the precise function of USP53 in TNBC remains unknown. Our study has identified USP53 as a molecular target that mediates the progression of TNBC. This not only deepens our understanding of the tumorigenesis and development of TNBC, but also provides new potential therapeutic targets for TNBC patients. Breast cancer (BC) ranks in the top five malignant tumors in terms of morbidity and mortality rates. Among BC subtypes, TNBC has a high recurrence rate and metastasis rate and the worst prognosis. However, the exact mechanism by which TNBC develops is unclear. Here, we show that the deubiquitinase USP53 contributes to tumor growth and metastasis in TNBC. USP53 is overexpressed in TNBC, and this phenotype is linked to a poor prognosis. Functionally, USP53 promotes TNBC cell proliferation, migration, invasion, and epithelial–mesenchymal transition (EMT). More importantly, USP53 decreases the chemosensitivity of BC cells by enhancing v-crk sarcoma virus CT10 oncogene homologue (avian)-like (CRKL) expression. Mechanistically, USP53 directly binds CRKL to stabilize and deubiquitinate it, thereby preventing CRKL degradation. Overall, we discovered that USP53 deubiquitinates CRKL, encourages tumor development and metastasis, and reduces chemosensitivity in TNBC. These findings imply that USP53 might represent a new therapeutic target for the treatment of TNBC. [ABSTRACT FROM AUTHOR]

Deubiquitinases (DUBs) are essential for bone remodeling by regulating the differentiation of osteoblast and osteoclast. USP17 encodes for a deubiquitinating enzyme, specifically known as ubiquitin-specific protease 17, which plays a critical role in regulating protein stability and cellular signaling pathways. However, the role of USP17 during osteoblast differentiation has not been elusive. In this study, we initially investigated whether USP17 could regulate the differentiation of osteoblasts. Moreover, USP17 overexpression experiments were conducted to assess the impact on osteoblast differentiation induced by bone morphogenetic protein 4 (BMP4). The positive effect was confirmed through alkaline phosphatase (ALP) expression and activity studies since ALP is a representative marker of osteoblast differentiation. To confirm this effect, Usp17 knockdown was performed, and its impact on BMP4-induced osteoblast differentiation was examined. As expected, knockdown of Usp17 led to the suppression of both ALP expression and activity. Mechanistically, it was observed that USP17 interacted with Osterix (Osx), which is a key transcription factor involved in osteoblast differentiation. Furthermore, overexpression of USP17 led to an increase in Osx protein levels. Thus, to investigate whether this effect was due to the intrinsic function of USP17 in deubiquitination, protein stabilization experiments and ubiquitination analysis were conducted. An increase in Osx protein levels was attributed to an enhancement in protein stabilization via USP17-mediated deubiquitination. In conclusion, USP17 participates in the deubiquitination of Osx, contributing to its protein stabilization, and ultimately promoting the differentiation of osteoblasts. [ABSTRACT FROM AUTHOR]

ETS2 is a member of the ETS family of transcription factors and has been implicated in the regulation of cell proliferation, differentiation, apoptosis, and tumorigenesis. The aberrant activation of ETS2 is associated with various human cancers, highlighting its importance as a therapeutic target. Understanding the regulatory mechanisms and interacting partners of ETS2 is crucial for elucidating its precise role in cellular processes and developing novel strategies to modulate its activity. In this study, we conducted binding assays using a human deubiquitinase (DUB) library and identified USP39 as a novel ETS2-binding DUB. USP39 interacts with ETS2 through their respective amino-terminal regions, and the zinc finger and PNT domains are not required for this binding. USP39 deubiquitinates ETS2 without affecting its protein stability. Interestingly, however, USP39 significantly suppresses the transcriptional activity of ETS2. Furthermore, we demonstrated that USP39 leads to a reduction in the nuclear localization of ETS2. Our findings provide valuable insights into the intricate regulatory mechanisms governing ETS2 function. Understanding the interplay between USP39 and ETS2 may have implications for therapeutic interventions targeting ETS2-related diseases, including cancer, where the dysregulation of ETS2 is frequently observed. [ABSTRACT FROM AUTHOR]

Aim: Oral cancer is one of the top three types of cancer and is of significant public health importance in India. A common post-translational modification in cells is ubiquitination/deubiquitination, and its dysregulation is closely associated with the development of cancer. Studies on the role of ubiquitination in oral squamous cell carcinoma (OSCC) are lacking. Increased expression of usp36 has been observed in various types of cancer, and this study aimed to check the gene expression of usp36 in OSCC patients. In this study, we analyzed the expression of ubiquitin-specific proteases (USPs) 36 in OSCC. Materials and Methods: A total of 15 OSCC patients at different stages of tumor differentiation and age- and sex-matched controls were recruited for the study. The patients were categorized based on their differentiation patterns. RNA was extracted from the tissues, and usp36 gene expression was checked in these samples using a quantitative real-time PCR technique. Results: Our study showed increased expression of usp36 gene in OSCC patients. The usp36 mRNA was 231.8 ± 137.94 folds higher in well-differentiated squamous cell carcinoma patients, 38.18 ± 3.77 folds higher in moderately differentiated squamous cell carcinoma patients, and 25.49 ± 7.30 folds higher in poorly differentiated squamous cell carcinoma patients compared to control tissues. Conclusion: Our study reports, for the first time, an increased gene expression of usp36 in OSCC tissues. [ABSTRACT FROM AUTHOR]

Ubiquitination is a specific protein degradation and reversible post-translational modification process that can be reversed by deubiquitinase (DUBs). DUBs can hydrolyze and release ubiquitin in the substrate protein so that the substrate can avoid degradation or change its activity, and it has an impact on plant growth and development, cell cycle, abiotic stress response, and other biological processes. Transcript sequences of potato varieties "DM1-3", "Atlantic" and "Cooperation-88" downloaded from Potato Genome Resources were used for genome-wide identification of the DUB gene family using Hidden Markov Models and verified in the NCBI CD-Search tool. The characteristics of DUB genes from different potato varieties were analyzed including subcellular localization, gene structural motifs, phylogenetic tree, and sequence homology. Polyethylene glycol 6000 (PEG6000) induced drought stress transcriptome analysis was performed on the "Atlantic", and differentially expressed genes were screened, with emphasis on the characterization of deubiquitinase. DUB genes have a complex gene structure, often with a large number of exons and alternative splicing. Their promoters contain abundant abiotic stress-responsive elements, such as 425 MYC, 325 ABRE, and 320 MYB. There are also a large number of orthologous genes in the DUBs of the three potato varieties, and these genes are often clustered in similar regions on the genome. We performed transcriptome sequencing of the potato under PEG-induced drought stress and analyzed it for the first time using the Atlantic as a reference genome. We identified a total of 6067 down-regulated differentially expressed genes (DEGs) and 4950 upregulated DEGs under PEG-induced drought stress. We screened the expression of DUBs and observed that 120 DUBs were up-regulated where most of them functioned in the nucleus, and the interacting proteins of DUBs were also localized in the nucleus. We have comprehensively identified and analyzed potato DUBs, and the accurately aligned transcriptome data which will further deepen the understanding of DUBs involved in the regulation of osmotic stress. [ABSTRACT FROM AUTHOR]

Simple Summary: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the world, with a heterogeneous characteristic. We firstly systematically screened the critical gene ubiquitin-specific protease 14 (USP14) in HNSCC from the DUB library after building a risk signature. Our data suggested that USP14 regulated heat shock transcription factor 1 (HSF1) stabilization by its deubiquitination and increased its downstream proteins, such as heat shock protein 60 (HSP60), HSP70, and HSP90, to promote HNSCC proliferation and lung metastasis in vivo and in vitro, and the overexpression of HSF1 reversed the inhibitory effect of USP14 depletion in vivo. Forty-five tissue samples from patients with HNSCC cancer were collected to determine the correlation of USP14 and HSF1 expression in the diagnosis of HNSCC, which is a novel mechanism of USP14 as a carcinogenic gene in HNSCC. Our results emphasized that USP14 and its downstream HSF1 have therapeutic potential in targeting HNSCC. The ubiquitin-proteasome system is a pivotal intracellular proteolysis process in posttranslational modification. It regulates multiple cellular processes. Deubiquitinating enzymes (DUBs) are a stabilizer in proteins associated with tumor growth and metastasis. However, the link between DUBs and HNSCC remains incompletely understood. In this study, therefore, we identified USP14 as a tumor proliferation enhancer and a substantially hyperactive deubiquitinase in HNSCC samples, implying a poor prognosis prediction. Silencing USP14 in vitro conspicuously inhibited HNSCC cell proliferation and migration. Consistently, defective USP14 in vivo significantly diminished HNSCC tumor growth and lung metastasis compared to the control group. Luciferase assays indicated that HSF1 was downstream from USP14, and an evaluation of the cellular effects of HSF1 overexpression in USP14-dificient mice tumors showed that elevated HSF1 reversed HNSCC growth and metastasis predominantly through the HSF1-HSP pathway. Mechanistically, USP14 encouraged HSF1 expression by deubiquitinating and stabilizing HSF1, which subsequently orchestrated transcriptional activation in HSP60, HSP70, and HSP90, ultimately leading to HNSCC progression and metastasis. Collectively, we uncovered that hyperactive USP14 contributed to HNSCC tumor growth and lung metastasis by reinforcing HSF1-depedent HSP activation, and our findings provided the insight that targeting USP14 could be a promising prognostic and therapeutic strategy for HSNCC. [ABSTRACT FROM AUTHOR]

The nuclear factor E2-related factor 1 (Nrf1) transcription factor performs a critical role in regulating cellular homeostasis as part of the cellular stress response and drives the expression of antioxidants and detoxification enzymes among many other functions. Ubiquitination plays an important role in controlling the abundance and thus nuclear accumulation of Nrf1 proteins, but the regulatory enzymes that act on Nrf1 are not fully defined. Here, we identified ubiquitin specific protease 7 (USP7), a deubiquitinating enzyme, as a novel regulator of Nrf1 activity. We found that USP7 interacts with Nrf1a and TCF11-the two long protein isoforms of Nrf1. Expression of wildtype USP7, but not its catalytically defective mutant, resulted in decreased ubiquitination of TCF11 and Nrf1a, leading to their increased stability and increased transactivation of reporter gene expression by TCF11 and Nrf1a. In contrast, knockdown or pharmacologic inhibition of USP7 dramatically increased ubiquitination of TCF11 and Nrf1a and reduction of their steady state levels. Loss of USP7 function attenuated the induction of Nrf1 protein expression in response to treatment with arsenic and other toxic metals, and inhibition of USP7 activity significantly sensitized cells to arsenic treatment. Collectively, these findings suggest that USP7 may act to modulate abundance of Nrf1 protein to induce gene expression in response to toxic metal exposure.

Ubiquitin-specific protease 18 (USP18) is a protein recognized for its dual enzymatic and non-enzymatic nature. It is involved in many physiological processes like the cell cycle and cell signaling. It also suppresses heart muscle remodeling upon an increase in the afterload. The role of USP18 in kidney pathology remains unknown. The objective of the study was to assess the relationship between serum and urine USP18 levels, the factors contributing to cardiovascular risk, and the markers of kidney disease activity at different stages of chronic kidney disease (CKD). One hundred participants, aged between 24 and 85 years (mean 53.1 ± 17.1 years), were included. Five groups (n = 20 each) were recruited according to their renal status (healthy individuals, patients with proteinuric glomerulonephritis, patients with non-proteinuric CKD, patients who were treated with hemodialysis, and kidney transplant recipients). The measurements of serum and urine USP18 levels were performed using ELISA. The median serum USP18 level was the highest in healthy participants (1143.0 pg/mL) and kidney transplant recipients (856.6 pg/mL), whereas, in individuals with different forms of CKD, it fitted within the range of 402.1–471.9 pg/mL. Urinary USP18 reached the highest level in the group of CKD patients not yet on dialysis (303.3 pg/mL). Only in this group did it correlate with serum creatinine and urea concentrations. Our results suggest the inhibition of cardioprotective USP18 signaling when kidney function is impaired. Moreover, an increased level of urinary USP18 may indicate chronic tubular damage.

Deubiquitination is an important form of post-translational modification that regulates protein homeostasis. Ovarian tumor domain-containing proteins (OTUDs) subfamily member OTUD3 was identified as a deubiquitinating enzyme involved in the regulation of various physiological processes such as immunity and inflammation. Disturbances in these physiological processes trigger diseases in humans and animals, such as cancer, neurodegenerative diseases, diabetes, mastitis, etc. OTUD3 is aberrantly expressed in tumors and is a double-edged sword, exerting tumor-promoting or anti-tumor effects in different types of tumors affecting cancer cell proliferation, metastasis, and metabolism. OTUD3 is regulated at the transcriptional level by a number of MicroRNAs, such as miR-520h, miR-32, and miR101-3p. In addition, OTUD3 is regulated by a number of post-translational modifications, such as acetylation and ubiquitination. Therefore, understanding the regulatory mechanisms of OTUD3 expression can help provide insight into its function in human immunity and disease, offering the possibility of its use as a therapeutic target to diagnose or treat disease.

Ubiquitin-specific protease 2 (USP2) is a deubiquitinase belonging to the USPs subfamily. USP2 has been known to display various biological effects including tumorigenesis and inflammation. Therefore, we aimed to examine the sensitization effect of USP2 in TRAIL-mediated apoptosis. The pharmacological inhibitor (ML364) and siRNA targeting USP2 enhanced TNF-related apoptosis-inducing ligand (TRAIL)-induced cancer cell death, but not normal cells. Mechanistically, USP2 interacted with survivin, and ML364 degraded survivin protein expression by increasing the ubiquitination of survivin. Overexpression of survivin or USP2 significantly prevented apoptosis through cotreatment with ML364 and TRAIL, whereas a knockdown of USP2 increased sensitivity to TRAIL. Taken together, our data suggested that ML364 ubiquitylates and degrades survivin, thereby increasing the reactivity to TRAIL-mediated apoptosis in cancer cells. [ABSTRACT FROM AUTHOR]

Background: Sepsis is a systemic inflammatory response syndrome characterized by persistent inflammation and immunosuppression, leading to septic shock and multiple organ dysfunctions. Ubiquitin-specific peptidase 10 (USP10), a deubiquitinase enzyme, plays a vital role in cancer and arterial restenosis, but its involvement in sepsis is unknown. Objective: In this study, we investigated the significance of USP10 in lipopolysaccharide (LPS)-stimulated macrophages and its biological roles in LPS-induced sepsis. Methods: Lipopolysaccharides (LPS) were used to establish sepsis models in vivo and in vitro. We use western blot to identify USP10 expression in macrophages. Spautin-1 and USP10-siRNA were utilized for USP10 inhibition. ELISA assays were used to assess for TNF‐α and IL-6 in vitro and in vivo. Nuclear and cytoplasmic protein extraction and Confocal microscopy were applied to verify the translocation of NF‐κB. Mechanically, co-immunoprecipitation and rescue experiments were used to validate the regulation of USP10 and NEMO. Results: In macrophages, we found that LPS induced USP10 upregulation. The inhibition or knockdown of USP10 reduced the pro‐inflammatory cytokines TNF-α and IL-6 and suppressed LPS‐induced NF‐κB activation by regulating the translocation of NF‐κB. Furthermore, we found that NEMO, the regulatory subunit NF-κB essential modulator, was essential for the regulation of LPS-induced inflammation by USP10 in macrophages. NEMO protein evidently interacted with USP10, whereby USP10 inhibition accelerated the degradation of NEMO. Suppressing USP10 significantly attenuated inflammatory responses and improved the survival rate in LPS-induced sepsis mice. Conclusions: Overall, USP10 was shown to regulate inflammatory responses by stabilizing the NEMO protein, which may be a potential therapeutic target for sepsis-induced lung injury. [ABSTRACT FROM AUTHOR]