Notoginsenoside Ft1 inhibits colorectal cancer growth by increasing CD8+ T cell proportion in tumor-bearing mice through the USP9X signaling pathway.

The management of colorectal cancer (CRC) poses a significant challenge, necessitating the development of innovative and effective therapeutics. Our research has shown that notoginsenoside Ft1 (Ng-Ft1), a small molecule, markedly inhibits subcutaneous tumor formation in CRC and enhances the proportion of CD8+ T cells in tumor-bearing mice, thus restraining tumor growth. Investigation into the mechanism revealed that Ng-Ft1 selectively targets the deubiquitination enzyme USP9X, undermining its role in shielding ß-catenin. This leads to a reduction in the expression of downstream effectors in the Wnt signaling pathway. These findings indicate that Ng-Ft1 could be a promising small-molecule treatment for CRC, working by blocking tumor progression via the Wnt signaling pathway and augmenting CD8+ T cell prevalence within the tumor environment.

USP25 Deficiency Exacerbates Acute Pancreatitis via Up-Regulating TBK1-NF-κB Signaling in Macrophages.

BACKGROUND & AIMS: Severe acute pancreatitis can easily lead to systemic inflammatory response syndrome and death. Macrophages are known to be involved in the pathophysiology of acute pancreatitis (AP), and macrophage activation correlates with disease severity. In this study, we examined the role of ubiquitin-specific protease 25, a deubiquitinating enzyme and known regulator of macrophages, in the pathogenesis of AP. METHODS: We used L-arginine, cerulein, and choline-deficient ethionine-supplemented diet-induced models of AP in Usp25-/- mice and wild-type mice. We also generated bone marrow Usp25-/- chimeric mice and initiated L-arginine-mediated AP. Primary acinar cells and bone marrow-derived macrophages were isolated from wild-type and Usp25-/- mice to dissect molecular mechanisms. RESULTS: Our results show that Usp25 deficiency exacerbates pancreatic and lung injury, neutrophil and macrophage infiltration, and systemic inflammatory responses in L-arginine, cerulein, and choline-deficient ethionine-supplemented diet-induced models of AP. Bone marrow Usp25-/- chimeric mice challenged with L-arginine show that Usp25 deficiency in macrophages exaggerates AP by up-regulating the TANK-binding kinase 1 (TBK1)-nuclear factor-κB (NF-κB) signaling pathway. Similarly, in vitro data confirm that Usp25 deficiency enhances the TBK1-NF-κB pathway, leading to increased expression of inflammatory cytokines in bone marrow-derived macrophages. CONCLUSIONS: Usp25 deficiency in macrophages enhances TBK1-NF-κB signaling, and the induction of inflammatory chemokines and type I interferon-related genes exacerbates pancreatic and lung injury in AP.

Upregulation of USP22 and ABCC1 during Sorafenib Treatment of Hepatocellular Carcinoma Contribute to Development of Resistance.

Sorafenib is a small molecule that blocks tumor proliferation by targeting the activity of multi-kinases for the treatment of advanced hepatocellular carcinoma (HCC). Increasing sorafenib resistance following long-term treatment is frequently encountered. Mechanisms underlying sorafenib resistance remain not completely clear. To further understand the mechanism of sorafenib resistance in HCC, we established sorafenib-resistant cell lines by slowly increasing sorafenib concentration in cell culture medium. Upregulation of USP22 and ABCC1 were found in Sorafenib-resistant cells. Sorafenib-resistant cells treated with USP22 siRNA showed significant reduction in endogenous mRNA and protein levels of ABCC1. During sorafenib treatment, upregulation of USP22 increases ABCC1 expression and subsequently contributes to sorafenib resistance in HCC cells. Immunohistochemical analysis revealed a positive correlation between USP22 and ABCC1 expression in tissue samples from sorafenib-resistant patients (Pearson's correlation = 0.59, p = 0.03). Our findings indicate that upregulation of USP22 and ABCC1 expression during treatment contribute to sorafenib resistance in HCC cells and that USP22 has strong potential as a therapeutic target for overcoming sorafenib resistance in HCC patients.

BAP1 and YY1 regulate expression of death receptors in malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is a rare, aggressive, and incurable cancer arising from the mesothelial lining of the pleura, with few available treatment options. We recently reported that loss of function of the nuclear deubiquitinase BRCA1-associated protein 1 (BAP1), a frequent event in MPM, is associated with sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. As a potential underlying mechanism, here we report that BAP1 negatively regulates the expression of TRAIL receptors: death receptor 4 (DR4) and death receptor 5 (DR5). Using tissue microarrays of tumor samples from MPM patients, we found a strong inverse correlation between BAP1 and TRAIL receptor expression. BAP1 knockdown increased DR4 and DR5 expression, whereas overexpression of BAP1 had the opposite effect. Reporter assays confirmed wt-BAP1, but not catalytically inactive BAP1 mutant, reduced promoter activities of DR4 and DR5, suggesting deubiquitinase activity is required for the regulation of gene expression. Co-immunoprecipitation studies demonstrated direct binding of BAP1 to the transcription factor Ying Yang 1 (YY1), and chromatin immunoprecipitation assays revealed BAP1 and YY1 to be enriched in the promoter regions of DR4 and DR5. Knockdown of YY1 also increased DR4 and DR5 expression and sensitivity to TRAIL. These results suggest that BAP1 and YY1 cooperatively repress transcription of TRAIL receptors. Our finding that BAP1 directly regulates the extrinsic apoptotic pathway will provide new insights into the role of BAP1 in the development of MPM and other cancers with frequent BAP1 mutations.

SARS-CoV-2 Papain-Like Protease Potential Inhibitors-In Silico Quantitative Assessment.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encodes the papain-like protease (PLpro). The protein not only plays an essential role in viral replication but also cleaves ubiquitin and ubiquitin-like interferon-stimulated gene 15 protein (ISG15) from host proteins, making it an important target for developing new antiviral drugs. In this study, we searched for novel, noncovalent potential PLpro inhibitors by employing a multistep in silico screening of a 15 million compound library. The selectivity of the best-scored compounds was evaluated by checking their binding affinity to the human ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), which, as a deubiquitylating enzyme, exhibits structural and functional similarities to the PLpro. As a result, we identified 387 potential, selective PLpro inhibitors, from which we retrieved the 20 best compounds according to their IC50 values toward PLpro estimated by a multiple linear regression model. The selected candidates display potential activity against the protein with IC50 values in the nanomolar range from approximately 159 to 505 nM and mostly adopt a similar binding mode to the known, noncovalent SARS-CoV-2 PLpro inhibitors. We further propose the six most promising compounds for future in vitro evaluation. The results for the top potential PLpro inhibitors are deposited in the database prepared to facilitate research on anti-SARS-CoV-2 drugs.

Antileukemic effect of caffeic acid 3,4-dihydroxyphenetyl ester. Evidences for its mechanisms of action.

BACKGROUND: Caffeic acid 3,4-dihydroxyphenethyl ester (CADPE) is a natural polyphenolic ester isolated as a minor component from a water extract of the Chinese medicine Zhongjiefeng [Sarcandra glabra (Thunb.) Nakai (Chloranthaceae)] and has previously shown to have activity against solid tumors through the modulation of multiple targets or signal pathways. However, the activity and potential mechanism of CADPE against leukemia cells have not yet been characterized. PURPOSE: To investigate whether and how CADPE kills leukemia cells. METHOD: (1) The activity of CADPE inhibiting the growth of different leukemia cell lines was evaluated by MTT assay; (2) Cell cycle arrest and apoptosis induced by CADPE were determined by flow cytometry with FlowJo software for quantification; (3) The protein levels were analyzed by Western blot and ubiquitin-binding c-Myc was acquired by co-immunoprecipitation. RESULTS: CADPE exerted potent activity against different leukemia cell lines with low toxicity in normal cells. In terms of mechanism of action, CADPE promoted ubiquitin-proteasome-dependent degradation of c-Myc through activating glycogen synthase kinase-3ß (GSK3ß) and downregulating deubiquitinating enzyme USP28 to trigger the interaction of c-Myc with ubiquitin ligase Fbw7, resulting in the downregulation of cell cycle regulators and anti-apoptotic proteins and consequently, cell cycle arrest and cell apoptosis. CONCLUSION: CADPE is a novel c-Myc inhibitor with high activity and a unique mechanism for killing leukemia cells.

Identification of novel human USP2 inhibitor and its putative role in treatment of COVID-19 by inhibiting SARS-CoV-2 papain-like (PLpro) protease.

Human ubiquitin carboxyl-terminal hydrolase-2 (USP2) inhibitors, such as thiopurine analogs, have been reported to inhibit SARS-CoV papain-like proteases (PLpro). The PLpro have significant functional implications in the innate immune response during SARS-CoV-2 infection and considered an important antiviral target. Both proteases share strikingly similar USP fold with right-handed thumb-palm-fingers structural scaffold and conserved catalytic triad Cys-His-Asp/Asn. In this urgency situation of COVID-19 outbreak, there is a lack of in-vitro facilities readily available to test SARS-CoV-2 inhibitors in whole-cell assays. Therefore, we adopted an alternate route to identify potential USP2 inhibitor through integrated in-silico efforts. After an extensive virtual screening protocol, the best compounds were selected and tested. The compound Z93 showed significant IC50 value against Jurkat (9.67 µM) and MOTL-4 cells (11.8 µM). The binding mode of Z93 was extensively analyzed through molecular docking, followed by MD simulations, and molecular interactions were compared with SARS-CoV-2. The relative binding poses of Z93 fitted well in the binding site of both proteases and showed consensus π-π stacking and H-bond interactions with histidine and aspartate/asparagine residues of the catalytic triad. These results led us to speculate that compound Z93 might be the first potential chemical lead against SARS-CoV-2 PLpro, which warrants in-vitro evaluations.

Blockage of UCHL1 activity attenuates cardiac remodeling in spontaneously hypertensive rats.

Cardiac remodeling is an important pathological process ultimately leading to heart failure. Ubiquitin carboxy-terminal hydrolase 1 (UCHL1) is a deubiquitinase that plays a critical role in neurodegenerative diseases and cancer. However, its role in cardiac remodeling in spontaneously hypertensive rats remains unclear. Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs) were administered the UCHL1 inhibitor LDN-57444 (20 µg/kg/day) from 2 months of age for 4 months. Blood pressure, cardiac hypertrophy, fibrosis, inflammation, and oxidative stress were evaluated by the tail-cuff system, echocardiography, and histological analysis. Gene and protein expression levels were examined by real-time PCR and immunoblotting analysis. At 6 months of age, the expression of UCHL at the mRNA and protein levels was significantly upregulated in SHRs compared with WKYs. Moreover, systolic blood pressure, cardiac performance, left ventricular (LV) hypertrophy, fibrosis, inflammation, and superoxide production were significantly increased in SHRs compared with WKYs, and these effects were markedly attenuated by LDN-57444 after 4 months of administration. These beneficial actions were possibly associated with a reduction in blood pressure and inactivation of multiple signaling pathways, including AKT, ERK1/2, STAT3, calcineurin A, TGF-ß/Smad2/3, and NF-κB. In conclusion, the results indicate that UCHL1 is involved in hypertensive cardiac remodeling in SHRs, and targeting UCHL1 activity may be a novel potential therapeutic approach for the treatment of hypertensive heart diseases.

The Timing and Extent of Motor Neuron Vulnerability in ALS Correlates with Accumulation of Misfolded SOD1 Protein in the Cortex and in the Spinal Cord.

Understanding the cellular and molecular basis of selective vulnerability has been challenging, especially for motor neuron diseases. Developing drugs that improve the health of neurons that display selective vulnerability relies on in vivo cell-based models and quantitative readout measures that translate to patient outcome. We initially developed and characterized UCHL1-eGFP mice, in which motor neurons are labeled with eGFP that is stable and long-lasting. By crossing UCHL1-eGFP to amyotrophic lateral sclerosis (ALS) disease models, we generated ALS mouse models with fluorescently labeled motor neurons. Their examination over time began to reveal the cellular basis of selective vulnerability even within the related motor neuron pools. Accumulation of misfolded SOD1 protein both in the corticospinal and spinal motor neurons over time correlated with the timing and extent of degeneration. This further proved simultaneous degeneration of both upper and lower motor neurons, and the requirement to consider both upper and lower motor neuron populations in drug discovery efforts. Demonstration of the direct correlation between misfolded SOD1 accumulation and motor neuron degeneration in both cortex and spinal cord is important for building cell-based assays in vivo. Our report sets the stage for shifting focus from mice to diseased neurons for drug discovery efforts, especially for motor neuron diseases.

Radix Tetrastigma Hemsleyani Flavone Suppresses Cutaneous Squamous Cell Carcinoma A431 Cells via Proteasome Inhibition.

BACKGROUND Radix Tetrastigma Hemsleyani Flavone (RTHF) has detoxification and anti- inflammation activity and is widely used. Here, we report that RTHF inhibits cell proliferation and induces apoptosis in cutaneous squamous cell carcinoma A431 cells and is a potential strategy for cancer therapy. MATERIAL AND METHODS A431 cells were cultured in different concentrations of RTHF. The inhibition of cell proliferation was assessed by MTT assay, cell apoptosis was shown through FCM, and cell invasion was assessed by Transwell methods. Enzyme proteasome assay was used to detect the activity of proteasome and DUB. Expression of apoptosis-related and ubiquitin proteasome pathway-associated proteins were assessed by PCR and Western blot. RESULTS RTHF obviously suppressed the proliferation and induced apoptosis of A431 cells in a dose-dependent manner. Transwell assay showed that RTHF inhibited the cell metastasis significantly. Enzyme proteasome assay show that the RTHF treatment of activity of proteasome and DUB was significantly lower than in control. RTHF increased the expression of Bax and inhibited Bcl-2, pro-caspase3, and pro-caspase9 activity. The expression of USP14, UCHL5, and POH1 decreased and ub-prs increased significantly in the treatment group. CONCLUSIONS Our study reveals that RTHF-mediated inhibition of DUBs and proteasome may provide a potential strategy for cancer therapy.

Cai's Neiyi Prescription promotes apoptosis and inhibits inflammation in endometrial stromal cells with endometriosis through inhibiting USP10.

To observe the effect of Cai's Neiyi Prescription (CNYP) on the apoptosis and inflammation in endometrial stromal cells with endometriosis (EM) both in vivo and in vitro, EM model rats and endometrial stromal cells were treated with CNYP and the level of USP10, p-ERK1/2, ERK1/2, and apoptosis-related protein as well as the levels of proinflammatory factors were measured by Western blotting and ELISA, respectively. Rats with surgically induced EM showed increased USP10 expression and ERK/2 activation. Intragastric administration of CNYP granule significantly inhibited EM-induced ERK1/2 activation and expression of USP10 and Bcl-2, but increased the expression of Bax and Caspase-7 in EM-induced rats. CNYP granule administration also inhibited EM-induced inflammation in rats. Moreover, the ectopic endometrial stromal cells isolated from EM patients demonstrated decreased ERK1/2 activation and expression of USP10 and Bcl-2 and increased expression of Bax and Caspase-7 after cultured in DMEM containing CNYP-medicated rat serum, which were reversed by USP10 overexpression and were enhanced by USP10 siRNA. USP10 overexpression also inhibited while USP10 siRNA enhanced the CNYP-induced inhibition of inflammation in ectopic endometrial stromal cells. Taken together, our results suggest that CNYP granule promotes apoptosis and inhibits inflammation in endometrial stromal cells with EM through inhibiting USP10.

The Prader-Willi syndrome proteins MAGEL2 and necdin regulate leptin receptor cell surface abundance through ubiquitination pathways.

In Prader-Willi syndrome (PWS), obesity is caused by the disruption of appetite-controlling pathways in the brain. Two PWS candidate genes encode MAGEL2 and necdin, related melanoma antigen proteins that assemble into ubiquitination complexes. Mice lacking Magel2 are obese and lack leptin sensitivity in hypothalamic pro-opiomelanocortin neurons, suggesting dysregulation of leptin receptor (LepR) activity. Hypothalamus from Magel2-null mice had less LepR and altered levels of ubiquitin pathway proteins that regulate LepR processing (Rnf41, Usp8, and Stam1). MAGEL2 increased the cell surface abundance of LepR and decreased their degradation. LepR interacts with necdin, which interacts with MAGEL2, which complexes with RNF41 and USP8. Mutations in the MAGE homology domain of MAGEL2 suppress RNF41 stabilization and prevent the MAGEL2-mediated increase of cell surface LepR. Thus, MAGEL2 and necdin together control LepR sorting and degradation through a dynamic ubiquitin-dependent pathway. Loss of MAGEL2 and necdin may uncouple LepR from ubiquitination pathways, providing a cellular mechanism for obesity in PWS.

Cell Lysate-Based AlphaLISA Deubiquitinase Assay Platform for Identification of Small Molecule Inhibitors.

The deubiquitinases, or DUBs, are associated with various human diseases, including neurological disorders, cancer, and viral infection, making them excellent candidates for pharmacological intervention. Drug discovery campaigns against DUBs require enzymatic deubiquitination assays amenable for high-throughput screening (HTS). Although several DUB substrates and assays have been developed in recent years, they are largely limited to recombinantly purified DUBs. Many DUBs are large multidomain proteins that are difficult to obtain recombinantly in sufficient quantities for HTS. Therefore, an assay that obviates the need of recombinant protein generation and also recapitulates a physiologically relevant environment is highly desirable. Such an assay will open doors for drug discovery against many therapeutically relevant, but currently inaccessible, DUBs. Here, we report a cell lysate DUB assay based on AlphaLISA technology for high throughput screening. This assay platform uses a biotin-tagged ubiquitin probe and a HA-tagged DUB expressed in human cells. The assay was validated and adapted to a 1536-well format, which enabled a screening against UCHL1 as proof of principle using a library of 15 000 compounds. We expect that the new platform can be readily adapted to other DUBs to allow the identification of more potent and selective small molecule inhibitors and chemical probes.

Comparative efficacy of alpha-linolenic acid and gamma-linolenic acid to attenuate valproic acid-induced autism-like features

The present study was undertaken to elucidate the effect of alpha-linolenic acid (ALA, 18:3, ω-3) and gamma-linolenic acid (GLA, 18:3, ω-6) on experimental autism features induced by early prenatal exposure to valproic acid (VPA) in albino wistar pups. The pups were scrutinized on the accounts of behavioral, biochemical, and inflammatory markers, and the results suggested that the GLA can impart significant protection in comparison to ALA against VPA-induced autism features. When scrutinized histopathologically, the cerebellum of the GLA-treated animals was evident for more marked protection toward neuronal degeneration and neuronal loss in comparison to ALA. Concomitant administration of ALA and GLA with VPA demonstrated a marked cutdown in the Pgp 9.5 expression with GLA having more pronounced effect. Henceforth, it can be concluded that ALA and GLA can impart favorable protection against the VPA-induced autism-like features with GLA having pronounced effect (AU)

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Thymosin α1 represents a potential potent single-molecule-based therapy for cystic fibrosis.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) that compromise its chloride channel activity. The most common mutation, p.Phe508del, results in the production of a misfolded CFTR protein, which has residual channel activity but is prematurely degraded. Because of the inherent complexity of the pathogenetic mechanisms involved in CF, which include impaired chloride permeability and persistent lung inflammation, a multidrug approach is required for efficacious CF therapy. To date, no individual drug with pleiotropic beneficial effects is available for CF. Here we report on the ability of thymosin alpha 1 (Tα1)-a naturally occurring polypeptide with an excellent safety profile in the clinic when used as an adjuvant or an immunotherapeutic agent-to rectify the multiple tissue defects in mice with CF as well as in cells from subjects with the p.Phe508del mutation. Tα1 displayed two combined properties that favorably opposed CF symptomatology: it reduced inflammation and increased CFTR maturation, stability and activity. By virtue of this two-pronged action, Tα1 has strong potential to be an efficacious single-molecule-based therapeutic agent for CF.

Targeting BAP1: a new paradigm for mesothelioma.

New treatment strategies for malignant pleural mesothelioma (MPM) are important. BAP1 mutations are present in 47-67% of the MPM tumors, making this a good target for treatment. Multiple functions of BAP1 are investigated in the preclinical situation. Due to many functions of BAP1, the phenotypic effect of BAP1 is diverse. Preclinical data on inhibitors reversing these phenotypic effects are promising. However, the mechanism of BAP1 is not fully elucidated yet and further research about the mechanism and possible inhibitors is necessary.

Comparative efficacy of alpha-linolenic acid and gamma-linolenic acid to attenuate valproic acid-induced autism-like features.

The present study was undertaken to elucidate the effect of alpha-linolenic acid (ALA, 18:3, ω-3) and gamma-linolenic acid (GLA, 18:3, ω-6) on experimental autism features induced by early prenatal exposure to valproic acid (VPA) in albino wistar pups. The pups were scrutinized on the accounts of behavioral, biochemical, and inflammatory markers, and the results suggested that the GLA can impart significant protection in comparison to ALA against VPA-induced autism features. When scrutinized histopathologically, the cerebellum of the GLA-treated animals was evident for more marked protection toward neuronal degeneration and neuronal loss in comparison to ALA. Concomitant administration of ALA and GLA with VPA demonstrated a marked cutdown in the Pgp 9.5 expression with GLA having more pronounced effect. Henceforth, it can be concluded that ALA and GLA can impart favorable protection against the VPA-induced autism-like features with GLA having pronounced effect.

α-Chymotrypsin regulates free fatty acids and UCHL-1 to ameliorate N-methyl nitrosourea induced mammary gland carcinoma in albino wistar rats.

This study was undertaken to investigate the effect of α-chymotrypsin on methyl nitrosourea (MNU) induced mammary gland carcinoma in albino wistar rats. Animals were randomized into four groups (six animals in each). Group I (sham control 0.9 % normal saline p.o.); Group II (toxic control, MNU 47 mg/kg, i.v.); Group III (α-chymotrypsin, 5 mg/kg, p.o.); Group IV (α-chymotrypsin, 10 mg/kg p.o.). Toxicity was induced by single i.v. injection of MNU followed by α-chymotrypsin supplementation therapy for 100 days. MNU treatment was evident with increased alveolar bud count, differentiation score, upregulated inflammatory enzymes markers (COX, LOX and NO) antioxidative stress markers (TBARs, SOD, catalase and GSH).MNU associated toxicity was also ascertained by PGP 9.5 and NF-κB expression in the mammary gland tissue followed by FAME analysis for fatty acid profiling. α-chymotrypsin afforded significant protection against the deleterious effects of MNU.

Electroacupuncture with high frequency at acupoint ST-36 induces regeneration of lost enteric neurons in diabetic rats via GDNF and PI3K/AKT signal pathway.

Background electroacupuncture (EA) at acupoint ST-36 (Zusanli) has been used to alleviate gastrointestinal symptoms and improve gastrointestinal motility, but the effects and mechanisms of EA on enteric nervous system (ENS) have scarcely been investigated. SD rats were randomly divided into eight groups: normal control group, diabetes mellitus group (DM), chronic high-frequency EA (C-HEA), chronic low-frequency EA (C-LEA), chronic sham stimulation group (C-SEA), acute high-frequency EA group (A-HEA), acute low-frequency EA group (A-LEA), and diabetic with acute sham stimulation group (A-SEA). The parameters of HEA included a frequency of 100 Hz and an amplitude of 1 mA, while the parameters for LEA were 10 Hz and 1 mA. The expressions of PGP9.5, neuronal nitric oxide synthase neurons, CHAT neurons, glia cell line-derived neurotrophic factor (GDNF) and p-Akt were measured by immunofluorescence or immunohistochemistry, real-time PCR, and Western blotting methods in colon tissues of each rat. The total neurons and the two types of enteric neurons (neuronal nitric oxide synthase and choline acetyl transferase neurons), together with GDNF and p-Akt in the mRNA and protein level were significantly decreased in DM group compared with the normal control group in colon (P < 0.01). Compared with DM or all other DM with EA groups, the chronic HEA could induce a more significant quantitative increase in the mRNA and protein level of the enteric neurons and GDNF and p-Akt in colon (P < 0.01). EA with high-frequency and long-term stimuli at acupoint ST-36 can induce regeneration of lost enteric neurons in diabetic rats, and GDNF and PI3K/Akt signal pathway may play an important role in EA-induced regeneration of impaired enteric neurons.

Cutting edge: Ubiquitin-specific protease 4 promotes Th17 cell function under inflammation by deubiquitinating and stabilizing RORγt.

RORγt is a key transcription factor that controls the development and function of inflammatory Th17. The mechanisms that regulate RORγt stability remain unclear. We report that Th17 cells highly express the deubiquitinase ubiquitin-specific protease (USP)4, which is essential for maintaining RORγt and Th17 cell function. Inhibition of the catalytic activity of USP4 with vialinin A, a compound derived from Chinese traditional medicine, dampened Th17 differentiation. USP4 interacted and deubiquitinated K48-linked polyubiquitination of RORγt, thereby promoting RORγt function and IL-17A transcription. Interestingly, TGF-ß plus IL-6 enhanced USP4-mediated deubiquitination of RORγt. Moreover, USP4 and IL-17 mRNA, but not RORγt mRNA, were significantly elevated in CD4(+) T cells from patients with rheumatic heart disease. Thus, USP4 could be a novel therapeutic target for the treatment of Th17-modulated autoimmune diseases.