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Background: Spinal cord injury (SCI) can cause destructive neurological dysfunctions. Objectives: The present study investigates the role and mechanism of methyltransferase like 3 (METTL3) in neural functional recovery post-SCI. Results: SCI mice showed neurological function impairment, with significantly elevated METTL3 expression and m6A content. METTL3 inhibition improved motor and sensory injury in SCI mice, alleviated tissue injury, up-regulated BDNF, GDNF, and IL-10 levels, and down-regulated IL-1β and TNF-α levels. Mechanistically, METTL3 mediated m6A modification of USP4 mRNA and enhanced YTHDF2 enrichment on USP4 mRNA, thus reducing USP4 mRNA stability and expression. Combined experiments confirmed that METTL3 exacerbated neurological impairment in SCI mice by reducing USP4 expression. Conclusion: METTL3-dominated m6A modification enhances YTHDF2 enrichment on USP4 mRNA and thereby reduces USP4 mRNA stability, eventually aggravating neurological impairment in SCI mice. [ABSTRACT FROM AUTHOR]

In this study, we aimed to explore the effects of curcumin on the progression of colorectal cancer and its underlying mechanisms involved. Cell proliferation, apoptosis and invasion were determined through CCK-8 assay, colony formation assay, EdU assay, flow cytometry, and transwell invasion assay, respectively. The protein expression of Bax, MMP2, USP4 and LAMP3 was measured using western blot. Pearson correlation coefficient was used to evaluate the relationship between USP4 and LAMP3. Co-IP was also conducted to determine the interaction between USP4 and LAMP3. Xenograft tumor model was established to explore the role of curcumin in colorectal cancer in vivo. IHC was utilized to measure the expression of Bax, MMP2, USP4 and LAMP3 in tumor tissues from mice. Curcumin significantly accelerated cell apoptosis, and inhibited cell proliferation and invasion in LoVo and HCT-116 cells. LAMP3 was augmented in colorectal cancer tissues and cells, and curcumin could reduce the expression of LAMP3. Curcumin decreased LAMP3 expression to exhibit the inhibition role in the progression of colorectal cancer. USP4 interacted with LAMP3, and positively regulated LAMP3 expression in colorectal cancer cells. LAMP3 overexpression could reverse the suppressive effects of USP4 knockdown on the development of colorectal cancer. Curcumin downregulated USP4 to impeded the progression of colorectal cancer via repressing LAMP3 expression. In addition, curcumin obviously restrained tumor growth in mice through downregulating USP4 and LAMP3 expression. These data indicated that curcumin exert the anti-tumor effects on the development of colorectal cancer through modulating the USP4/LAMP3 pathway. [ABSTRACT FROM AUTHOR]

Introduction: Head and neck squamous cell carcinoma (HNSCC) is among the most common cancers in the world with a low survival rate and common diagnosis at late stages. Deubiquitination of proteins is involved in tumor growth, metastasis, apoptosis, and immunosuppressive pathways. The impact of the ubiquitin-specific protease (USP4) on survival was only scarcely investigated so far. The goal of our research was to analyze the association of USP4 expression with prognosis and clinicopathological features in HNSCC. Methods: USP4 mRNA levels were derived from The Cancer Genome Atlas (TCGA) for a cohort of 510 patients. Protein expression of USP4 was analyzed by immunohistochemistry in a second cohort of 113 patients. Associations between USP4 levels and overall survival, disease-free survival and clinicopathological data were analyzed. Results: High levels of USP4 mRNA were associated with prolonged overall survival in univariable analysis. There was no more association with survival after correction for the confounders HPV, stage and smoker status. High USP4 mRNA levels were linked to a lower T-stage, the patient's age at diagnosis, and a positive HPV status. USP4 protein levels were not associated with prognosis or other features. Conclusion: Since high USP4 mRNA was not an independent prognostic marker, we assume that the association is a result of the correlation of high USP4 mRNA with an HPV-positive status. Therefore, further investigation of USP4 mRNA and its association with the HPV status of HNSCC patients is warranted. [ABSTRACT FROM AUTHOR]

Abstract Background Long non-coding RNAs (lncRNAs) are closely related to the occurrence and development of cancer. Abnormally expressed lncRNA can be used as a diagnostic marker for cancer. In this study, we aim to investigate the clinical significance of MIR99AHG expression in lung adenocarcinoma (LUAD), and its biological roles in LUAD progression. Methods The relative expression of MIR99AHG in LUAD tissues and cell lines was analyzed using public databases and RT-qPCR. The biological functions of MIR99AHG were investigated using a loss-of-function approach. The effect of MIR99AHG on lung fibrosis was assessed by scratch assay, invasion assay and lung fibrosis rat model. FISH, luciferase reporter assay and immunofluorescence were performed to elucidate the underlying molecular mechanisms. Results LncRNA MIR99AHG expression level was downregulated in LUAD tissues and cell lines. Low MIR99AHG levels were associated with poorer patient overall survival. Functional analysis showed that MIR99AHG is associated with the LUAD malignant phenotype in vitro and in vivo. Further mechanistic studies showed that, MIR99AHG functions as a competitive endogenous RNA (ceRNA) to antagonize miR-136-5p-mediated ubiquitin specific protease 4 (USP4) degradation, thereby unregulated the expression of angiotensin-converting enzyme 2 (ACE2), a downstream target gene of USP4, which in turn affected alveolar type II epithelial cell fibrosis and epithelial–mesenchymal transition (EMT). In summary, the MIR99AHG/miR-136-5p/USP4/ACE2 signalling axis regulates lung fibrosis and EMT, thus inhibiting LUAD progression. Conclusion This study showed that downregulated MIR99AHG leads to the development of pulmonary fibrosis. Therefore, overexpression of MIR99AHG may provide a new approach to preventing LUAD progression.

Ubiquitin‐specific protease 4 (USP4) is highly overexpressed in colon cancer and acts as a potent protooncogenic protein by deubiquitinating β‐catenin. However, its prominent roles in tumor formation and migration in cancer cells are not fully understood by its deubiquitinating enzyme (DUB) activity on β‐catenin. Thus, we investigated an additional role of USP4 in cancer. In this study, we identified cortactin (CTTN), an actin‐binding protein involved in the regulation of cytoskeleton dynamics and a potential prognostic marker for cancers, as a new cellular interacting partner of USP4 from proximal labeling of HCT116 cells. Additionally, the role of USP4 in CTTN activation and promotion of cell dynamics and migration was investigated in HCT116 cells. We confirmed that interacting of USP4 with CTTN increased cell movement. This finding was supported by the fact that USP4 overexpression in HCT116 cells with reduced expression of CTTN was insufficient to promote cell migration. Additionally, we observed that USP4 overexpression led to a significant increase in CTTN phosphorylation, which is a requisite mechanism for cell migration, by regulating Src/focal adhesion kinase (FAK) binding to CTTN and its activation. Our results suggest that USP4 plays a dual role in cancer progression, including stabilization of β‐catenin as a DUB and interaction with CTTN to promote cell dynamics by inducing CTTN phosphorylation. Therefore, this study demonstrates that USP4 is important for cancer progression and is a good target for treating or preventing cancer. [ABSTRACT FROM AUTHOR]

Ubiquitin-specific protease 4 (USP4) is pivotal in negatively regulating the Toll-like receptor (TLR) signaling-mediated innate immune response. Although USP4 has been well studied in mammals, its role in TLR signaling pathways in fish remains largely unknown. In this study, we investigated the role of USP4 (OmUSP4) in regulating TLR response in rainbow trout Oncorhynchus mykiss. OmUSP4 contained the characteristic domains conserved in other USP4s: domain in USP (DUSP), ubiquitin-like (UBL), and the bi-part catalytic domain known as USP. OmUSP4 expression was increased in RTH-149 cells by stimulation with fish-pathogenic bacteria and bacterial ligands. Gain- and loss-of-function experiments revealed that OmUSP4 mitigated the activation of MAPKs and NF-κB, as well as the expression of pro-inflammatory cytokines in LPS-stimulated cells. OmUSP4 interacted with TAK1, a critical mediator in TLR-mediated NF-κB signaling pathways. LPS stimulation increased the K63-linked polyubiquitination of TAK1, which was significantly suppressed when OmUSP4 was compelled to be overexpressed. These results imply that OmUSP4 might function like mammals to downregulate LPS-induced inflammation in rainbow trout by removing the K63-linked ubiquitin chain on TAK1. • Rainbow trout USP4 (OmUSP4) is a functional homolog of human USP4. • OmUSP4 decreased LPS-induced activation of MAPKs and NF-κB. • OmUSP4 decreased LPS-induced expression of pro-inflammatory cytokines. • OmUSP4 interacted directly with TAK1. • OmUSP4 removed the K63-linked ubiquitin chain on TAK1. [ABSTRACT FROM AUTHOR]

Lysyl‐oxidase‐like 3 (LOXL3) was reported to be essential in epithelial‐mesenchymal transition (EMT) of cancers. However, the role of LOXL3 in hepatocellular carcinoma (HCC) remained unclear. In this study, we explored clinical significance, biological functions, and regulatory mechanisms of LOXL3 in HCC. Our study found that LOXL3 expression was markedly associated with the tumor size and clinical stage of HCC, and it was highly expressed in tumor tissues of metastatic HCC patients. High expression of LOXL3 predicted a poor prognosis of HCC. TGF‐β1 treatment elevated LOXL3 protein expression and cell invasion, and reduced cell apoptosis in HCC cell lines (SMMC‐7721 and Huh‐7), while downregulation of LOXL3 reversed the promotive effects of TGF‐β1 treatment on LOXL3 protein expression and cell invasion, and the inhibitory effect on cell apoptosis. Mechanistically, LOXL3 interacted with snail family transcriptional repressor 1 (Snail1) through STRING database and RIP assay, and Snail1 bound to ubiquitin‐specific peptidase 4 (USP4) promoter by JASPAR database, luciferase reporter gene and Co‐IP assays. Overexpression of USP4 reversed the inhibitory effect of LOXL3 silence on EMT in HCC cells through deubiquitinating and stabilizing the expression of Snail1. Moreover, LOXL3‐promoted HCC EMT through Wnt/β‐catenin/Snail1 signaling pathway. In vivo study revealed that silence of LOXL3‐inhibited HCC tumor growth. In conclusion, LOXL3 silence inhibited HCC invasion and EMT through Snail1/USP4‐mediated circulation loop and Wnt/β‐catenin signaling pathway. [ABSTRACT FROM AUTHOR]

Background: Long non-coding RNAs (lncRNAs) are closely related to the occurrence and development of cancer. Abnormally expressed lncRNA can be used as a diagnostic marker for cancer. In this study, we aim to investigate the clinical significance of MIR99AHG expression in lung adenocarcinoma (LUAD), and its biological roles in LUAD progression.Methods: The relative expression of MIR99AHG in LUAD tissues and cell lines was analyzed using public databases and RT-qPCR. The biological functions of MIR99AHG were investigated using a loss-of-function approach. The effect of MIR99AHG on lung fibrosis was assessed by scratch assay, invasion assay and lung fibrosis rat model. FISH, luciferase reporter assay and immunofluorescence were performed to elucidate the underlying molecular mechanisms.Results: LncRNA MIR99AHG expression level was downregulated in LUAD tissues and cell lines. Low MIR99AHG levels were associated with poorer patient overall survival. Functional analysis showed that MIR99AHG is associated with the LUAD malignant phenotype in vitro and in vivo. Further mechanistic studies showed that, MIR99AHG functions as a competitive endogenous RNA (ceRNA) to antagonize miR-136-5p-mediated ubiquitin specific protease 4 (USP4) degradation, thereby unregulated the expression of angiotensin-converting enzyme 2 (ACE2), a downstream target gene of USP4, which in turn affected alveolar type II epithelial cell fibrosis and epithelial-mesenchymal transition (EMT). In summary, the MIR99AHG/miR-136-5p/USP4/ACE2 signalling axis regulates lung fibrosis and EMT, thus inhibiting LUAD progression.Conclusion: This study showed that downregulated MIR99AHG leads to the development of pulmonary fibrosis. Therefore, overexpression of MIR99AHG may provide a new approach to preventing LUAD progression. [ABSTRACT FROM AUTHOR]

The para-terphenyl derivative vialinin A (Vi-A), isolated from Thelephora fungi, has been characterized as a potent inhibitor of the ubiquitin-specific protease 4 (USP4). Blockade of USP4 contributes to the anti-inflammatory and anticancer properties of the natural product. We have investigated the interaction of Vi-A with USP4 by molecular modeling, to locate the binding site (around residue V98 within the domain in USP segment) and to identify the binding process and interaction contacts. From this model, a series of 32 p-terphenyl compounds were tested as potential USP4 binders, mainly in the vialinin, terrestrin and telephantin series. We identified 11 compounds presenting a satisfactory USP4 binding capacity, including two fungal products, vialinin B and aurantiotinin A, with a more favorable empirical energy of USP4 interaction (ΔE) than the reference product Vi-A. The rare p-terphenyl aurantiotinin A, isolated from the basidiomycete T. aurantiotincta, emerged as a remarkable USP4 binder. Structure-binding relationships have been identified and discussed, to guide the future design of USP4 inhibitors based on the p-terphenyl skeleton. The docking study should help the identification of other protease inhibitors from fungus. [ABSTRACT FROM AUTHOR]

Ubiquitin-specific protease 4 (USP4) has been identified as a promising oncogenic factor implicated in various human malignancies. However, the exact biological functions and underlying mechanisms of USP4 in the progression of papillary thyroid carcinoma (PTC) remain elusive. In this study, we observed a marked upregulation of USP4 expression in PTC tumor tissues. Elevated levels of USP4 were significantly correlated with aggressive clinicopathological features and poor prognosis. Functional assays for loss-of-function demonstrated that silencing USP4 hindered the proliferation of PTC cells. Furthermore, our investigation revealed a specific interaction between USP4 and lactate dehydrogenase A (LDHA), wherein USP4 played a crucial role in stabilizing LDHA protein levels via deubiquitination in PTC cells. Notably, this study demonstrated that USP4 promotes PTC proliferation by modulating the MAPK and AKT signaling pathways. In summary, our findings elucidate the critical involvement of the USP4/LDHA axis in driving PTC progression through the modulation of MAPK and AKT pathways, thereby identifying USP4 as a potential therapeutic target for the treatment of PTC.

Esophageal cancer (EC) is a highly aggressive disease, and its progression involves a complex gene regulation network. Transcription factor SOX2 is amplified in various cancers including EC. A pathway involving SOX2 regulation of microRNAs (miRNAs) and their target genes has been previously revealed. This study aims to delineate the ability of SOX2 to influence the EC progression, with the involvement of miR-30e/USP4/SMAD4/CK2 axis. SOX2 expression was first examined in the clinical tissue samples from 30 EC patients. Effects of SOX2 on proliferation, migration, and invasion alongside tumorigenicity of transfected cells were examined by means of gain- and loss-of-function experiments. EC tissues and cells exhibited high expression of SOX2, miR-30e, and CK2 and poor expression of USP4 and SMAD4. Mechanistically, SOX2 was positively correlated with miR-30e and upregulated the expression of miR-30e. miR-30e specifically targeted USP4, which induced deubiquitination of SMAD4 and promoted its expression. Meanwhile, SMAD4 was enriched in the CK2 promoter region and thus inhibited its expression. SOX2 stimulated EC cell proliferative, invasive, and migratory capacities in vitro and tumor growth in vivo by regulating the miR-30e/USP4/SMAD4/CK2 axis. Collectively, our work reveals a novel SOX2-mediated regulatory network in EC that may be a viable target for EC treatment.

Abstract Cancer remains one of the major culprits causing disease-related deaths and leads to a high morbidity and similar mortality. Insidious onset, difficult early detection and a lack of broad-spectrum and effective multi-cancer therapeutic targets have limited the prolongation of cancer patients’ survival for decades. Therefore, a versatile therapeutic target which is involved in various cancer-related signaling pathways and different cancers may be more effective for cancer targeted therapy. USP4, one of the DUBs members which participates in deubiquitination, an inverse process of ubiquitination, can regulate various classical cancer-related signaling pathways, and thereby plays a vital role in some pathological and physiological processes including tumor initiation and progression. Recently, USP4 has been found to exert versatile influences on cells proliferation, migration and invasion, also apoptosis of various tumors. Moreover, USP4 can also act as a prognostic biomarker in several cancers. This review will give a comprehensive introduction of USP4 about its regulatory mechanisms, related signaling pathways, pathophysiological functions and the roles in various cancers which may help us better understand its biological functions and improve future studies to construct suitable USP4-targeted cancer therapy system.

As a severe metabolic disease, type 2 diabetes mellitus (T2DM) has become a serious threat to human health in recent years. Gastrodin, as a primary chemical constituent in Gastrodia elata Blume, has antidiabetic effects. However, the possible mechanisms are unclear. The aim of the present study was to investigate the effects and possible mechanisms of gastrodin on the treatment of T2DM. In vivo , after treatment with gastrodin for 6 weeks, fasting blood glucose levels, blood lipid metabolism, and insulin sensitivity index values were remarkably reduced compared with those of the diabetic control group. The values of aspartate aminotransferase and alanine aminotransferase also showed that gastrodin alleviates liver toxicity caused by diabetes. Moreover, gastrodin relieved pathological damage to the pancreas in T2DM rats. In vitro , gastrodin alleviated insulin resistance by increasing glucose consumption, glucose uptake, and glycogen content in dexamethasone-induced HepG2 cells. The Western blotting results showed that gastrodin upregulated the expression of insulin receptors and ubiquitin-specific protease 4 (USP4) and increased the phosphorylation of GATA binding protein 1 (GATA1) and protein kinase B (AKT) in vivo and in vitro. Furthermore, gastrodin decreased the ubiquitin level of the insulin receptor via UPS4 and increased the binding of GATA1 to the USP4 promoter. Additionally, administration of the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway inhibitors MK-2206 and LY294002 abolished the beneficial effects of gastrodin. Our results indicate that gastrodin promotes the phosphorylation of GATA1 via the PI3K/AKT pathway, enhances the transcriptional activity of GATA1, and then increases the expression level of USP4, thereby reducing the ubiquitination and degradation of insulin receptors and ultimately improving insulin resistance. Our study provides scientific evidence for the beneficial actions and underlying mechanism of gastrodin in the treatment of T2DM. [ABSTRACT FROM AUTHOR]

As a severe metabolic disease, type 2 diabetes mellitus (T2DM) has become a serious threat to human health in recent years. Gastrodin, as a primary chemical constituent in Gastrodia elata Blume, has antidiabetic effects. However, the possible mechanisms are unclear. The aim of the present study was to investigate the effects and possible mechanisms of gastrodin on the treatment of T2DM. In vivo, after treatment with gastrodin for 6 weeks, fasting blood glucose levels, blood lipid metabolism, and insulin sensitivity index values were remarkably reduced compared with those of the diabetic control group. The values of aspartate aminotransferase and alanine aminotransferase also showed that gastrodin alleviates liver toxicity caused by diabetes. Moreover, gastrodin relieved pathological damage to the pancreas in T2DM rats. In vitro, gastrodin alleviated insulin resistance by increasing glucose consumption, glucose uptake, and glycogen content in dexamethasone-induced HepG2 cells. The Western blotting results showed that gastrodin upregulated the expression of insulin receptors and ubiquitin-specific protease 4 (USP4) and increased the phosphorylation of GATA binding protein 1 (GATA1) and protein kinase B (AKT) in vivo and in vitro. Furthermore, gastrodin decreased the ubiquitin level of the insulin receptor via UPS4 and increased the binding of GATA1 to the USP4 promoter. Additionally, administration of the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway inhibitors MK-2206 and LY294002 abolished the beneficial effects of gastrodin. Our results indicate that gastrodin promotes the phosphorylation of GATA1 via the PI3K/AKT pathway, enhances the transcriptional activity of GATA1, and then increases the expression level of USP4, thereby reducing the ubiquitination and degradation of insulin receptors and ultimately improving insulin resistance. Our study provides scientific evidence for the beneficial actions and underlying mechanism of gastrodin in the treatment of T2DM.

Background: It has been reported that ubiquitin specific peptidase 4 (USP4) was functional in several tumors, but its function and mechanism in gastric cancer were still unknown. Methods: Bioinformatic tools were used to predict the prognosis of gastric cancer patients and the expression levels of USP4 in gastric cancer. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunoblotting were carried out to detect the messenger RNA (mRNA) and protein levels. Cell viability of gastric cancer was evaluated by Cell Counting Kit-8 (CCK-8) assay. Cell line-derived xenograft models were established to evaluate the tumor growth of gastric cancer. Luciferase assay and immunoblotting were used to determine the activation of nuclear factor kappa B (NF-κB) signaling. Results: The public database Kaplan-Meier Plotter showed that gastric cancer patients with high USP4 expression had a shorter overall survival or post-progression survival than the patients with decreased USP4. Further studies indicated that USP4 was elevated in gastric cancer tumor tissues. In contrast, knockdown of USP4 markedly inhibited gastric cancer cell growth, and suppressed the tumor growth of gastric cancer. Further studies revealed that USP4 knockdown significantly suppressed NF-κB-driven luciferase activity, and inhibited the phosphorylation of NF-κB p65 in gastric cancer cells. Additionally, qRT-PCR analysis showed that USP4 knockdown significantly downregulated the expressions of cyclin D2 (CCND2) and B cell leukemia/lymphoma 2 (BCL2). We also found that USP4 knockdown decreased the expressions of phosphatase of regenerating liver-3 (PRL-3), in contrast, overexpression of PRL-3 attenuated the inhibitory effects of USP4 knockdown on NF-κB signaling and cell viability in gastric cancer cells. Finally, PR-619, which has been proven to inhibit the activities of USP4 and other deubiquitinases, could inhibit cell viability and NF-κB signaling in gastric cancer cells. Conclusions: This study indicated that elevated USP4 predicted a poor index for gastric cancer patients, and mediated gastric cancer cell growth by regulating PRL-3/NF-κB signaling, which suggested USP4 may be a novel therapeutic target for gastric cancer.

The para-terphenyl derivative vialinin A (Vi-A), isolated from Thelephora fungi, has been characterized as a potent inhibitor of the ubiquitin-specific protease 4 (USP4). Blockade of USP4 contributes to the anti-inflammatory and anticancer properties of the natural product. We have investigated the interaction of Vi-A with USP4 by molecular modeling, to locate the binding site (around residue V98 within the domain in USP segment) and to identify the binding process and interaction contacts. From this model, a series of 32 p-terphenyl compounds were tested as potential USP4 binders, mainly in the vialinin, terrestrin and telephantin series. We identified 11 compounds presenting a satisfactory USP4 binding capacity, including two fungal products, vialinin B and aurantiotinin A, with a more favorable empirical energy of USP4 interaction (ΔE) than the reference product Vi-A. The rare p-terphenyl aurantiotinin A, isolated from the basidiomycete T. aurantiotincta, emerged as a remarkable USP4 binder. Structure-binding relationships have been identified and discussed, to guide the future design of USP4 inhibitors based on the p-terphenyl skeleton. The docking study should help the identification of other protease inhibitors from fungus.

Despite the approval of immune checkpoint blockade (ICB) therapy for various tumor types, its effectiveness is limited to only approximately 15% of patients with microsatellite instability-high (MSI-H) or mismatch repair deficiency (dMMR) colorectal cancer (CRC). Approximately 80%–85% of CRC patients have a microsatellite stability (MSS) phenotype, which features a rare T-cell infiltration. Thus, elucidating the mechanisms underlying resistance to ICB in patients with MSS CRC is imperative. In this study, we demonstrate that ubiquitin-specific peptidase 4 (USP4) is upregulated in MSS CRC tumors and negatively regulates the immune response against tumors in CRC. Additionally, USP4 represses the cellular interferon (IFN) response and antigen presentation and impairs PRR signaling-mediated cell death. Mechanistically, USP4 impedes the nuclear localization of interferon regulator Factor 3 (IRF3) by deubiquitinating the K63-polyubiquitin chain of TRAF6 and IRF3. Knockdown of USP4 enhances the infiltration of T cells in CRC tumors and overcomes ICB resistance in an MC38 syngeneic mouse model. Moreover, published datasets revealed that patients showing higher USP4 expression exhibited decreased responsiveness to anti-PD-L1 therapy. These findings highlight an essential role of USP4 in the suppression of antitumor immunity in CRC. • USP4 is upregulated in MSS CRC tumors and negatively regulates anti-tumor immune response. • USP4 removes K63 linked polyubiquitination of IRF3 and inhibits IRF3 activation. • USP4 suppresses the interferon response and antigen presentation pathways depending on its enzyme activity. • Inhibition of USP4 enhances the anti-tumor immune response and improves the efficacy of anti-PD-L1 therapy in CRC. [ABSTRACT FROM AUTHOR]

Context can determine whether a given gene acts as an oncogene or a tumor suppressor. Deubiquitinating enzymes (DUBs) regulate the stability of many components of the pathways dictating cell fate so it would be expected that alterations in the levels or activity of these enzymes may have oncogenic or tumor suppressive consequences. In the current review we survey publications reporting that genes encoding DUBs are oncogenes or tumor suppressors. For many DUBs both claims have been made. For such "double agents," the effects of gain or loss of function will depend on the overall status of a complex of molecular signaling networks subject to extensive crosstalk. As the TGF‐β paradox makes clear context is critical in cell fate decisions, and the disconnect between experimental findings and patient survival outcomes can in part be attributed to disparities between culture conditions and the microenvironment in vivo. Convincing claims for oncogene or tumor suppressor roles require the documentation of gene alterations in patient samples; survival curves are alone inadequate. [ABSTRACT FROM AUTHOR]

The deubiquitinating enzyme (DUB)–mediated cleavage of ubiquitin plays a critical role in balancing protein synthesis and degradation. Ubiquitin-specific protease 4 (USP4), a member of the largest subfamily of cysteine protease DUBs, removes monoubiquitinated and polyubiquitinated chains from its target proteins. USP4 contains a DUSP (domain in USP)–UBL (ubiquitin-like) domain and a UBL-insert catalytic domain, sharing a common domain organization with its paralogs USP11 and USP15. USP4 plays a critical role in multiple cellular and biological processes and is tightly regulated under normal physiological conditions. When its expression or activity is aberrant, USP4 is implicated in the progression of a wide range of pathologies, especially cancers. In this review, we comprehensively summarize the current knowledge of USP4 structure, biological functions, pathological roles, and cellular regulation, highlighting the importance of exploring effective therapeutic interventions to target USP4.

Inflammation and fibrosis are major consequences of autoimmune hepatitis, however, the therapeutic mechanism remains to be investigated. USP4 is a deubiquitinating enzyme and plays an important role in tissue fibrosis and immune disease. Vialinin A is an extract from mushroom and is a specific USP4 inhibitor. However, there is lack of evidences that Vialinin A plays a role in autoimmune hepatitis. By employing S100‐induced autoimmune hepatitis in mice and AML12 cell line, therapeutic mechanism of Vialinin A was examined. Inflammation was documented by liver histological staining and inflammatory cytokines. Fibrosis was demonstrated by Masson, Sirius red staining and fibrotic cytokines with western blot and real‐time RT‐PCR. In experimental animal, there were increases in inflammation and fibrosis as well as USP4, and which were reduced after treatment of Vialinin A. Vialinin A also reduced Rheb and phosphorylated mTOR. Moreover, in LPS‐treated AML12 cells, LPS‐induced USP4, inflammatory and fibrotic cytokines, phosphorylated mTOR and Rheb. Specific inhibitory siRNA of USP4 reduced USP4 level and the parameters mentioned above. In conclusion, USP4 was significantly elevated in autoimmune hepatitis mice and Vialinin A reduced USP4 level and attenuate inflammation and fibrosis in the liver. The mechanism may be related to regulation of Rheb/mTOR signalling. [ABSTRACT FROM AUTHOR]

BackgroundLiver cancer is a frequent malignancy with poor prognosis and high mortality all over the world. It has been reported many lncRNAs could modulate the tumorigenesis of liver cancer. To identify novel potential targets for liver cancer, the differential expressed lncRNAs between liver cancer and adjacent normal tissues was analyzed with bioinformatics tool.MethodsThe differential expressed lncRNAs between liver cancer and adjacent normal tissues were analyzed with bioinformatics tool. Cell viability and proliferation was tested by CCK8 and Ki67, respectively. Apoptosis of liver cancer cells was tested by flow cytometry. Gene and protein expressions in liver cancer cells were measured by qRT-PCR and western blot, respectively. In vivo model of liver cancer was established to detect the effect of LINC01234 on liver cancer in vivo.ResultsLINC01234 was found to be negatively correlated with the survival rate of patients with liver cancer. Moreover, knockdown of LINC01234 significantly suppressed the proliferation and invasion of liver cancer cells via inducing the apoptosis. Meanwhile, miR-513a-5p was sponged by LINC01234, and USP4 was found to be a direct target of miR-513a-5p. In addition, LINC01234 knockdown inhibited the tumorigenesis of liver cancer via inactivating TGF-β signaling. Furthermore, silencing of LINC01234 notably inhibited the tumor growth of liver cancer in vivo.ConclusionDownregulation of LINC01234 could inhibit the tumorigenesis of liver cancer via mediation of miR-513a-5p/USP4/TGF-β axis. Thus, LINC01234 might serve as a new target for the treatment of liver cancer.

Abstract Background Although numerous studies have reported that tricho-rhino-phalangeal syndrome type I (TRPS1) protein, the only reported atypical GATA transcription factor, is overexpressed in various carcinomas, the underlying mechanism(s) by which it contributes to cancer remain unknown. Methods Both overexpression and knockdown of TRPS1 assays were performed to examine the effect of TRPS1 on histone deacetylase 2 (HDAC2) protein level and luminal breast cancer cell proliferation. Also, RT-qRCR, luciferase reporter assay and RNA-sequencing were used for transcription detection. Chromatin immunoprecipitation (ChIP) using H4K16ac antibody in conjunction with qPCR was used for determining H4K16ac levels in targeted genes. Furthermore, in vitro cell proliferation assay and in vivo tumor xenografts were used to detect the effect of TRPS1 on tumor growth. Results We found that TRPS1 scaffolding recruits and enhances interaction between USP4 and HDAC2 leading to HDAC2 de-ubiquitination and H4K16 deacetylation. We detected repression of a set of cellular growth-related genes by the TRPS1-USP4-HDAC2 axis indicating it is essential in tumor growth. In vitro and in vivo experiments confirmed that silencing TRPS1 reduced tumor growth, whereas overexpression of HDAC2 restored tumor growth. Conclusion Our study deciphered the TRPS1-USP4-HDAC2 axis as a novel mechanism that contributes to tumor growth. Significantly, our results revealed the scaffolding function of TPRS1 in USP4-directed HDAC2 de-ubiquitination and provided new mechanistic insights into the crosstalk between TRPS1, ubiquitin, and histone modification systems leading to tumor growth.

Background: Liver cancer is a frequent malignancy with poor prognosis and high mortality all over the world. It has been reported many lncRNAs could modulate the tumorigenesis of liver cancer. To identify novel potential targets for liver cancer, the differential expressed lncRNAs between liver cancer and adjacent normal tissues was analyzed with bioinformatics tool. Methods: The differential expressed lncRNAs between liver cancer and adjacent normal tissues were analyzed with bioinformatics tool. Cell viability and proliferation was tested by CCK8 and Ki67, respectively. Apoptosis of liver cancer cells was tested by flow cytometry. Gene and protein expressions in liver cancer cells were measured by qRT-PCR and western blot, respectively. In vivo model of liver cancer was established to detect the effect of LINC01234 on liver cancer in vivo. Results: LINC01234 was found to be negatively correlated with the survival rate of patients with liver cancer. Moreover, knockdown of LINC01234 significantly suppressed the proliferation and invasion of liver cancer cells via inducing the apoptosis. Meanwhile, miR-513a-5p was sponged by LINC01234, and USP4 was found to be a direct target of miR-513a-5p. In addition, LINC01234 knockdown inhibited the tumorigenesis of liver cancer via inactivating TGF-β signaling. Furthermore, silencing of LINC01234 notably inhibited the tumor growth of liver cancer in vivo. Conclusion: Downregulation of LINC01234 could inhibit the tumorigenesis of liver cancer via mediation of miR-513a-5p/USP4/TGF-β axis. Thus, LINC01234 might serve as a new target for the treatment of liver cancer. [ABSTRACT FROM AUTHOR]

Cancer remains one of the major culprits causing disease-related deaths and leads to a high morbidity and similar mortality. Insidious onset, difficult early detection and a lack of broad-spectrum and effective multi-cancer therapeutic targets have limited the prolongation of cancer patients' survival for decades. Therefore, a versatile therapeutic target which is involved in various cancer-related signaling pathways and different cancers may be more effective for cancer targeted therapy. USP4, one of the DUBs members which participates in deubiquitination, an inverse process of ubiquitination, can regulate various classical cancer-related signaling pathways, and thereby plays a vital role in some pathological and physiological processes including tumor initiation and progression. Recently, USP4 has been found to exert versatile influences on cells proliferation, migration and invasion, also apoptosis of various tumors. Moreover, USP4 can also act as a prognostic biomarker in several cancers. This review will give a comprehensive introduction of USP4 about its regulatory mechanisms, related signaling pathways, pathophysiological functions and the roles in various cancers which may help us better understand its biological functions and improve future studies to construct suitable USP4-targeted cancer therapy system. [ABSTRACT FROM AUTHOR]

Although clinically associated with the progression of multiple cancers, the biological function of p21‐activated kinase 5 (PAK5) in breast cancer remains largely unknown. Here, we reveal that the PAK5–aspartyl aminopeptidase (DNPEP)–ubiquitin‐specific protease 4 (USP4) axis is involved in breast cancer progression. We show that PAK5 interacts with and phosphorylates DNPEP at serine 119. Functionally, we demonstrate that DNPEP overexpression suppresses breast cancer cell proliferation and invasion and restricts breast cancer growth and metastasis in mice. Furthermore, we identify USP4 as a downstream target of the PAK5–DNPEP pathway; DNPEP mediates USP4 downregulation. Importantly, we verify that DNPEP expression is frequently downregulated in breast cancer tissues and is negatively correlated with PAK5 and USP4 expression. PAK5 decreases DNPEP abundance via the ubiquitin–proteasome pathway. Consistently, analyses of clinical breast cancer specimens revealed significantly increased PAK5 and USP4 levels and an association between higher PAK5 and USP4 expression and worse breast cancer patient survival. These findings suggest a pivotal role for PAK5‐elicited signaling in breast cancer progression. What's new? Increasing lines of evidence suggest that PAK5 is an oncogenic protein that is commonly overexpressed in many cancer tissues and plays multiple functions in cancer progression. However, the underlying molecular mechanisms and the biological significance of PAK5 in breast cancer remain to be fully explored. Here, the authors reveal that DNPEP links the upstream PAK5 kinase to the downstream target USP4 through a phosphorylation‐based regulatory mechanism in malignant breast cancer. Moreover, they identify an association between elements of the PAK5‐DNPEP‐USP4 pathway and clinicopathological features in breast cancer patients, altogether providing potential prognostic markers and therapeutic targets for breast cancer intervention. [ABSTRACT FROM AUTHOR]

• Vialinin A protects neurons against oxidative stress injury after ischaemic stroke. • Vialinin A enhances the activation of Keap1-Nrf2-ARE signaling pathway. • USP4 binds to and mediates the deubiquitination modification of Keap1. • Vialinin A enhances the ubiquitination of Keap1 and promoting its degradation. • USP4 upregulation reverses the neuron protection of vialinin A on ischaemic stroke. Oxidative stress is known as a hallmark of cerebral ischaemia‒reperfusion injury and it exacerbates the pathologic progression of ischaemic brain damage. Vialinin A, derived from a Chinese edible mushroom, possesses multiple pharmacological activities in cancer, Kawasaki disease, asthma and pathological scarring. Notably, vialinin A is an inhibitor of ubiquitin-specific peptidase 4 (USP4) that shows anti-inflammatory and antioxidative properties. However, the precise effect of vialinin A in ischaemic stroke, as well as its underlying mechanisms, remains largely unexplored. The present research focuses on the impacts of vialinin A on oxidative stress and explores the underlying mechanisms involved while also examining its potentiality as a therapeutic candidate for ischaemic stroke. Mouse ischaemic stroke was conducted by MCAO surgery. Vialinin A was administered via lateral ventricular injection at a dose of 2 mg/kg after reperfusion. Subsequent experiments were meticulously conducted at the appropriate time points. Stroke outcomes were evaluated by TTC staining, neurological score, Nissl staining and behavioural analysis. Co-IP assays were operated to examine the protein-protein interactions. Immunoblot analysis, qRT-PCR, and luciferase reporter assays were conducted to further investigate its underlying mechanisms. In this study, we initially showed that administration of vialinin A alleviated cerebral ischaemia‒reperfusion injury-induced neurological deficits and neuronal apoptosis. Furthermore, vialinin A, which is an antioxidant, reduced oxidative stress injury, promoted the activation of the Keap1-Nrf2-ARE signaling pathway and increased the protein degradation of Keap1. The substantial neuroprotective effects of vialinin A against ischaemic stroke were compromised by the overexpression of USP4. Mechanistically, vialinin A inhibited the deubiquitinating enzymatic activity of USP4, leading to enhanced ubiquitination of Keap1 and subsequently promoting its degradation. This cascade caused the activation of Nrf2-dependent antioxidant response, culminating in a reduction of neuronal apoptosis and the amelioration of neurological dysfunction following ischaemic stroke. This study demonstrates that inhibition of USP4 to activate Keap1-Nrf2-ARE signaling pathway may represent a mechanism by which vialinin A conferred protection against cerebral ischaemia‒reperfusion injury and sheds light on its promising prospects as a therapeutic intervention for ischaemic stroke. [Display omitted] [ABSTRACT FROM AUTHOR]

Background/Aims: Dysregulation of deubiquitinating enzymes (DUBs), which regulate the stability of key proteins, has been implicated in many human diseases, including cancers. Thus, DUBs can be considered as potential therapeutic targets for many diseases. Among them, USP4 has been proposed as a promising target for colon cancer drugs since USP4 controls the stability of β-catenin, a key factor in the Wnt signaling involved in the tumorigenesis of colorectal cancer. However, developing potential DUB inhibitors has been hindered because many DUBs harbor similar active site structures and show broad substrate specificities. Methods: By performing in vitro deubiquitinating activity assays using a chemical library, we identified several potential DUB inhibitors. Among them, only neutral red (NR) showed selective inhibitory activity on USP4 in a cell-based assay system. In colon cancer cells, NR affected the protein stability of β-catenin, as shown by immunoblotting, and it affected the target gene expression of β-catenin, as shown by quantitative real-time PCR. NR’s potential as an anticancer drug was further estimated by colony formation and cell migration assays and by using a mouse xenograft model. Results: We identified NR as an uncompetitive inhibitor of USP4 and validated its effects in colorectal cancer. NR-treated cells showed decreased β-catenin stability and reduced expression of β-catenin target genes. Additionally, treating colon cancer cells with NR significantly reduced colony formation and cell migration, and injecting NR into a mouse xenograft model reduced the tumor volume. Conclusion: The current results suggest that NR could be developed as an anticancer drug targeting USP4, and they support the possibility of developing specific DUB inhibitors as therapeutic agents. [ABSTRACT FROM AUTHOR]

Background: Glioblastoma multiforme (GBM) is one of the most common brain tumors in adults. Current treatments cannot increase survival to a large extent, as the glioblastoma development mechanisms remain unknown. It has been well documented that ubiquitination contributes to tumor initiation and/or progression in many kinds of cancer. Ubiquitin-specific protease 4 (USP4), a member of deubiquitinating enzymes (DUBs) family, can remove ubiquitin residues and play a role in cancer development. Methods: In the current study, lentiviruses were used to manipulate the expression of USP4. Real-time PCR and Western blot were used to measure the expression level of USP4. Then, CCK-8 and annexin-V staining were used to detect cell proliferation and cell apoptosis, respectively. Results: First, we found that USP4 was highly upregulated in GBM tissues in comparison with that in normal tissues and high level of USP4 correlated with poor prognosis. Moreover, knockdown of USP4 could significantly inhibit cell proliferation and increase cell apoptosis in U87 and T98G cells. Cells with stable USP4 reduction exhibited slower tumor growth rate and smaller tumor size than the control group cells in a xenograft mouse model. Inhibition of USP4 downregulated the expression of PCNA, Bcl-2 and p-ERK1/2, but upregulated the expression of Bax both in vitro and in vivo. Inversely, USP4 overexpression could attenuate the effects contributed by ERK inhibitor. TGF-βR inhibition reduced level of TGF-βR1, p-smad2 and p-ERK1/2 which can partially be rescued by USP4 overexpression. Conclusion: USP4, as a potential novel oncogene, promotes GBM by activation of ERK pathway through regulating TGF-β. [ABSTRACT FROM AUTHOR]

Abstract: Ubiquitin specific protease 4 (USP4) is a member of the USPs family, which catalyzes the cleavage of ubiquitin from a series of protein substrates, thereby modulating a number of cellular signaling pathways. In this study, we aimed to explore the expression profile of USP4 in lung adenocarcinoma (LUAD) using large patient cohorts in the Cancer Genome Atlas and the International Cancer Genome Consortium and to investigate its prognostic value and the possible mechanisms of its dysregulation. Results showed that USP4 was significantly downregulated in LUAD tissues (N = 514) compared with the normal controls (N = 59). The high USP4 expression group had significantly better overall survival (OS) and recurrence‐free survival (RFS). Multivariate analysis showed that preserved USP4 expression was an independent prognostic factor of favorable OS (HR: 0.574, 95%CI: 0.427–0.771, P < 0.001) and RFS (HR: 0.625, 95%CI: 0.444–0.880, P = 0.007) in LUAD. In comparison, although USP4 was downregulated in lung squamous cell carcinoma, its expression had no prognostic value in term of OS and RFS. By examining USP4 DNA copy number alterations (CNAs) (N = 511) and DNA methylation (N = 453) in LUAD, we found that DNA shallow deletion was frequent (–1, N = 239, 46.8%) and was associated with significantly decreased USP4 expression compared with the copy‐neutral (0) cases. The methylation status of some CpG sites in USP4 DNA was negatively correlated with USP4 expression. Based on these findings, we infer that USP4 expression might be a favorable biomarker in terms of OS and RFS in LUAD patients. DNA shallow deletion and hypermethylation might be two important mechanisms of decreased USP4 in these patients. © 2018 IUBMB Life, 70(7):670–677, 2018 [ABSTRACT FROM AUTHOR]

Abstract: Melanoma is the most malignant skin cancer with increasing incidence worldwide. Although innovative therapies such as BRAF inhibitor and immune checkpoint inhibitor have gained remarkable advances, metastatic melanoma remains an incurable disease for its notorious aggressiveness. Therefore, further clarification of the underlying mechanism of melanoma pathogenesis is critical for the improvement of melanoma therapy. Ubiquitination is an important regulatory event for cancer hallmarks and melanoma development, and the deubiquitinating enzymes including ubiquitin‐specific peptidase (USP) families are greatly implicated in modulating cancer biology. Herein, we first found that the expression of the deubiquitinase USP4 was significantly up‐regulated in melanoma tissues and cell lines. Furthermore, although USP4 knockdown had little impact on melanoma cell proliferation, it could increase the sensitivity to DNA damage agent cisplatin. We subsequently showed that USP4 regulated cisplatin‐induced cell apoptosis via p53 signalling. More importantly, USP4 could accentuate the invasive and migratory capacity of melanoma cells by promoting epithelial‐mesenchymal transition. Altogether, our results demonstrate that the up‐regulated USP4 plays an oncogenic role in melanoma by simultaneously suppressing stress‐induced cell apoptosis and facilitating tumour metastasis. [ABSTRACT FROM AUTHOR]

[Display omitted] Currently there are no compendial assays for testing drug release from rectal suppositories. It is therefore essential to study different in vitro release testing (IVRT) and in vitro permeation testing (IVPT) methods for identifying a suitable technique to compare in vitro drug release and to predict in vivo performance of rectal suppositories. In the present study, three different rectal suppository formulations of mesalamine (CANASA, Generic, and In-house) were studied for in vitro bioequivalence. All the different suppository products were characterized by performing weight variation, content uniformity, hardness, melting time, and pH tests. Viscoelastic behavior of the suppositories was also tested both in presence and absence of mucin. Four different IVRT techniques such as Dialysis, Horizontal Ussing Chamber, Vertical Franz cell, and USP apparatus 4. IVPT studies were performed using Horizontal Ussing chamber and Vertical Franz cell methods. Q1/Q2 equivalent products (CANASA, Generic) and a half-strength product were studied to understand the reproducibility, bio relevance, and discriminatory ability of the IVRT and IVPT methods. This study is the first of its kind where molecular docking studies were performed to determine the potential interactions of drug (mesalamine) with mucin, IVRT studies were conducted with and without the presence of mucin, and porcine rectal mucosa was used to perform IVPT tests. The USP 4 method and Horizontal Ussing chamber methods were found to be suitable IVRT and IVPT techniques, respectfully, for rectal suppositories. RLD (Reference Listed Drug) and Generic rectal suppositories were found to exhibit similar release rate and permeation profiles obtained from USP 4, and the IVPT studies, respectfully. Wilcoxon Rank Sum/Mann-Whitney rank test, conducted for the IVRT profiles obtained using USP 4 method, proved the sameness of RLD and Generic suppository products. [ABSTRACT FROM AUTHOR]

NF-κB is involved in the activation of microglia, which induces secondary spinal cord injury (SCI). This process involves the activation of NF-κB signaling pathway by TRAF6 through its polyubiquitination function. We know that deubiquitination of TRAF6 mediated by deubiquitinating enzyme (DUB) significantly inhibits activation of NF-κB pathway. The ubiquitin-specific protease 4 (USP4) belongs to the deubiquitinase family. Therefore, we hypothesize that USP4 is involved in the microglial activation and subsequent neuronal inflammation after SCI. In this study, we examined the expression and the role of USP4 after SCI. Western blot analysis showed that the expression of USP4 was downregulated and the expression of p-p65 was upregulated in the spinal cord after SCI. Immunohistochemical and immunofluorescence staining showed that USP4 was expressed in microglia but its expression decreased after SCI. In vitro LPS-induced activation of microglia showed decreased expression of USP4 and increased expression of p-p65 and TRAF6. USP4 silencing in LPS-induced activation of microglia promoted the expression of p-p65 and TRAF6 and the secretion of TNF-α and IL-1β. In conclusion, our study provides the first evidence that in microglial cells expression of USP4 decreases after SCI in rats. The downregulation of USP4 expression may promote microglial activation and subsequent neuronal inflammation through NF-κB by attenuating the deubiquitination of TRAF6. This mechanism is of great significance in the pathophysiology of secondary SCI. [ABSTRACT FROM AUTHOR]

Our study mainly investigated ubiquitin-specific protease 4 (USP4) expression in pathogenesis of esophageal cancer. The data showed significantly increased expression of USP4 in cancer tissues compared to that in para-carcinoma tissues (68.38% ± 25.60% vs 13.04% ± 9.95%, P = 0.000) and positive correlation between USP4 and pathology grade (r = 0.249, P = 0.014), although survival analysis revealed that USP4 expression was positively associated with the prognosis (32.4% vs 10.9%, P = 0.043). Grouped analysis revealed that the prognosis of patients with high USP4 expression were significantly better only in the small tumor subgroup (diameter ≤ 5 cm) (52.6% VS 8.6%, P = 0.001) and the early stage subgroup (stages 1 and 2) (60.0% VS 16.7%, P = 0.006). Moreover, in the subgroup of clinical stages 1 and 2 with tumor diameter ≤ 5 cm, high USP4 expression prolonged the survival time of esophageal cancer patients more significantly (75.5% VS 5.9%, P = 0.000). Based on these results, we speculated that it was possible to significantly improve the prognosis of patients with low USP4 expression by targeted therapy in early esophageal cancer. Taken together, our study uncovered a previously unknown function of USP4 in esophageal cancer and more investigations would be carried out to further study its regulation gene network and molecular biological mechanism in esophageal cancer. [ABSTRACT FROM AUTHOR]

CD4+ CD25+ regulatory T (Treg) cells comprise a unique subset of T cells required for maintaining immune homeostasis. However, the molecular mechanisms associated with the functional variety of Treg cells are not fully delineated. In the present study, we demonstrate that ubiquitin-specific protease ( USP)4 physically interacted with interferon regulatory factor 8 ( IRF8) function via a K48-linked deubiquitinase, which stabilized IRF8 protein levels in Treg cells. Depletion of USP4 promoted the polyubiquitination of IRF8 and the upregulation of type 2 inflammatory cytokine gene expression in Treg cells. Consistently, treatment of Treg cells with USP4 inhibitor facilitated the polyubiquitination of IRF8. In addition, the deficiency of USP4 alleviated the suppressive function of Treg cells. Taken together, our results suggest that USP4 interacts with and stabilizes IRF8 to promote the suppressive function of Treg cells. [ABSTRACT FROM AUTHOR]

SMAD4 is a common intracellular effector for TGF-β family cytokines, but the mechanism by which its activity is dynamically regulated is unclear. We demonstrated that ubiquitin-specific protease ( USP) 4 strongly induces activin/ BMP signaling by removing the inhibitory monoubiquitination from SMAD4. This modification was triggered by the recruitment of the E3 ligase, SMURF2, to SMAD4 following ligand-induced regulatory (R)- SMAD- SMAD4 complex formation. Whereas the interaction of the negative regulator c- SKI inhibits SMAD4 monoubiquitination, the ligand stimulates the recruitment of SMURF2 to the c- SKI- SMAD2 complex and triggers c- SKI ubiquitination and degradation. Thus, SMURF2 has a role in termination and initiation of TGF-β family signaling. An increase in monoubiquitinated SMAD4 in USP4-depleted mouse embryonic stem cells ( mESCs) decreased both the BMP- and activin-induced changes in the embryonic stem cell fate. USP4 sustained SMAD4 activity during activin- and BMP-mediated morphogenic events in early zebrafish embryos. Moreover, zebrafish depleted of USP4 exhibited defective cell migration and slower coordinated cell movement known as epiboly, both of which could be rescued by SMAD4. Therefore, USP4 is a critical determinant of SMAD4 activity. [ABSTRACT FROM AUTHOR]

Ubiquitinating enzymes catalyze protein ubiquitination, a reversible process countered by deubiquitinating enzyme (DUB) action. Ubiquitin-specific protease 4 (USP4) is a member of the ubiquitin-specific protease (USP) family of DUBs that has a role in spliceosome regulation. In the present study, we demonstrated that USP4 may be involved in neuronal apoptosis in the processes of intracerebral hemorrhage (ICH). We obtained a significant up-regulation of USP4 in neurons adjacent to the hematoma following ICH by the results of Western blot, immunohistochemistry, and immunofluorescence. Increasing USP4 level was found to be accompanied by the up-regulation of active caspase-3, γH2AX, Bax, and decreased expression of Bcl-2. In addition, USP4 co-localized well with γH2AX in the nucleus in the ICH model and hemin-induced apoptosis model. Moreover, in vitro study, knocking down USP4 by USP4-specific siRNA in PC12 cells reduced active caspase-3 expression. All these results above suggested that USP4 may be involved in neuronal apoptosis after ICH. [ABSTRACT FROM AUTHOR]

ABSTRACT The canonical Wnt/β-catenin signaling pathway plays a pivotal role and is essentially required for the osteoblast differentiation and bone formation. In this study, we found ubiquitin-specific peptidase 4 (USP4) to strongly inhibit the Wnt/β-catenin signaling by removing Lysine-63 linked poly-ubiquitin chain from Dishevelled (Dvl). Ectopic expression of USP4 promoted β-catenin poly-ubiquitination and thus inhibited Wnt-induced accumulation of cytosolic β-catenin and counteracted Wnt-induced transcriptional activity. Moreover, USP4 knockdown or USP4 knockout led to an increase in the active β-catenin levels and in activation of Wnt/β-catenin-induced transcription. Functional studies in C2C12 myoblasts and KS483 osteoprogenitor cells showed that ectopic expression of USP4 resulted in impaired activation of endogenous Wnt3a-induced genes and decreased osteoblast differentiation and mineralization, whereas USP4 depletion showed the opposite effect. These results identify USP4 as a novel regulator of Dvl in Wnt/β-catenin signal and show its involvement in Wnt3a-induced osteoblast differentiation. © 2016 American Society for Bone and Mineral Research. [ABSTRACT FROM AUTHOR]

Background: Transforming growth factor-β1 (TGF-β1) plays a key role in mesothelial-to-mesenchymal transition (MMT) during peritoneal dialysis (PD). However, the role of Akt in MMT transformation in PD is not clear. Results: In this study, we observed that the phosphorylated form of protein kinase B (Akt), termed as pAkt, was up-regulated in the peritoneum of mice undergoing PD. It was associated with thickening of the peritoneum and up-regulation of TGF-β1. Upregulation of pAkt paralleled with the increased expression of Smad ubiquitination regulatory factor 2 (Smurf2), Vimentin and fibronectin (FN), and decreased expression of mothers against decapentaplegic homolog 7 (Smad7) and Zonula Occludens protein 1(ZO-1) in mice undergoing PD treatment and in TGF-β1 induced human peritoneal mesothelial cells (HPMCs). These changes were reversed with the treatment of a PI3K/Akt inhibitor LY294002 in vivo or in cells transfected with Akt dominant-negative (Akt-DN) plasmids in vitro. Increased Smurf2 expression in HPMCs, induced by TGF-β1 was accompanied with altered expression of Transforming growth factor receptor I (TβR-I), Smad7, ZO-1, Vimentin and FN via Akt modulation. In addition, inhibition of Ubiquitin carboxyl-terminal hydrolase 4 (USP4) decreased TGF- β1-induced expression of TβR-I and reversed the altered expression of Smad7, Smurf2, ZO-1 and Vimentin. Moreover, TGF-β1 accentuated the interactions between Smurf2 and Smad7, while reduced the association between TβR-I and Smurf2. These interactions were reversed by the treatment of Akt-DN and USP4 siRNA, respectively. Conclusions: These data implied that Akt mediated MMT in PD via Smurf2 modulation/and or Smad7 degradation while conceivably maintaining the TβRI stability, most likely by the USP4. [ABSTRACT FROM AUTHOR]

We identified six patients presenting with a strikingly similar clinical phenotype of profound syndromic intellectual disability of unknown etiology. All patients lived in the same village. Extensive genealogical work revealed that the healthy parents of the patients were all distantly related to a common ancestor from the 17th century, suggesting autosomal recessive inheritance. In addition to intellectual disability, the clinical features included hypotonia, strabismus, difficulty to fix the eyes to an object, planovalgus in the feet, mild contractures in elbow joints, interphalangeal joint hypermobility and coarse facial features that develop gradually during childhood. The clinical phenotype did not fit any known syndrome. Genome-wide SNP genotyping of the patients and genetic mapping revealed the longest shared homozygosity at 3p22.1-3p21.1 encompassing 11.5 Mb, with no other credible candidate loci emerging. Single point parametric linkage analysis showed logarithm of the odds score of 11 for the homozygous region, thus identifying a novel intellectual disability predisposition locus. Whole-genome sequencing of one affected individual pinpointed three genes with potentially protein damaging homozygous sequence changes within the predisposition locus: transketolase ( TKT ), prolyl 4-hydroxylase transmembrane ( P4HTM ), and ubiquitin specific peptidase 4 ( USP4 ). The changes were found in heterozygous form with 0.3–0.7% allele frequencies in 402 whole-genome sequenced controls from the north-east of Finland. No homozygotes were found in this nor additional control data sets. Our study facilitates clinical and molecular diagnosis of patients with this novel autosomal recessive intellectual disability syndrome. However, further studies are needed to unambiguously identify the underlying genetic defect. [ABSTRACT FROM AUTHOR]

Background: It has been reported that ubiquitin specific peptidase 4 (USP4) was functional in several tumors, but its function and mechanism in gastric cancer were still unknown., Methods: Bioinformatic tools were used to predict the prognosis of gastric cancer patients and the expression levels of USP4 in gastric cancer. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunoblotting were carried out to detect the messenger RNA (mRNA) and protein levels. Cell viability of gastric cancer was evaluated by Cell Counting Kit-8 (CCK-8) assay. Cell line-derived xenograft models were established to evaluate the tumor growth of gastric cancer. Luciferase assay and immunoblotting were used to determine the activation of nuclear factor kappa B (NF-κB) signaling., Results: The public database Kaplan-Meier Plotter showed that gastric cancer patients with high USP4 expression had a shorter overall survival or post-progression survival than the patients with decreased USP4. Further studies indicated that USP4 was elevated in gastric cancer tumor tissues. In contrast, knockdown of USP4 markedly inhibited gastric cancer cell growth, and suppressed the tumor growth of gastric cancer. Further studies revealed that USP4 knockdown significantly suppressed NF-κB-driven luciferase activity, and inhibited the phosphorylation of NF-κB p65 in gastric cancer cells. Additionally, qRT-PCR analysis showed that USP4 knockdown significantly downregulated the expressions of cyclin D2 (CCND2) and B cell leukemia/lymphoma 2 (BCL2). We also found that USP4 knockdown decreased the expressions of phosphatase of regenerating liver-3 (PRL-3), in contrast, overexpression of PRL-3 attenuated the inhibitory effects of USP4 knockdown on NF-κB signaling and cell viability in gastric cancer cells. Finally, PR-619, which has been proven to inhibit the activities of USP4 and other deubiquitinases, could inhibit cell viability and NF-κB signaling in gastric cancer cells., Conclusions: This study indicated that elevated USP4 predicted a poor index for gastric cancer patients, and mediated gastric cancer cell growth by regulating PRL-3/NF-κB signaling, which suggested USP4 may be a novel therapeutic target for gastric cancer., Competing Interests: The authors declare no conflict of interest., (© 2022 The Author(s). Published by IMR Press.)