Differential Oligomerization of the Deubiquitinases USP25 and USP28 Regulates Their Activities.

Deubiquitinases have emerged as promising drug targets for cancer therapy. The two DUBs USP25 and USP28 share high similarity but vary in their cellular functions. USP28 is known for its tumor-promoting role, whereas USP25 is a regulator of the innate immune system and, recently, a role in tumorigenesis was proposed. We solved the structures of the catalytic domains of both proteins and established substantial differences in their activities. While USP28 is a constitutively active dimer, USP25 presents an auto-inhibited tetramer. Our data indicate that the activation of USP25 is not achieved through substrate or ubiquitin binding. USP25 cancer-associated mutations lead to activation in vitro and in vivo, thereby providing a functional link between auto-inhibition and the cancer-promoting role of the enzyme. Our work led to the identification of significant differences between USP25 and USP28 and provided the molecular basis for the development of new and highly specific anti-cancer drugs.

Structural basis for the bi-specificity of USP25 and USP28 inhibitors.

The development of cancer therapeutics is often hindered by the fact that specific oncogenes cannot be directly pharmaceutically addressed. Targeting deubiquitylases that stabilize these oncogenes provides a promising alternative. USP28 and USP25 have been identified as such target deubiquitylases, and several small-molecule inhibitors indiscriminately inhibiting both enzymes have been developed. To obtain insights into their mode of inhibition, we structurally and functionally characterized USP28 in the presence of the three different inhibitors AZ1, Vismodegib and FT206. The compounds bind into a common pocket acting as a molecular sink. Our analysis provides an explanation why the two enzymes are inhibited with similar potency while other deubiquitylases are not affected. Furthermore, a key glutamate residue at position 366/373 in USP28/USP25 plays a central structural role for pocket stability and thereby for inhibition and activity. Obstructing the inhibitor-binding pocket by mutation of this glutamate may provide a tool to accelerate future drug development efforts for selective inhibitors of either USP28 or USP25 targeting distinct binding pockets.

Heterozygous variants in USP25 cause genetic generalized epilepsy.

USP25 encodes ubiquitin-specific proteases 25, a key member of deubiquitinating enzyme family and is involved in neural fate determination. Although abnormal expression in Down's syndrome was reported previously, the specific role of USP25 in human diseases has not been defined. In this study, we performed trio-based whole exome sequencing in a cohort of 319 cases (families) with generalized epilepsy of unknown etiology. Five heterozygous USP25 variants including two de novo and three co-segregated variants were determined in eight individuals affected by generalized seizures and/or febrile seizures from five unrelated families. The frequency of USP25 variants showed a significantly high aggregation in this cohort compared to the East Asian population and all populations in the gnomAD database. The mean onset ages of febrile and afebrile seizures were 10 months (infancy) and 11.8 years (juvenile), respectively. The patients achieved seizure freedom except one had occasional nocturnal seizures at the last follow-up. Two patients exhibited intellectual disability. Usp25 was ubiquitously expressed in mouse brain with two peaks on embryonic days (E14‒E16) and postnatal day 21, respectively. Similarly, USP25 expressed in fetus/early childhood stage with a second peak at approximately 12‒20 years old in human brain, consistent with the seizure onset age at infancy and juvenile in the patients. To investigate the functional impact of USP25 deficiency in vivo, we established Usp25 knock-out mice, which showed increased seizure susceptibility compared to wild-type mice in pentylenetetrazol-induced seizure test. To explore the impact of USP25 variants, we employed multiple functional detections. In HEK293T cells, the severe phenotype associated variant (p.Gln889Ter) led to a significant reduction of mRNA and protein expressions but formed a stable truncated dimers with increment of deubiquitinating enzyme activities and abnormal cellular aggregations, indicating a gain-of-function effect. The p.Gln889Ter and p.Leu1045del increased neuronal excitability in mice brain, with a higher firing ability in p.Gln889Ter. These functional impairments align with the severity of the observed phenotypes, suggesting a genotype-phenotype correlation. Hence, a moderate association between USP25 and epilepsy was noted, indicating USP25 is potentially a predisposing gene for epilepsy. Our results from Usp25 null mice and the patient-derived variants indicated that USP25 would play epileptogenic role via loss-of-function or gain-of-function effects. The truncated variant p.Gln889Ter would have profoundly different effect on epilepsy. Together, our results underscore the significance of USP25 heterozygous variants in epilepsy, thereby highlighting the critical role of USP25 in the brain.

USP25 regulates Wnt signaling by controlling the stability of tankyrases.

Aberrant activation of the Wnt signaling pathway plays an important role in human cancer development. Wnt signaling is negatively regulated by Axin, a scaffolding protein that controls a rate-limiting step in the destruction of ß-catenin, the central activator of the Wnt pathway. In Wnt-stimulated cells, Axin is rapidly modified by tankyrase-mediated poly(ADP-ribosyl)ation, which promotes the proteolysis of Axin and consequent stabilization of ß-catenin. Thus, regulation of the levels and activity of tankyrases is mechanistically important in controlling Wnt signaling. Here, we identify ubiquitin-specific protease 25 (USP25) as a positive regulator of Wnt/ß-catenin signaling. We found that USP25 directly interacted with tankyrases to promote their deubiquitination and stabilization. We demonstrated that USP25 deficiency could promote the degradation of tankyrases and consequent stabilization of Axin to antagonize Wnt signaling. We further characterized the interaction between TNKS1 and USP25 by X-ray crystal structure determination. Our results provide important new insights into the molecular mechanism that regulates the turnover of tankyrases and the possibility of targeting the stability of tankyrases by antagonizing their interaction with USP25 to modulate the Wnt/ß-catenin pathway.

USP25 Promotes the Antimycobacterial Response of Macrophages Through Stabilizing B-Raf and C-Raf.

Ubiquitin-specific peptidase 25 (USP25) is one of the best-characterized deubiquitinating enzymes and plays a vital regulatory role in various biological processes, especially in cancer development and immune regulation. However, the exact role of USP25 and its underlying mechanisms in macrophage activation and immunogenicity during Mycobacterium tuberculosis infection remain unclear. In this study, we found that M tuberculosis infection induced USP25 expression in human and mouse macrophages. In particular, USP25 expression is elevated in multiple cell types, especially monocytes, in patients with tuberculosis. Additionally, USP25 deficiency in macrophages and mice resulted in compromised immunity against M tuberculosis infection, accompanied by reduced expressions of various proinflammatory cytokines and chemokines. Mechanistically, USP25 in macrophages promoted the activation of the ERK signaling pathway through deubiquitination and stabilization of B-Raf and C-Raf. These findings collectively suggest the critical roles of USP25 in M tuberculosis infection and its potential as a therapeutic target.

USP25 contributes to defective neurogenesis and cognitive impairments.

Both Down syndrome (DS) individuals and animal models exhibit hypo-cellularity in hippocampus and neocortex indicated by enhanced neuronal death and compromised neurogenesis. Ubiquitin-specific peptidase 25 (USP25), a human chromosome 21 (HSA21) gene, encodes for a deubiquitinating enzyme overexpressed in DS patients. Dysregulation of USP25 has been associated with Alzheimer's phenotypes in DS, but its role in defective neurogenesis in DS has not been defined. In this study, we found that USP25 upregulation impaired cell cycle regulation during embryonic neurogenesis and cortical development. Overexpression of USP25 in hippocampus promoted the neural stem cells to glial cell fates and suppressed neuronal cell fate by altering the balance between cyclin D1 and cyclin D2, thus reducing neurogenesis in the hippocampus. USP25-Tg mice showed increased anxiety/depression-like behaviors and learning and memory deficits. These results suggested that USP25 overexpression resulted in defective neurogenesis and cognitive impairments, which could contribute to the pathogenesis of DS. USP25 may be a potential pharmaceutical target for DS.

USP25 attenuates anti-GBM nephritis in mice by negative feedback regulation of Th17 cell differentiation.

PURPOSE: This study aimed to elucidate the role of USP25 in a mouse model of anti-glomerular basement membrane glomerulonephritis (anti-GBM GN). METHODS: USP25-deficient anti-GBM GN mice were generated, and their nephritis progression was monitored. Naïve CD4+ T cells were isolated from spleen lymphocytes and stimulated to differentiate into Th1, Th2, and Th17 cells. This approach was used to investigate the impact of USP25 on CD4+ T lymphocyte differentiation in vitro. Furthermore, changes in USP25 expression were monitored during Th17 differentiation, both in vivo and in vitro. RESULTS: USP25-/- mice with anti-GBM GN exhibited accelerated renal function deterioration, increased infiltration of Th1 and Th17 cells, and elevated RORγt transcription. In vitro experiments demonstrated that USP25-/- CD4+ T lymphocytes had a higher proportion for Th17 cell differentiation and exhibited higher RORγt levels upon stimulation. Wild-type mice with anti-GBM GN showed higher USP25 levels compared to healthy mice, and a positive correlation was observed between USP25 levels and Th17 cell counts. Similar trends were observed in vitro. CONCLUSION: USP25 plays a crucial role in mitigating renal histopathological and functional damage during anti-GBM GN in mice. This protective effect is primarily attributed to USP25's ability to inhibit the differentiation of naïve CD4+ T cells into Th17 cells. The underlying mechanism may involve the downregulation of RORγt. Additionally, during increased inflammatory responses or Th17 cell differentiation, USP25 expression is activated, forming a negative feedback regulatory loop that attenuates immune activation.

Upregulation of USP25 promotes progression of human diffuse large B-cell lymphoma through blocking the ubiquitinated degradation of MDM2.

Diffuse large B cell lymphoma (DLBCL) is a type of cancer that originates from abnormal B cells in the lymph nodes or other lymphoid tissues. Dysfunction of deubiquitinases is frequently implicated in malignant progression. This study planned to uncover the biological roles of deubiquitinase USP25 during DLBCL tumorigenesis. In this study we identified USP25 as a novel oncogene which is frequently upregulated in DLBCL and associated with dismal prognosis of patients. Moreover, USP25 silencing was found to inhibit DLBCL growth, migration, while induced an obvious increase in apoptosis in vitro. Meanwhile, USP25 could promote DLBCL tumour growth and lung metastasis in vivo. Mechanistically, the co-immunoprecipitation test provided a mechanistic explanation, showing that USP25 directly interacted with murine double minute 2 (MDM2) and MDM2 protein stability was maintained by USP25 mediated deubiquitination. In addition, overexpression of USP25 with C178A mutation failed to decrease its modification on MDM2 stability. Further mechanism-of-action studies demonstrated that USP25 promoted DLBCL progression via stabilizing MDM2 and consequently decreasing p53 expression. In addition, further analysis showed that the oncogenic effect of USP25 was relied on MDM2-p53 signaling pathway-mediated cell-cycle accelerating. Collective, USP25 was shown to be an important upstream regulator of the MDM2-p53 signaling pathway in DLBCL, and it has the potential to be employed as a novel target gene in the development of new therapeutic applications.

USP25 inhibits DNA damage by stabilizing BARD1 protein in a house dust mite-induced asthmatic model in vitro and in vivo.

House dust mites (HDM) can cause DNA double-strand breaks in the lungs of asthmatic patients. However, the molecular mechanisms driving DNA damage and repair in HDM-induced asthma are yet to be elucidated. Thus, in this study, HDM treatment was applied to BEAS-2B cells and mice to mimic the pathological process of asthma in vitro and in vivo, respectively. γ-H2AX foci and expression were measured by immunofluorescence staining and western blot, respectively. The levels of interleukin (IL)-4, IL-6, IL-13, and tumour necrosis factor α (TNFα) were measured using enzyme-linked immunoassay. The expression of USP25 and BARD1 was measured by reverse transcription quantitative PCR and western blot. Co-immunoprecipitation and ubiquitination assays were employed to detect the relationship between USP25 and BARD1. As per the results, it was found that the deubiquitylating enzyme USP25 repressed HDM-induced DNA damage and the production of proinflammatory cytokines, including TNF-α, IL-4, IL-8, and IL-13, in BEAS-2B cells; in contrast, the depletion of USP25 led to the opposite effects. USP25-mediated inhibition of DNA damage and inflammation was facilitated by the stabilizing protein BARD1, which is a tumor suppressor that principally functions by promoting DNA repair and replication in BEAS-2B cells. Furthermore, USP25 was found to robustly augment BARD1 protein abundance and prevent HDM-induced DNA damage and inflammation in vivo. Taken together, these results suggest a novel mechanism contributing to DNA damage and repair in HDM-induced asthma and that selectively modulating this pathway could lead to a novel therapeutic approach for controlling and managing asthma due to HDM exposure.

The miR-27a-3p/USP25 axis participates in the pathogenesis of recurrent miscarriage by inhibiting trophoblast migration and invasion.

Insufficient invasion ability of trophoblasts might be associated with the development of recurrent miscarriage (RM). Ubiquitin-specific protease 25 (USP25) can regulate the processes of invasion and migration in different types of cancer cells. However, the effect of USP25 on trophoblasts and its roles in the development of RM are unknown. In this study, we first analyzed the USP25 expression in placental villous tissues from RM patients, and then assessed the roles of USP25 in epithelial-to-mesenchymal transition (EMT), invasion and migration of trophoblasts. Furthermore, bioinformatics prediction and luciferase reporter assay were used to explore the mechanism of microRNA on USP25 expression, and regulation of USP25 expression in trophoblasts was assessed following transfection with microRNA mimics or inhibitor. The results showed that the expression of USP25 in the placental villous tissues was downregulated in RM patients. Knockdown of USP25 suppressed the EMT process, the invasion and migration capability of trophoblast cells, while overexpression of USP25 exhibited opposite results. Mechanistically, miR-27a-3p could regulate USP25 expression by binding to the 3'-untranslated region of USP25 in trophoblasts. Quantitative real-time polymerase chain reaction results found the expression of miR-27a-3p were negatively related to USP25 in RM patients. MiR-27a-3p mimics inhibited but miR-27a-3p inhibitor enhanced the migration and invasion capability of trophoblasts. Furthermore, sh-USP25 counteracted the promotion of invasion and migration mediated by the miR-27a-3p inhibitor. Taken together, these data indicate that USP25 downregulation by miR-27a-3p contributes to the EMT process, thereby inhibiting the migration and invasion of trophoblast cells, and these findings might provide potential biomarkers for RM.

Smurf1 restricts the antiviral function mediated by USP25 through promoting its ubiquitination and degradation.

Protein ubiquitination and deubiquitination enzymes are widely involved in innate immune responses. The ubiquitin specific protease 25 (USP25), a deubiquitinating enzyme, has been demonstrated to play an important role in virus infection and immunity. However, how USP25 is degraded and regulated by E3 ubiquitin ligases remains poorly understood. Here, we identified Smad ubiquitin regulatory factor 1(Smurf1) as a first novel E3 ubiquitin ligase of USP25. Smurf1 overexpression decreases USP25 protein turnover, and the E3 ligase enzymatic activity of Smurf1 is required for USP25 degradation. Additionally, Smurf1-mediated degradation of USP25 is via promoting the K48-linkage polyubiquitination of USP25 in an ubiquitin proteasome dependent pathway. Importantly, USP25 overexpression restricts vesicular stomatitis virus (VSV) replication and the restriction of VSV replication by USP25 is enhanced in Smurf1 stable knock down cells. Therefore, our study firstly identified that Smurf1 negatively regulated the antiviral function mediated by USP25. Our findings revealed a previously unrecognized role of Smurf1 acting on USP25 and also their roles in the regulation of VSV replications.

Induction of USP25 by viral infection promotes innate antiviral responses by mediating the stabilization of TRAF3 and TRAF6.

Host pathogen-recognition receptors detect nucleic acid from invading viruses and initiate a series of signaling pathways that lead to the production of type I interferons (IFNs) and proinflammatory cytokines. Here, we found that a viral infection-induced deubiquitinase (DUB), ubiquitin-specific protease 25 (USP25) was required for host defense against RNA and DNA viruses. The activation of transcription factors IRF3 and NF-κB was impaired and the production of type I IFNs and proinflammatory cytokines was inhibited in Usp25-/- cells compared with the wild-type counterparts after RNA or DNA viruses infection. Consistently, USP25 deficient mice were more susceptible to H5N1 or HSV-1 infection compared with the wild-type mice. USP25 was associated with TRAF3 and TRAF6 after infection by RNA or DNA viruses and protected virus-induced proteasome-dependent or independent degradation of TRAF3 and TRAF6, respectively. Moreover, reconstitution of TRAF3 and TRAF6 into Usp25-/- MEFs restored virus-triggered production of type I IFNs and proinflammatory cytokines. Our findings thus reveal a previously uncovered positive feedback regulation of innate immune responses against RNA and DNA viruses by USP25.

The Type I Interferon-IRF7 Axis Mediates Transcriptional Expression of Usp25 Gene.

Viral infection or lipopolysaccharide (LPS) treatment induces expression of a large array of genes, the products of which play a critical role in host antipathogen immunity and inflammation. We have previously reported that the expression of ubiquitin-specific protease 25 (USP25) is significantly up-regulated after viral infection or LPS treatment, and this is essential for innate immune signaling. However, the mechanism behind this phenomenon is unclear. In this study, we found that viral infection-induced up-regulation of Usp25 is diminished in cells lacking interferon regulatory factor 7 (IRF7) or interferon α receptor 1 (IFNAR1) but not p65. Sendai virus- or type I interferon-induced up-regulation of Usp25 requires de novo protein synthesis of IRF7. Furthermore, IRF7 directly binds to the two conserved IRF binding sites on the USP25 promoter to drive transcription of Usp25, and mutation of these two sites abolished Sendai virus-induced IRF7-mediated activation of the USP25 promoter. Our study has uncovered a previously unknown mechanism by which viral infection or LPS induces up-regulation of USP25.

Putative role of SUMOylation in controlling the activity of deubiquitinating enzymes in cancer.

Deubiquitinating enzymes (DUBs) are specialized proteins that can recognize ubiquitinated proteins, and after direct interaction, deconjugate monomeric or polymeric ubiquitin chains, thus changing the fate of the substrates. This process is instrumental in mediating or changing downstream signaling pathways. Beside mutations and alterations in their expression levels, the activity and stability of deubiquitinating enzymes is vital for their function. SUMOylations consist of the conjugation of the small peptide SUMO to protein substrates which is very similar to ubiquitination in the mechanistic and machinery required. In this review, we will focus on how SUMOylation can regulate DUB enzymatic activity, stability or DUB interaction with partners and substrates, in cancer. Furthermore, we will discuss the impact of these recent findings in the identification of new potential tools for efficient anticancer treatment strategies.

USP25 Elevates SHLD2-Mediated DNA Double-Strand Break Repair and Regulates Chemoresponse in Cancer.

DNA damage plays a significant role in the tumorigenesis and progression of the disease. Abnormal DNA repair affects the therapy and prognosis of cancer. In this study, it is demonstrated that the deubiquitinase USP25 promotes non-homologous end joining (NHEJ), which in turn contributes to chemoresistance in cancer. It is shown that USP25 deubiquitinates SHLD2 at the K64 site, which enhances its binding with REV7 and promotes NHEJ. Furthermore, USP25 deficiency impairs NHEJ-mediated DNA repair and reduces class switch recombination (CSR) in USP25-deficient mice. USP25 is overexpressed in a subset of colon cancers. Depletion of USP25 sensitizes colon cancer cells to IR, 5-Fu, and cisplatin. TRIM25 is also identified, an E3 ligase, as the enzyme responsible for degrading USP25. Downregulation of TRIM25 leads to an increase in USP25 levels, which in turn induces chemoresistance in colon cancer cells. Finally, a peptide that disrupts the USP25-SHLD2 interaction is successfully identified, impairing NHEJ and increasing sensitivity to chemotherapy in PDX model. Overall, these findings reveal USP25 as a critical effector of SHLD2 in regulating the NHEJ repair pathway and suggest its potential as a therapeutic target for cancer therapy.

A novel TNKS/USP25 inhibitor blocks the Wnt pathway to overcome multi-drug resistance in TNKS-overexpressing colorectal cancer.

Modulating Tankyrases (TNKS), interactions with USP25 to promote TNKS degradation, rather than inhibiting their enzymatic activities, is emerging as an alternative/specific approach to inhibit the Wnt/ß-catenin pathway. Here, we identified UAT-B, a novel neoantimycin analog isolated from Streptomyces conglobatus, as a small-molecule inhibitor of TNKS-USP25 protein-protein interaction (PPI) to overcome multi-drug resistance in colorectal cancer (CRC). The disruption of TNKS-USP25 complex formation by UAT-B led to a significant decrease in TNKS levels, triggering cell apoptosis through modulation of the Wnt/ß-catenin pathway. Importantly, UAT-B successfully inhibited the CRC cells growth that harbored high TNKS levels, as demonstrated in various in vitro and in vivo studies utilizing cell line-based and patient-derived xenografts, as well as APCmin/+ spontaneous CRC models. Collectively, these findings suggest that targeting the TNKS-USP25 PPI using a small-molecule inhibitor represents a compelling therapeutic strategy for CRC treatment, and UAT-B emerges as a promising candidate for further preclinical and clinical investigations.

Ubiquitin-specific peptidase 25 exacerbated osteoarthritis progression through facilitating TXNIP ubiquitination and NLRP3 inflammasome activation.

Several members of the ubiquitin-specific proteases (USPs) family have been revealed to regulate the progression of osteoarthritis (OA). The current study aimed to investigate the role and the underlying mechanism of USP25 in IL-1ß-induced chondrocytes and OA rat model. It was discovered that IL-1ß stimulation upregulated USP25, increased ROS level, and suppressed cell viability in rat chondrocytes. Besides, USP25 knockdown alleviated IL-1ß-induced injury by decreasing ROS level, attenuating pyroptosis, and downregulating the expression of IL-18, NLRP3, GSDMD-N, active caspase-1, MMP-3, and MMP-13. Furthermore, we discovered that USP25 affected the IL-1ß-induced injury in chondrocytes in a ROS-dependent manner. Moreover, USP25 was revealed to interact with TXNIP, and USP25 knockdown increased the ubiquitination of TXNIP. The pro-OA effect of USP25 abundance could be overturned by TXNIP suppression in IL-1ß-induced chondrocytes. Finally, in vivo experiment results showed that USP25 inhibition alleviated cartilage destruction in OA rats. In conclusion, we demonstrated that USP25 stimulated the overproduction of ROS to activate the NLRP3 inflammasome via regulating TXNIP, resulting in increased pyroptosis and inflammation in OA.

USP25 Inhibits Neuroinflammatory Responses After Cerebral Ischemic Stroke by Deubiquitinating TAB2.

Cerebral ischemic stroke is a leading cause of morbidity and mortality globally. However, the mechanisms underlying ischemic stroke injury remain poorly understood. Here, it is found that deficiency of the ubiquitin-specific protease USP25 significantly aggravate ischemic stroke injury in mice. USP25 has no impact on neuronal death under hypoxic conditions, but reduced ischemic stroke-induced neuronal loss and neurological deficits by inhibiting microglia-mediated neuroinflammation. Mechanistically, USP25 restricts the activation of NF-κB and MAPK signaling by regulating TAB2. As a deubiquitinating enzyme, USP25 removeds K63-specific polyubiquitin chains from TAB2. AAV9-mediated TAB2 knockdown ameliorates ischemic stroke injury and abolishes the effect of USP25 deletion. In both mouse and human brains, USP25 is markedly upregulated in microglia in the ischemic penumbra, implying a clinical relevance of USP25 in ischemic stroke. Collectively, USP25 is identified as a critical inhibitor of ischemic stroke injury and this data suggest USP25 may serve as a therapeutic target for ischemic stroke.

Structure and function of the highly homologous deubiquitinases ubiquitin specific peptidase 25 and 28: Insights into their pathophysiological and therapeutic roles.

Deubiquitination is the reverse process of ubiquitination, an important protein post-translational modification. Deubiquitination is assisted by deubiquitinating enzymes (DUBs), which catalyze the hydrolysis and removal of ubiquitin chains from targeted proteins and play an important role in regulating protein stability, cell signaling transduction, and programmed cell death. Ubiquitin-specific peptidases 25 and 28 (USP25 and USP28), important members of the USP subfamily of DUBs, are highly homologous, strictly regulated, and closely associated with various diseases, such as cancer and neurodegenerative diseases. Recently, the development of inhibitors targeting USP25 and USP28 for disease treatment has garnered extreme attention. Several non-selective and selective inhibitors have shown potential inhibitory effects. However, the specificity, potency, and action mechanism of these inhibitors remain to be further improved and clarified. Herein, we summarize the structure, regulation, emerging physiological roles, and target inhibition of USP25 and USP28 to provide a basis for the development of highly potent and specific inhibitors for the treatment of diseases, such as colorectal cancer, breast cancer and so on.

USP25 inhibits renal fibrosis by regulating TGFß-SMAD signaling pathway in Ang II-induced hypertensive mice.

Renal fibrosis is a crucial pathological feature of hypertensive renal disease (HRD). In-depth analysis of the pathogenesis of fibrosis is of great significance for the development of new drugs for the treatment of HRD. USP25 is a deubiquitinase that can regulate the progression of many diseases, but its function in the kidney remains unclear. We found that USP25 was significantly increased in human and mice HRD kidney tissues. In the HRD model induced by Ang II, USP25-/- mice showed significant aggravation of renal dysfunction and fibrosis compared with the control mice. Consistently, AAV9-mediated overexpression of USP25 significantly improved renal dysfunction and fibrosis. Mechanistically, USP25 inhibited the TGF-ß pathway by reducing SMAD4 K63-linked polyubiquitination, thereby suppressing SMAD2 nuclear translocation. In conclusion, this study demonstrates for the first time that the deubiquitinase USP25 plays an important regulatory role in HRD.