The deubiquitinase Usp7 in Drosophila melanogaster is required for synaptonemal complex maintenance.

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

Coordination of transcription-coupled repair and repair-independent release of lesion-stalled RNA polymerase II.

Transcription-blocking lesions (TBLs) stall elongating RNA polymerase II (Pol II), which then initiates transcription-coupled repair (TCR) to remove TBLs and allow transcription recovery. In the absence of TCR, eviction of lesion-stalled Pol II is required for alternative pathways to address the damage, but the mechanism is unclear. Using Protein-Associated DNA Damage Sequencing (PADD-seq), this study reveals that the p97-proteasome pathway can evict lesion-stalled Pol II independently of repair. Both TCR and repair-independent eviction require CSA and ubiquitination. However, p97 is dispensable for TCR and Pol II eviction in TCR-proficient cells, highlighting repair's prioritization over repair-independent eviction. Moreover, ubiquitination of RPB1-K1268 is important for both pathways, with USP7's deubiquitinase activity promoting TCR without abolishing repair-independent Pol II release. In summary, this study elucidates the fate of lesion-stalled Pol II, and may shed light on the molecular basis of genetic diseases caused by the defects of TCR genes.

Exosomal circ-0100519 promotes breast cancer progression via inducing M2 macrophage polarisation by USP7/NRF2 axis.

BACKGROUND: Breast cancer (BC) is one of the most prevalent malignant tumours that threatens women health worldwide. It has been reported that circular RNAs (circRNAs) play an important role in regulating tumour progression and tumour microenvironment (TME) remodelling. METHODS: Differentially expression characteristics and immune correlations of circRNAs in BC were verified using high-throughput sequencing and bioinformatic analysis. Exosomes were characterised by nanoparticle transmission electron microscopy and tracking analysis. The biological function of circ-0100519 in BC development was demonstrated both in vitro and in vivo. Western blotting, RNA pull-down, RNA immunoprecipitation, flow cytometry, and luciferase reporter were conducted to investigate the underlying mechanism. RESULTS: Circ-0100519 was significant abundant in BC tumour tissues and related to poor prognosis. It can be encapsulated into secreted exosomes, thereby promoting BC cell invasion and metastasis via inducing M2-like macrophages polarisation.Mechanistically, circ-0100519 acted as a scaffold to enhance the interaction between the deubiquitinating enzyme ubiquitin-specific protease 7 (USP7) and nuclear factor-like 2 (NRF2) in macrophages, inducing the USP7-mediated deubiquitination of NRF2. Additionally, HIF-1α could function as an upstream effector to enhance circ-0100519 transcription. CONCLUSIONS: Our study revealed that exosomal circ-0100519 is a potential biomarker for BC diagnosis and prognosis, and the HIF-1α inhibitor PX-478 may provide a therapeutic target for BC.

Discovery of pyrrolo[2,3-d]pyrimidin-4-one derivative YCH3124 as a potent USP7 inhibitor for cancer therapy.

USP7 is one of the most studied deubiquitinating enzymes, which is involved in the regulation of multiple cell signaling pathways and has been shown to be associated with the occurrence and progression of a variety of cancers. Inhibitors targeting USP7 have been studied by several teams, but most of them lack selectivity and have low activities. Herein, we reported a serious of pyrrole[2,3-d]pyrimidin-4-one derivatives through scaffold hopping of recently reported 4-hydroxypiperidine compounds. The representative compound Z33 (YCH3124) exhibited highly potent USP7 inhibition activity as well as anti-proliferative activity against four kinds of cancer cell lines. Further study revealed that YCH3124 effectively inhibited the downstream USP7 pathway and resulted in the accumulation of both p53 and p21 in a dose-dependent manner. Notably, YCH3124 disrupted cell cycle progression through restricting G1 phase and induced significant apoptosis in CHP-212 cells. In summary, our efforts provided a series of novel pyrrole[2,3-d]pyrimidin-4-one analogs as potent USP7 inhibitors with excellent anti-cancer activity.

Artesunate attenuates osteoarthritis in mice by promoting MTA1 transcription through a USP7/FoxO1 axis.

Artesunate (ART) is a derivative of artemisinin and has anti-inflammatory, anti-tumor, and anti-angiogenic properties. Although ART has been implicated in osteoarthritis (OA), the mechanism needs to be further dissected. Here, we explored the effects of ART on the development of OA and the underlying mechanism using destabilization of the medial meniscus (DMM) surgical instability model. Mice with OA were developed using DMM and treated with ART. The pathological morphology of knee joint tissues was examined, and the degeneration of joint cartilage was assessed. Mouse knee chondrocytes were isolated and induced with IL-1ß, followed by ART treatment. ART alleviates OA in mice by elevating ubiquitin carboxyl-terminal hydrolase 7 (USP7) expression, and USP7 inhibitor (P22077) treatment mitigated the protective effects of ART on chondrocytes. We also showed that USP7 mediated the deubiquitination of forkhead box protein O1 (FoxO1), while FoxO1 alleviated chondrocyte injury. In addition, FoxO1 promoted metastasis-associated protein MTA1 (MTA1) transcription, and downregulation of MTA1 exacerbated chondrocyte injury. Our study identifies that USP7/FoxO1/MTA1 is a key signaling cascade in the treatment of ART on OA.

Oxidative stress reprograms the transcriptional coactivator Yki to suppress cell proliferation.

The transcriptional coactivator Yorkie (Yki) regulates organ size by promoting cell proliferation. It is unclear how cells control Yki activity when exposed to harmful stimuli such as oxidative stress. In this study, we show that oxidative stress inhibits the binding of Yki to Scalloped (Sd) but promotes the interaction of Yki with another transcription factor, forkhead box O (Foxo), ultimately leading to a halt in cell proliferation. Mechanistically, Foxo normally exhibits a low binding affinity for Yki, allowing Yki to form a complex with Sd and activate proliferative genes. Under oxidative stress, Usp7 deubiquitinates Foxo to promote its interaction with Yki, thereby activating the expression of proliferation suppressors. Finally, we show that Yki is essential for Drosophila survival under oxidative stress. In summary, these findings suggest that oxidative stress reprograms Yki from a proliferation-promoting factor to a proliferation suppressor, forming a self-protective mechanism.

ATAD5 functions as a regulatory platform for Ub-PCNA deubiquitination.

Ubiquitination status of proliferating cell nuclear antigen (PCNA) is crucial for regulating DNA lesion bypass. After the resolution of fork stalling, PCNA is subsequently deubiquitinated, but the underlying mechanism remains undefined. We found that the N-terminal domain of ATAD5 (ATAD5-N), the largest subunit of the PCNA-unloading complex, functions as a scaffold for Ub-PCNA deubiquitination. ATAD5 recognizes DNA-loaded Ub-PCNA through distinct DNA-binding and PCNA-binding motifs. Furthermore, ATAD5 forms a heterotrimeric complex with UAF1-USP1 deubiquitinase, facilitating the deubiquitination of DNA-loaded Ub-PCNA. ATAD5 also enhances the Ub-PCNA deubiquitination by USP7 and USP11 through specific interactions. ATAD5 promotes the distinct deubiquitination process of UAF1-USP1, USP7, and USP11 for poly-Ub-PCNA. Additionally, ATAD5 mutants deficient in UAF1-binding had increased sensitivity to DNA-damaging agents. Our results ultimately reveal that ATAD5 and USPs cooperate to efficiently deubiquitinate Ub-PCNA prior to its release from the DNA in order to safely deactivate the DNA repair process.

USP7 Promotes TGF-ß1 Signaling by De-Ubiquitinating Smad2/Smad3 in Pulmonary Fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a long-term, progressive, and irreversible pulmonary interstitial disease. The activation of Smad family member 2 (Smad2) and Smad3 transcription factors by transforming growth factor ß-1 (TGF-ß1) is a critical event in the pathogenesis of IPF. However, there is still a lack of understanding regarding the molecular mechanisms governing Smad2 and Smad3 proteins. Ubiquitin-specific protease 7 (USP7) is a deubiquitinase that plays a vital role in regulating protein stability within cells. However, its regulation of the TGF-ß signaling pathway and its significance in IPF remain undiscovered. This study aims to clarify the function of USP7 in the TGF-ß signaling pathway, while simultaneously exploring the specific molecular mechanisms involved. Additionally, this study seeks to evaluate the therapeutic potential of targeted USP7 inhibitors in IPF, thereby providing novel insights for the diagnosis and management of IPF. METHODS: We first detected the expression of USP7 in lung tissues of mice with Bleomycin (BLM)-induced pulmonary fibrosis and in Beas-2B cells treated with or without TGF-ß1 through Western blot analysis. Subsequently, we explored the influence of USP7 on fibrotic processes and the TGF-ß1 signaling pathway, utilizing in vitro and in vivo studies. Finally, we assessed the effectiveness of USP7-specific inhibitors in an IPF murine model. RESULTS: In the present study, USP7 was found to de-ubiquitinate Smad2 and Smad3, consequently increasing their stability and promoting the TGF-ß1-induced production of profibrotic proteins including α-smooth muscle actin (α-SMA) and fibronectin 1 (FN-1). Inhibition or knockdown of USP7 resulted in decreased levels of Smad2 and Smad3 proteins, leading to reduced expression of FN-1, Collagen Type I Alpha 1 Chain (Col1A1), and α-SMA induced by TGF-ß1 in human pulmonary epithelial cells. These findings demonstrate that overexpression of USP7 reduces Smad2/3 ubiquitination, whereas inhibition or knockdown of USP7 enhances their ubiquitination. USP7 is abundantly expressed in IPF lungs. The expressions of USP7, Smad2, and Smad3 were upregulated in bleomycin-induced lung injury. The USP7 inhibitor P22077 reduced the expression of FN-1 and type I collagen as well as Smad2/3 and collagen deposition in lung tissue in a model of pulmonary fibrosis induced by bleomycin. CONCLUSIONS: This study demonstrates that USP7 promotes TGF-ß1 signaling by stabilizing Smad2 and Smad3. The contribution of USP7 to the progression of IPF indicates it may be a viable treatment target.

Evaluation of a Covalent Library of Diverse Warheads (CovLib) Binding to JNK3, USP7, or p53.

Purpose: Over the last few years, covalent fragment-based drug discovery has gained significant importance. Thus, striving for more warhead diversity, we conceived a library consisting of 20 covalently reacting compounds. Our covalent fragment library (CovLib) contains four different warhead classes, including five α-cyanoacacrylamides/acrylates (CA), three epoxides (EO), four vinyl sulfones (VS), and eight electron-deficient heteroarenes with a leaving group (SNAr/SN). Methods: After predicting the theoretical solubility of the fragments by LogP and LogS during the selection process, we determined their experimental solubility using a turbidimetric solubility assay. The reactivities of the different compounds were measured in a high-throughput 5,5'-dithiobis-(2-nitrobenzoic acid) DTNB assay, followed by a (glutathione) GSH stability assay. We employed the CovLib in a (differential scanning fluorimetry) DSF-based screening against different targets: c-Jun N-terminal kinase 3 (JNK3), ubiquitin-specific protease 7 (USP7), and the tumor suppressor p53. Finally, the covalent binding was confirmed by intact protein mass spectrometry (MS). Results: In general, the purchased fragments turned out to be sufficiently soluble. Additionally, they covered a broad spectrum of reactivity. All investigated α-cyanoacrylamides/acrylates and all structurally confirmed epoxides turned out to be less reactive compounds, possibly due to steric hindrance and reversibility (for α-cyanoacrylamides/acrylates). The SNAr and vinyl sulfone fragments are either highly reactive or stable. DSF measurements with the different targets JNK3, USP7, and p53 identified reactive fragment hits causing a shift in the melting temperatures of the proteins. MS confirmed the covalent binding mode of all these fragments to USP7 and p53, while additionally identifying the SNAr-type electrophile SN002 as a mildly reactive covalent hit for p53. Conclusion: The screening and target evaluation of the CovLib revealed first interesting hits. The highly cysteine-reactive fragments VS004, SN001, SN006, and SN007 covalently modify several target proteins and showed distinct shifts in the melting temperatures up to +5.1 °C and -9.1 °C.

PRMT1 promotes Warburg effect by regulating the PKM2/PKM1 ratio in non-small cell lung cancer.

Abnormal epigenetic modifications are involved in the regulation of Warburg effect in tumor cells. Protein arginine methyltransferases (PRMTs) mediate arginine methylation and have critical functions in cellular responses. PRMTs are deregulated in a variety of cancers, but their precise roles in Warburg effect in cancer is largely unknown. Experiments from the current study showed that PRMT1 was highly expressed under conditions of glucose sufficiency. PRMT1 induced an increase in the PKM2/PKM1 ratio through upregulation of PTBP1, in turn, promoting aerobic glycolysis in non-small cell lung cancer (NSCLC). The PRMT1 level in p53-deficient and p53-mutated NSCLC remained relatively unchanged while the expression was reduced in p53 wild-type NSCLC under conditions of glucose insufficiency. Notably, p53 activation under glucose-deficient conditions could suppress USP7 and further accelerate the polyubiquitin-dependent degradation of PRMT1. Melatonin, a hormone that inhibits glucose intake, markedly suppressed cell proliferation of p53 wild-type NSCLC, while a combination of melatonin and the USP7 inhibitor P5091 enhanced the anticancer activity in p53-deficient NSCLC. Our collective findings support a role of PRMT1 in the regulation of Warburg effect in NSCLC. Moreover, combination treatment with melatonin and the USP7 inhibitor showed good efficacy, providing a rationale for the development of PRMT1-based therapy to improve p53-deficient NSCLC outcomes.

Discovery of a Highly Potent, Selective and Efficacious USP7 Degrader for the Treatment of Acute Lymphoblastic Leukemia.

USP7 is an attractive therapeutic target for cancers, especially for acute lymphoblastic leukemia (ALL) with wild-type p53. Herein, we report the discovery of XM-U-14 as a highly potent, selective and efficacious USP7 proteolysis-targeting chimera degrader. XM-U-14 achieves DC50 values of 0.74 nM and Dmax of 93% in inducing USP7 degradation in RS4;11 cell lines, and also significantly inhibits ALL cell growth. XM-U-14 even at 5 mg/kg dosed daily effectively inhibits RS4;11 tumor growth with 64.7% tumor regressions and causes no signs of toxicity in mice. XM-U-14 is a promising USP7 degrader for further optimization for ALL treatment.

Identification of mechanism of the oncogenic role of FGFR1 in papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) is the most prevalent malignancy of the thyroid. Fibroblast growth factor receptor 1 (FGFR1) is highly expressed in PTC and works as an oncogenic protein in this disease. In this report, we wanted to uncover a new mechanism that drives overexpression of FGFR1 in PTC. Analysis of FGFR1 expression in clinical specimens and PTC cells revealed that FGFR1 expression was enhanced in PTC. Using siRNA/shRNA silencing experiments, we found that FGFR1 downregulation impeded PTC cell growth, invasion, and migration and promoted apoptosis in vitro, as well as suppressed tumor growth in vivo. Bioinformatic analyses predicted the potential USP7-FGFR1 interplay and the potential binding between YY1 and the FGFR1 promoter. The mechanism study found that USP7 stabilized FGFR1 protein via deubiquitination, and YY1 could promote the transcription of FGFR1. Our rescue experiments showed that FGFR1 re-expression had a counteracting effect on USP7 downregulation-imposed in vitro alterations of cell functions and in vivo suppression of xenograft growth. In conclusion, our study identifies the deubiquitinating enzyme USP7 and the oncogenic transcription factor YY1 as potent inducers of FGFR1 overexpression. Designing inhibitors targeting FGFR1 or its upstream inducers USP7 and YY1 may be foreseen as a promising strategy to control PTC development.

The phosphorylation-deubiquitination positive feedback loop of the CHK2-USP7 axis stabilizes p53 under oxidative stress.

p53 regulates multiple signaling pathways and maintains cell homeostasis under conditions of DNA damage and oxidative stress. Although USP7 has been shown to promote p53 stability via deubiquitination, the USP7-p53 activation mechanism has remained unclear. Here, we propose that DNA damage induces reactive oxygen species (ROS) production and activates ATM-CHK2, and CHK2 then phosphorylates USP7 at S168 and T231. USP7 phosphorylation is essential for its deubiquitination activity toward p53. USP7 also deubiquitinates CHK2 at K119 and K131, increasing CHK2 stability and creating a positive feedback loop between CHK2 and USP7. Compared to peri-tumor tissues, thyroid cancer and colon cancer tissues show higher CHK2 and phosphorylated USP7 (S168, T231) levels, and these levels are positively correlated. Collectively, our results uncover a phosphorylation-deubiquitination positive feedback loop involving the CHK2-USP7 axis that supports the stabilization of p53 and the maintenance of cell homeostasis.

USP7 promotes IgA class switching through stabilizing RUNX3 for germline transcription activation.

Class switch recombination (CSR) diversifies the effector functions of antibodies and involves complex regulation of transcription and DNA damage repair. Here, we show that the deubiquitinase USP7 promotes CSR to immunoglobulin A (IgA) and suppresses unscheduled IgG switching in mature B cells independent of its role in DNA damage repair, but through modulating switch region germline transcription. USP7 depletion impairs Sα transcription, leading to abnormal activation of Sγ germline transcription and increased interaction with the CSR center via loop extrusion for unscheduled IgG switching. Rescue of Sα transcription by transforming growth factor ß (TGF-ß) in USP7-deleted cells suppresses Sγ germline transcription and prevents loop extrusion toward IgG CSR. Mechanistically, USP7 protects transcription factor RUNX3 from ubiquitination-mediated degradation to promote Sα germline transcription. Our study provides evidence for active transcription serving as an anchor to impede loop extrusion and reveals a functional interplay between USP7 and TGF-ß signaling in promoting RUNX3 expression for efficient IgA CSR.

Biologically significant interaction of human herpesvirus 8 viral interferon regulatory factor 4 with ubiquitin-specific protease 7.

Human herpesvirus 8 (HHV-8), associated with Kaposi sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman disease, encodes four interferon regulatory factor homologs, vIRFs 1-4, that interact with and inhibit various mediators of host-cell defense against virus infection. A cellular protein targeted by all the vIRFs is ubiquitin-specific protease 7 (USP7); while replication-modulatory and latently infected PEL-cell pro-viability phenotypes of USP7 targeting have been identified for vIRFs 1-3, the significance of the interaction of vIRF-4 with USP7 has remained undetermined. Here we show, through genetic ablation of the vIRF-4-USP7 interaction in infected cells, that vIRF-4 association with USP7 is necessary for optimal expression of vIRF-4 and normal HHV-8 replication. Findings from experiments on transfected and infected cells identified ubiquitination of vIRF-4 via K48-linkage and USP7-binding-associated suppression of vIRF-4 ubiquitination and, in infected cells, increased vIRF-4 expression. Analysis of IFN-I induction and associated signaling as a function of vIRF-4 and its interaction with USP7 identified a role of each in innate-immune suppression. Finally, activation via K63-polyubiquitination of the innate-immune signaling mediator TRAF3 was found to be suppressed by vIRF-4 in a USP7-binding-associated manner in infected cells, but not in transfected cells, likely via binding-regulated expression of vIRF-4. Together, our data identify the first examples of vIRF ubiquitination and a vIRF substrate of USP7, enhanced expression of vIRF-4 via its interaction with USP7, and TRAF3-inhibitory activity of vIRF-4. The findings address, for the first time, the biological significance of the interaction of vIRF-4 with USP7 and reveal a mechanism of vIRF-4-mediated innate-immune evasion and pro-replication activity via TRAF3 regulation. IMPORTANCE: HHV-8 homologs of cellular interferon regulatory factors (IRFs), involved in host-cell defense against virus infection, interact in an inhibitory fashion with IRFs and other mediators of antiviral innate immunity. These interactions are of demonstrated or hypothesized importance for successful primary, productive (lytic), and latent (persistent) infection by HHV-8. While HHV-8 vIRF-4 is known to interact physically with USP7 deubiquitinase, a key regulator of various cellular proteins, the functional and biological significance of the interaction has not been addressed. The present study identifies the interaction as important for HHV-8 productive replication and, indeed, for vIRF-4 expression and reveals a new function of vIRF-4 via inhibition of the activity of TRAF3, a pivotal mediator of host-cell antiviral activity through activation of cellular IRFs and induction of type-I interferons. These findings identify potential targets for the development of novel anti-HHV-8 agents, such as those able to disrupt vIRF-4-USP7 interaction or vIRF-4-stabilizing USP7 activity.

USP7 cardiomyocyte specific knockout causes disordered mitochondrial biogenesis and dynamics and early neonatal lethality in mice.

BACKGROUND: Ubiquitination is an enzymatic modification involving ubiquitin chains, that can be reversed by deubiquitination (DUB) enzymes. Ubiquitin-specific protease 7 (USP7), which is also known as herpes virus-associated ubiquitin-specific protease (HAUSP), has been shown to play a vital role in cardiovascular diseases. However, the underlying molecular mechanism by which USP7 regulates cardiomyocyte function has not been reported. METHODS: To understand the physiological function of USP7 in the heart, we constructed cardiomyocyte-specific USP7 conditional knockout mice. RESULTS: We found that homozygous knockout mice died approximately three weeks after birth, while heterozygous knockout mice grew normally into adulthood. Severe cardiac dysfunction, hypertrophy, fibrosis, and cell apoptosis were observed in cardiomyocyte-specific USP7 knockout mice, and these effects were accompanied by disordered mitochondrial dynamics and cardiometabolic-related proteins. CONCLUSIONS: In summary, we investigated changes in the growth status and cardiac function of cardiomyocyte-specific USP7 knockout mice, and preliminarily explored the underlying mechanism.

Targeting senescent HDF with the USP7 inhibitor P5091 to enhance DFU wound healing through the p53 pathway.

OBJECTIVE: The objective of this study was to examine the potential of USP7 as a target for senolytic therapy and to investigate the molecular mechanism by which its inhibitor selectively induced apoptosis in senescent HDF and enhanced DFU wound healing. METHODS: Clinical samples of DFU were collected to detect the expression of USP7 and aging-related proteins using immunohistochemistry and Western blot. In addition, ß-galactosidase staining, qPCR, flow cytometry, ROS and MMP kits, and Western blot were used to analyze the biological functions of P5091 on senescence, cycle, and apoptosis. RNAseq was employed to further analyze the molecular mechanism of P5091. Finally, the DFU rat model was established to evaluate the effect of P5091 on wound healing. RESULTS: The expression of USP7 and p21 were increased in DFU clinical samples. After treatment with d-glucose (30 mM, 7 days), ß-galactosidase staining was deepened, proliferation rate decreased. USP7 inhibitors (P5091) could reduce the release of SASP factors, activate the production of ROS, and reduce MMP. In addition, it induced apoptosis and selectively clears senescent cells through the p53 signaling pathway. Finally, P5091 can improve diabetic wound healing in rats. CONCLUSION: This study clarified the molecular mechanism of USP7 inhibitor (P5091) selectively inducing apoptosis of high glucose senescent HDF cells. This provides a new senolytics target and experimental basis for promoting DFU wound healing.

USP7 promotes cardiometabolic disorders and mitochondrial homeostasis dysfunction in diabetic mice via stabilizing PGC1ß.

Diabetic cardiomyopathy (DCM) is a major complication of diabetes and is characterized by left ventricular dysfunction. Currently, there is a lack of effective treatments for DCM. Ubiquitin-specific protease 7 (USP7) plays a key role in various diseases. However, whether USP7 is involved in DCM has not been established. In this study, we demonstrated that USP7 was upregulated in diabetic mouse hearts and NMCMs co-treated with HG+PA or H9c2 cells treated with PA. Abnormalities in diabetic heart morphology and function were reversed by USP7 silencing through conditional gene knockout or chemical inhibition. Proteomic analysis coupled with biochemical validation confirmed that PCG1ß was one of the direct protein substrates of USP7 and aggravated myocardial damage through coactivation of the PPARα signaling pathway. USP7 silencing restored the expression of fatty acid metabolism-related proteins and restored mitochondrial homeostasis by inhibiting mitochondrial fission and promoting fusion events. Similar effects were also observed in vitro. Our data demonstrated that USP7 promoted cardiometabolic metabolism disorders and mitochondrial homeostasis dysfunction via stabilizing PCG1ß and suggested that silencing USP7 may be a therapeutic strategy for DCM.

P53 upregulation by USP7-engaging molecular glues.

Molecular glues are typically small chemical molecules that act at the interface between a target protein and degradation machinery to trigger ternary complex formation. Identifying molecular glues is challenging. There is a scarcity of target-specific upregulating molecular glues, which are highly anticipated for numerous targets, including P53. P53 is degraded in proteasomes through polyubiquitination by specific E3 ligases, whereas deubiquitinases (DUBs) remove polyubiquitination conjugates to counteract these E3 ligases. Thus, small-molecular glues that enhance P53 anchoring to DUBs may stabilize P53 through deubiquitination. Here, using small-molecule microarray-based technology and unbiased screening, we identified three potential molecular glues that may tether P53 to the DUB, USP7, and elevate the P53 level. Among the molecular glues, bromocriptine (BC) is an FDA-approved drug with the most robust effects. BC was further verified to increase P53 stability via the predicted molecular glue mechanism engaging USP7. Consistent with P53 upregulation in cancer cells, BC was shown to inhibit the proliferation of cancer cells in vitro and suppress tumor growth in a xenograft model. In summary, we established a potential screening platform and identified potential molecular glues upregulating P53. Similar strategies could be applied to the identification of other types of molecular glues that may benefit drug discovery and chemical biology studies.

The deubiquitinase USP7 and E3 ligase TRIM21 regulate vasculogenic mimicry and malignant progression of RMS by balancing SNAI2 homeostasis.

BACKGROUND: Rhabdomyosarcoma (RMS) is a rare malignancy and the most common soft tissue sarcoma in children. Vasculogenic mimicry (VM) is a novel tumor microcirculation model different from traditional tumor angiogenesis, which does not rely on endothelial cells to provide sufficient blood supply for tumor growth. In recent years, VM has been confirmed to be closely associated with tumor progression. However, the ability of RMS to form VM has not yet been reported. METHODS: Immunohistochemistry, RT-qPCR and western blot were used to test the expression level of SNAI2 and its clinical significance. The biological function in regulating vasculogenic mimicry and malignant progression of SNAI2 was examined both in vitro and in vivo. Mass spectrometry, co-immunohistochemistry, immunofluorescence staining, and ubiquitin assays were performed to explore the regulatory mechanism of SNAI2. RESULTS: Our study indicated that SNAI2 was abnormally expressed in patients with RMS and RMS cell lines and promoted the proliferation and metastasis of RMS. Through cell tubule formation experiments, nude mice Matrigel plug experiments, and immunohistochemistry (IHC), we confirmed that RMS can form VM and that SNAI2 promotes the formation of VM. Due to SNAI2 is a transcription factor that is not easily drugged, we used Co-IP combined with mass spectrometry to screen for the SNAI2-binding protein USP7 and TRIM21. USP7 depletion inhibited RMS VM formation, proliferation and metastasis by promoting SNAI2 degradation. We further demonstrated that TRIM21 is expressed at low levels in human RMS tissues and inhibits VM in RMS cells. TRIM21 promotes SNAI2 protein degradation through ubiquitination in the RMS. The deubiquitinase USP7 and E3 ligase TRIM21 function in an antagonistic rather than competitive mode and play a key role in controlling the stability of SNAI2 to determine the VM formation and progression of RMS. CONCLUSION: Our findings reveal a previously unknown mechanism by which USP7 and TRIM21 balance the level of SNAI2 ubiquitination, determining RMS vasculogenic mimicry, proliferation, and migration. This new mechanism may provide new targeted therapies to inhibit the development of RMS by restoring TRIM21 expression or inhibiting USP7 expression in RMS patients with high SNAI2 protein levels.