Targeted therapy for NRAS mutant melanoma remains an unmet clinical need. We found that inhibiting Ubiquitin Specific Peptidase 7 (USP7) with the selective USP7 inhibitor (USP7i) FT671 inhibited cell proliferation in NRAS mutant melanoma cell lines. In addition, we identified and validated the knockout of TP53BP1, TP53 or CDKN1A conferred resistance to FT671, suggesting the activation of a functional p53 signaling pathway is essential for the efficacy of USP7i. In Nras mutant melanoma isograft models, FT671 treatment delayed tumor growth. Moreover, the combinatorial treatment with FT671 and MEK1/2 inhibitor (MEKi) was synergistic and induced pyroptosis in vitro. In immunocompetent mice, the combined treatment profoundly suppressed tumor growth, prolonged survival and enhanced intratumoral immune cell infiltration, particularly increasing the ratios of CD8+ T cells and mature dendritic cells, indicative of activated antitumor immunity. Notably, the triple combination of USP7i, MEKi, and anti-PD-1 antibody resulted in durable tumor regression, with effects persisting beyond 80 days after treatment cessation. These findings establish USP7i+MEKi as a promising strategy for targeting NRAS, an 'undruggable' mutation in melanoma, and provide a strong rationale for the clinical development of USP7i plus MEKi as an adjuvant therapy to enhance anti-PD-1 immunotherapy in NRAS mutant melanoma patients.

Proteins play a pivotal role in maintaining cellular homeostasis. Their degradation primarily orchestrated through the ubiquitin-proteasome system (UPS) and cellular autophagy. Dysfunction of the UPS is associated with various human diseases, including cancer, autoimmune disorders, and neurodegenerative conditions. Consequently, the UPS has emerged as a promising therapeutic target. Deubiquitinases (DUBs) have garnered significant attention as potential targets for therapeutic intervention due to their role in modulating protein stability and function. This review focuses on recent advancements of DUBs, particularly their relevance in the UPS and their potential as drug targets. Notably, inhibitors targeting specific DUBs, such as USP1, USP7, USP14, and USP30 have shown promise in preclinical and clinical studies for cancer therapy. Additionally, DUB inhibitors have been involved in novel therapeutic approaches lately, including as targets for proteolysis-targeting chimeras (PROTACs) or as tools in deubiquitinase-targeting chimeras (DUBTACs).

The ubiquitin-specific protease (USP) family of deubiquitinases (DUBs) are regulators of Ub signaling that share a common catalytic-domain fold. The dynamic nature of this domain is important for controlling the function of USPs, with inter- and intramolecular interactions often influencing the structure and enzymatic activity of these DUBs. This conformational flexibility, in combination with the high sequence conservation of the USP active site, has made it challenging to readily identify potent and selective inhibitors for individual USPs. Here, we demonstrate how a naive, macrocycle-mRNA display selection rapidly yielded high-affinity binders to USP7 that specifically inhibit the DUB with nanomolar half-maximal inhibitory concentration (IC50) values. Structural analysis of the macrocycles bound to USP7 revealed a variety of binding modes and identified inhibition hotspots on the enzyme that mirror those used by small-molecule inhibitors. Together, these data suggest that initial macrocyclic hits could serve as pivotal tools in developing USP-specific inhibitors and probing USP biology.

Crimean-Congo hemorrhagic fever (CCHF) is a viral tick-borne disease with fatality rates of up to 30 %. Currently, there are no vaccines or specific antivirals available. The genome of the CCHF virus (CCHFV) encodes an ovarian tumor (OTU) protease with a deubiquitinating activity that is responsible for the evasion of the innate immune response. Therefore, the inhibition of the OTU protease could provide a strategy for the treatment of CCHFV infections. In this study, we screened for small-molecule inhibitors of CCHFV OTU using a fluorescent ubiquitin rhodamine 110 assay. We identified and validated a 2-aminothiazole hit compound (IC50 = 42.3 μM) followed by structure-activity relationships (SAR) studies resulting in a new inhibitor of the CCHFV OTU protease. The most active derivative is a competitive CCHFV OTU inhibitor with an IC50 value of 10.7 μM. Selectivity studies revealed that the ubiquitin-specific peptidase 7 (USP7), ubiquitin C-terminal hydrolase 5 (UCHL5), OTU deubiquitinase 1 (OTUD1), and Cezanne are also inhibited by this newly developed inhibitor indicating binding to conserved regions of the ubiquitin-binding site within the deubiquitinase superfamilies. Molecular docking into the active site of CCHFV OTU proposes starting points for further structural modifications to improve activity and selectivity. These structure-activity relationships are the first to our knowledge to be reported for the CCHFV OTU protease and will help guide further drug discovery efforts.

FDA-approved KRASG12C inhibitors, like Sotorasib, target G12C-mutated KRAS in NSCLC. However, issues with insensitivity and drug resistance have emerged, requiring the development of new combination therapies to overcome these limitations. USP47 has been identified as a regulator of cancer-related signaling pathways such as Wnt, Hippo, and p53. However, its role in the KRAS signaling pathway remains largely unexplored and USP47 inhibitors are less developed than those targeting its homolog, USP7. Here, we identify USP47 as a novel therapeutic target in KRASG12C-mutated NSCLC and report K-552, a selective USP47 inhibitor, as a potential treatment strategy. We demonstrate that USP47 stabilizes c-Myc by preventing its proteasomal degradation through deubiquitination, thereby promoting NSCLC cell proliferation. Additionally, the compound K-552, a USP47 inhibitor identified through virtual screening, effectively destabilizes c-Myc and inhibits KRASG12C-mutated NSCLC cell proliferation. Furthermore, USP47 inhibition-either by siRNA knockdown or K-552 treatment-enhances the efficacy of Sotorasib in vitro and in vivo. Together, our findings establish USP47 as a promising therapeutic target in KRASG12C-mutated NSCLC and introduce K-552 as a USP47 inhibitor with potential for combination therapy with KRASG12C inhibitors.

Ubiquitin-specific peptidase 13 (USP13) has emerged as a key regulator of proteins critical to the hallmarks of cancer, playing an essential role in cellular regulation. This deubiquitinating enzyme, often overexpressed in malignancies, wields its molecular scissors precisely, snipping ubiquitin tags to rescue oncoproteins from degradation. Our review highlights the dual role of USP13 in cancer biology: while it predominantly fuels tumor growth and metastasis, USP13 occasionally functions as a tumor suppressor. USP13 is as a formidable factor in cancer therapy, fortifying tumors against an arsenal of treatments. It bolsters DNA repair mechanisms, ignites prosurvival autophagy, and even reprograms cell lineages to evade targeted therapies. However, USP13 is also a promising target in the treatment of cancer. We highlight burgeoning strategies to neutralize USP13, from small molecule inhibitors to innovative protein degraders, which may disarm cancer resistance mechanisms. We also offer suggestions for future USP13 research, emphasizing the need for structural insights and more potent inhibitors. This review highlights the critical role of USP13 in cancer and underscores its potential as a therapeutic target for advancing cancer treatment.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common chronic liver disease worldwide, with a prevalence as high as 32.4%. MASLD encompasses a spectrum of liver pathologies, ranging from steatosis to metabolic dysfunction-associated steatohepatitis (MASH), fibrosis, and, in some cases, progression to end-stage liver disease (cirrhosis and hepatocellular carcinoma). A comprehensive understanding of the pathogenesis of this highly prevalent liver disease may facilitate the identification of novel targets for the development of improved therapies. E3 ubiquitin ligases and deubiquitinases (DUBs) are key regulatory components of the ubiquitin‒proteasome system (UPS), which plays a pivotal role in maintaining intracellular protein homeostasis. Emerging evidence implicates that aberrant expression of E3 ligases and DUBs is involved in the progression of MASLD. Here, we review abnormalities in E3 ligases and DUBs by (1) discussing their targets, mechanisms, and functions in MASLD; (2) summarizing pharmacological interventions targeting these enzymes in preclinical and clinical studies; and (3) addressing challenges and future therapeutic strategies. This review synthesizes current evidence to highlight the development of novel therapeutic strategies based on the UPS for MASLD and progressive liver disease.

The ubiquitin-specific protease 7 (USP7), also known as herpes-associated ubiquitin-specific protease (HAUSP) is an interesting target due to its role in the tumor suppressor p53 pathway. In recent years targeted covalent inhibitors have gained significant importance in pharmaceutical research. Thus, we have investigated a small library of 129 ligands bearing different types of covalent reactive groups ("warheads") from various kinase drug discovery projects for their reactivity towards the catalytic cysteine of USP7, as well as their influence on its melting temperature. These compounds mainly encompassed α,β-unsaturated amides specifically acrylamides, SNAr reacting compounds, aryl fluorosulfates and sulfonyl fluorides.

We analyzed an array of 129 electrophilic compounds which had been designed as covalent kinase inhibitors in a DSF-based (differential scanning fluorimetry) screen against USP7. The hits were evaluated for their ability to cause similar thermal shifts for a CYS-deficient USP7 control mutant (USP7asoc), where only the catalytic Cys223 was retained. Additionally, covalent binding was evaluated by intact protein mass spectrometry (MS).

The DSF screen revealed that, predominantly 18 of the 129 tested compounds decreased the melting temperature of USP7 and its mutant USP7asoc. For 8 of these, the hypothesized covalent binding mode was corroborated with native and mutant USP7 by intact protein MS. Nearly all identified hits have a covalent warhead that reacts via nucleophilic aromatic substitution (SNAr).

The screening and evaluation of the kinase library revealed several initial hits of interest. Seven SNAr warheads and one acrylamide warhead compound covalently modified the target protein (USP7) and showed clear shifts in the melting temperatures ranging from -6.0 °C to +1.7 °C.

Deubiquitylases (DUBs) are crucial in cell signaling and are often regulated by interactions within protein complexes. The BRCC36 isopeptidase complex (BRISC) regulates inflammatory signaling by cleaving K63-linked polyubiquitin chains on type I interferon receptors (IFNAR1). As a Zn2+-dependent JAMM/MPN (JAB1, MOV34, MPR1, Pad1 N-terminal) DUB, BRCC36 is challenging to target with selective inhibitors. Here, we discover first-in-class inhibitors, termed BRISC molecular glues (BLUEs), which stabilize a 16-subunit human BRISC dimer in an autoinhibited conformation, blocking active sites and interactions with the targeting subunit, serine hydroxymethyltransferase 2. This unique mode of action results in selective inhibition of BRISC over related complexes with the same catalytic subunit, splice variants and other JAMM/MPN DUBs. BLUE treatment reduced interferon-stimulated gene expression in cells containing wild-type BRISC and this effect was abolished when using structure-guided, inhibitor-resistant BRISC mutants. Additionally, BLUEs increase IFNAR1 ubiquitylation and decrease IFNAR1 surface levels, offering a potential strategy to mitigate type I interferon-mediated diseases. Our approach also provides a template for designing selective inhibitors of large protein complexes by promoting rather than blocking protein-protein interactions.

Ubiquitin-specific protease 7 (USP7) is a deubiquitylase essential for cell homeostasis, DNA repair, and regulation of both tumor suppressors and oncogenes. Inactivating USP7 mutations have been associated with Hao-Fountain Syndrome (HAFOUS), a rare neurodevelopmental disorder. Although a range of USP7 inhibitors have been developed over the last decade, in the context of HAFOUS as well as oncogene regulation, USP7 activators may represent a more relevant approach. To address this challenge, we report the discovery and characterization of a small-molecule activator of USP7 called MS-8. We showed that MS-8 activates USP7 by engaging the allosteric C-terminal binding pocket of USP7, thus mimicking the allosteric autoactivation by the USP7 C-terminal tail. We observed that MS-8 engages and activates mutant USP7 in a cellular context, impacting downstream proteins. Taken together, our study provides validation of the USP7 activator that paves the way towards novel activation-driven USP7 pharmacology.

Anticipating and addressing resistance is essential for maximizing the potential of an oncology target and effectively addressing clinical needs. In this study, we aimed to proactively outline the resistance mechanisms of USP7 inhibitors. We discovered a key treatment-emergent heterozygous mutation (V517F) in USP7 in the binding pocket of compounds as the primary cause of resistance to the USP7 inhibitor USP7-797. Our structural analysis, supported by AlphaFold2 predictions, indicates that the V517F mutation altered the conformation of the compound binding pocket, causing steric hindrance and reducing the affinity between USP7 and its inhibitors. Consistent with these predictions, the affinity between V517F mutant and USP7 inhibitors was found to reduce significantly. Conversely, substitutions at position V517 with smaller side chains, such as V517G, V517A, and V517I, do not significantly impact binding affinity. In contrast, replacement with the bulkier side chain V517Y leads to reduced binding affinity and diminished inhibitor efficacy. Furthermore, the engineered cell lines harboring the V517F mutation exhibited substantial resistance to USP7 inhibition. These data provide rationales for patient selection and the development of next-generation USP7 inhibitors designed to overcome treatment-emergent mutations.

Pathogenic variants in the ubiquitin-specific protease 7 (USP7) gene cause a neurodevelopmental disorder called Hao-Fountain syndrome. However, it remains unclear which of USP7's pleiotropic functions are relevant for neurodevelopment. Here, we present a combination of quantitative proteomics, transcriptomics, and epigenomics to define the USP7 regulatory circuitry during neuronal differentiation. USP7 activity is required for the transcriptional programs that direct both the differentiation of embryonic stem cells into neural stem cells and the neuronal differentiation of SH-SY5Y neuroblastoma cells. USP7 controls the dosage of the Polycomb monubiquitylated histone H2A lysine 119 (H2AK119ub1) ubiquitin ligase complexes ncPRC1.1 and ncPRC1.6. Loss-of-function experiments revealed that BCOR-ncPRC1.1, but not ncPRC1.6, is a key effector of USP7 during neuronal differentiation. Indeed, BCOR-ncPRC1.1 mediates a major portion of USP7-dependent gene regulation during this process. Besides providing a detailed map of the USP7 regulome during neurodifferentiation, our results suggest that USP7- and ncPRC1.1-associated neurodevelopmental disorders involve dysregulation of a shared epigenetic network.

Persistent infection with high-risk (HR) human papillomaviruses (HPVs) is responsible for approximately 5% of cancer cases worldwide, including a growing number of oropharyngeal and anogenital cancers. The major HPV oncoproteins, E6 and E7, act together to manipulate cellular pathways involved in the regulation of proliferation, the cell cycle and cell survival, ultimately driving malignant transformation. Protein ubiquitination and the ubiquitin proteasome system (UPS) is often deregulated upon viral infection and in oncogenesis. HPV E6 and E7 interact with and disrupt multiple components of the ubiquitination machinery to promote viral persistence, which can also result in cellular transformation and the formation of tumours. This review highlights the ways in which HPV manipulates protein ubiquitination and the ubiquitin-like protein pathways and how this contributes to tumour development. Furthermore, we discuss how understanding the interactions between HPV and the protein ubiquitination could lead to novel therapeutic targets that are of urgent need in HPV+ carcinomas.

Ubiquitin-specific protease 7 (USP7) is a promising prognostic and druggable target for cancer therapy. Inhibition of USP7 can activate the MDM2-P53 signaling pathway, thereby promoting cancer cell apoptosis. This study based on watvina molecular docking of virtual screening method and biological evaluation found the new USP7 inhibitors targeting catalytic active site. Three hits were screened from 3760 natural products and validated as USP7 inhibitors by enzymatic and kinetic assays. The IC50 values of scutellarein (Scu), semethylzeylastera (DML) and salvianolic acid C (SAC) were 3.017, 6.865 and 8.495 μM, respectively. Further, we reported that the hits could downregulate MDM2 and activate p53 signal pathway in HCT116 cells. Molecular dynamics simulation was used to investigate the binding mechanism of USP7 to Scu, the compound with the best performance, which formed stable contact with Val296, Gln297, Phe409, Tyr465 and Tyr514. These interactions are essential for maintaining the biological activity of Scu. Three natural products are suitable as lead compounds for the development of novel USP7 inhibitors, especially anti-colon cancer drugs.

The MDM2/MDMX-p53 circuitry is essential for controlling the development, apoptosis, immune response, angiogenesis, senescence, cell cycle progression, and proliferation of cancer cells. Research has demonstrated that USP7 exerts strong control over p53, MDM2, and MDMX stability, with multiple mediator proteins influencing the USP7-p53-MDM2/MDMX axis to modify p53 expression level and function. In cases where p53 is of the wild type (Wt-p53) in tumors, inhibiting USP7 promotes the degradation of MDM2/MDMX, leading to the activation of p53 signaling. This, in turn, results in cell cycle arrest and apoptosis. Hence, targeting USP7 presents a promising avenue for cancer therapy. Targeting USP7 in tumors that harbor mutant p53 (Mut-p53) is unlikely and remains largely unexplored due to the existence of numerous USP7 targets that function independently of p53. Considering that Mut-p53 exhibits resistance to degradation by MDM2 and other E3 ligases and also shares the same signaling pathways as Wt-p53, it is reasonable to suggest that USP7 may play a role in stabilizing Mut-p53. However, there is still much to be done in this area. If the hypothesis is correct, USP7 may be a potent target in cancers containing both Wt-p53 and Mut-p53.

Mutation or deletion of the deubiquitinase USP7 causes Hao-Fountain syndrome (HAFOUS), which is characterized by speech delay, intellectual disability, and aggressive behavior and highlights important unknown roles of USP7 in the nervous system. Here, we conditionally delete USP7 in glutamatergic neurons in the mouse forebrain, triggering disease-relevant phenotypes, including sensorimotor deficits, impaired cognition, and aggressive behavior. Although USP7 deletion induces p53-dependent neuronal apoptosis, most behavioral abnormalities in USP7 conditional knockout mice persist following p53 loss. Strikingly, USP7 deletion perturbs the synaptic proteome and dendritic spinogenesis independent of p53. Integrated proteomics and biochemical analyses identify the RNA splicing factor Ppil4 as a key substrate of USP7. Ppil4 knockdown phenocopies the effect of USP7 loss on dendritic spines. Accordingly, USP7 loss disrupts splicing of synaptic genes. These findings reveal that USP7-Ppil4 signaling regulates neuronal connectivity in the developing brain with implications for our understanding of HAFOUS pathogenesis and other neurodevelopmental disorders.

The N-Myc transcription factor, encoded by MYCN, is a mechanistically validated, yet challenging, target for neuroblastoma (NB) therapy development. In normal neuronal progenitors, N-Myc undergoes rapid degradation, while, in MYCN-amplified NB cells, Aurora kinase A (Aurora-A) binds to and stabilizes N-Myc, resulting in elevated protein levels. Here, we demonstrate that targeted protein degradation of Aurora-A decreases N-Myc levels. A potent Aurora-A degrader, HLB-0532259 (compound 4), was developed from an Aurora-A-binding ligand that engages the Aurora-A/N-Myc complex. HLB-0532259 promotes the degradation of Aurora-A, which elicits concomitant N-Myc degradation, with nanomolar potency and excellent selectivity. HLB-0532259 surpasses the cellular efficacy of established allosteric Aurora-A inhibitors, exhibits favorable pharmacokinetic properties, and elicits tumor reduction in a murine xenograft NB model. This study broadly delineates a strategy for targeting "undruggable" proteins that are reliant on accessory proteins for cellular stabilization.

Inhibition of the mitochondrial deubiquitinating (DUB) enzyme USP30 is neuroprotective and presents therapeutic opportunities for the treatment of idiopathic Parkinson's disease and mitophagy-related disorders. We integrated structural and quantitative proteomics with biochemical assays to decipher the mode of action of covalent USP30 inhibition by a small-molecule containing a cyanopyrrolidine reactive group, USP30-I-1. The inhibitor demonstrated high potency and selectivity for endogenous USP30 in neuroblastoma cells. Enzyme kinetics and hydrogen-deuterium eXchange mass spectrometry indicated that the inhibitor binds tightly to regions surrounding the USP30 catalytic cysteine and positions itself to form a binding pocket along the thumb and palm domains of the protein, thereby interfering its interaction with ubiquitin substrates. A comparison to a noncovalent USP30 inhibitor containing a benzosulfonamide scaffold revealed a slightly different binding mode closer to the active site Cys77, which may provide the molecular basis for improved selectivity toward USP30 against other members of the DUB enzyme family. Our results highlight advantages in developing covalent inhibitors, such as USP30-I-1, for targeting USP30 as treatment of disorders with impaired mitophagy.

Cancer management has traditionally depended on chemotherapy as the mainstay of treatment; however, recent advancements in targeted therapies and immunotherapies have offered new options. Ubiquitin-specific proteases (USPs) have emerged as promising therapeutic targets in cancer treatment due to their crucial roles in regulating protein homeostasis and various essential cellular processes. This review covers the following: (1) the structural and functional characteristics of USPs, highlighting their involvement in key cancer-related pathways, and (2) the discovery, chemical structures, mechanisms of action, and potential clinical implications of USP inhibitors in cancer therapy. Particular attention is given to the role of USP inhibitors in enhancing cancer immunotherapy, e.g., modulation of the tumor microenvironment, effect on regulatory T cell function, and influence on immune checkpoint pathways. Furthermore, this review summarizes the current progress and challenges of clinical trials involving USP inhibitors as cancer therapy. We also discuss the complexities of achieving target selectivity, the ongoing efforts to develop more specific and potent USP inhibitors, and the potential of USP inhibitors to overcome drug resistance and synergize with existing cancer treatments. We finally provide a perspective on future directions in targeting USPs, including the potential for personalized medicine based on specific gene mutations, underscoring their significant potential for enhancing cancer treatment. By elucidating their mechanisms of action, clinical progress, and potential future applications, we hope that this review could serve as a useful resource for both basic scientists and clinicians in the field of cancer therapeutics.

Cancer cells often display centrosome amplification, requiring the kinesin KIFC1/HSET for centrosome clustering to prevent multipolar spindles and cell death. In parallel siRNA screens of deubiquitinase enzymes, we identify OTUD6B as a positive regulator of KIFC1 expression that is required for centrosome clustering in triple-negative breast cancer (TNBC) cells. OTUD6B can localise to centrosomes and the mitotic spindle and interacts with KIFC1. In OTUD6B-deficient cells, we see increased KIFC1 polyubiquitination and premature KIFC1 degradation during mitosis. Depletion of OTUD6B increases multipolar spindles without inducing centrosome amplification. Phenotypic rescue is dependent on OTUD6B catalytic activity and evident upon KIFC1 overexpression. OTUD6B is commonly overexpressed in breast cancer, correlating with KIFC1 protein expression and worse patient survival. TNBC cells with centrosome amplification, but not normal breast epithelial cells, depend on OTUD6B to proliferate. Indeed CRISPR-Cas9 editing results in only OTUD6B-/+ TNBC cells which fail to divide and die. As a deubiquitinase that supports KIFC1 expression, allowing pseudo-bipolar cell division and survival of cancer cells with centrosome amplification, OTUD6B has potential as a novel target for cancer-specific therapies.

Regeneration plays a key role in energy recycling and homeostasis maintenance. Planarians, as ideal model animals for studying regeneration, stem cell proliferation, and apoptosis, have the strong regenerative abilities. Considerable evidence suggests that ubiquitin plays an important role in maintaining homeostasis and regulating regeneration, but the function of Ubiquitin specific proteases 7 (Usp7) on regeneration in planarians remains elusive.

We identified an evolutionarily conserved gene, Usp7, and utilized RNA interference (RNAi), Quantitative real-time PCR (qRT-PCR), Whole-mount immunofluorescence, Tunnel, Whole-mount in situ hybridization (WISH), and western blotting to detect the function of Usp7 during the planarian regeneration.

In this study, we found that the regenerative trunk fragments in the Usp7 RNAi worms could not regenerate missing tails; meanwhile, the level of cell proliferation was decreased, while cell apoptosis was increased. Furthermore, the expression of Islet was inhibited in the Usp7 RNAi worms during planarian regeneration. The hybridization signal of wnt1/P-1 exhibited the dot-like pattern at the posterior of the regenerating planarians after Usp7 RNAi at regenerative 1 day (R 1 d). However, the concentrated expression pattern wnt1/P-1 dramatically declined at regenerative 3 days (R 3 d) and disappeared at regenerative 7 days (R 7 d). In addition, activating the Wnt pathway partially rescued regenerative defects induced by inhibition of Usp7.

Collectively, Usp7 is necessary for tissue regeneration and tail blastema formation partially by regulating the cell proliferation and apoptosis during planarian regeneration. It could also promote the posterior polarity reconstruction of the regenerative planarians via the Islet/Wnt1 axis.

Adenocarcinoma of the esophagogastric junction (AEG) has received widespread attention because of its increasing incidence. However, the molecular mechanism underlying tumor progression remains unclear. Here, we report that the downregulation of ubiquitin-specific peptidase 49 (USP49) promotes ferroptosis in OE33 and OE19 cells, thereby inhibiting cell proliferation in vitro and in vivo, whereas the overexpression of USP49 had the opposite effect. In addition, USP49 downregulation promoted AEG cell radiotherapy sensitivity. Moreover, overexpression of Glutathione PeroXidase 4 reversed the ferroptosis and proliferation inhibition induced by USP49 knockdown. Mechanistically, USP49 deubiquitinates and stabilizes Shc SH2-domain-binding protein 1, subsequently facilitating the entry of β-catenin into the nucleus to enhance Glutathione PeroXidase 4 transcriptional expression. Finally, high USP49 expression was correlated with shorter overall survival in patients with AEG. In summary, our findings identify USP49 as a novel regulator of ferroptosis in AEG cells, indicating that USP49 may be a potential therapeutic target in AEG.

The immune checkpoint regulator CTLA4 is an unusually short-lived membrane protein. Here, we show that its lysosomal degradation is dependent on ubiquitylation at lysine residues 203 and 213. Inhibition of the v-ATPase partially restores CTLA4 levels following cycloheximide treatment, but also reveals a fraction that is secreted in exosomes. The endosomal deubiquitylase, USP8, interacts with CTLA4, and its loss enhances CTLA4 ubiquitylation in cancer cells, mouse CD4+ T cells, and cancer cell-derived exosomes. Depletion of the USP8 adapter protein, HD-PTP, but not ESCRT-0 recapitulates this cellular phenotype but shows distinct properties vis-à-vis exosome incorporation. Re-expression of wild-type USP8, but neither a catalytically inactive nor a localization-compromised ΔMIT domain mutant can rescue delayed degradation of CTLA4 or counteract its accumulation in clustered endosomes. UbiCRest analysis of CTLA4-associated ubiquitin chain linkages identifies a complex mixture of conventional Lys63- and more unusual Lys27- and Lys29-linked polyubiquitin chains that may underly the rapidity of protein turnover.

Epstein-Barr virus (EBV) is a ubiquitous human ɣ-herpesvirus implicated in various malignancies, including Burkitt's lymphoma and gastric carcinomas. In most EBV-associated cancers, the viral genome is maintained as an extrachromosomal episome by the EBV nuclear antigen-1 (EBNA1). EBNA1 is considered to be a highly stable protein that interacts with the ubiquitin-specific protease 7 (USP7). Here, we show that pharmacological inhibitors and small interfering RNA (siRNA) targeting USP7 reduce EBNA1 protein levels in a proteosome-dependent manner. Proteomic analysis revealed that USP7 inhibitor GNE6776 altered the EBNA1 protein interactome, including disrupting USP7 association with EBNA1. GNE6776 also inhibited EBNA1 binding to EBV oriP DNA and reduced viral episome copy number. Transcriptomic studies revealed that USP7 inhibition affected chromosome segregation and mitotic cell division pathways in EBV+ cells. Finally, we show that GNE6776 selectively inhibited EBV+ gastric and lymphoid cell proliferation in cell culture and slowed EBV+ tumor growth in mouse xenograft models. These findings suggest that USP7 inhibitors perturb EBNA1 stability and function and may be exploited to treat EBV latent infection and tumorigenesis.

Kidney renal clear cell carcinoma (KIRC) is recognized as an immunogenic tumor, and immunotherapy is incorporated into its treatment landscape for decades. The acquisition of a tumor mesenchymal phenotype through epithelial-to-mesenchymal transition (EMT) is associated with immune evasion and can contribute to immunotherapy resistance. Here, the involvement of STAM Binding Protein Like 1 (STAMBPL1) is reported in the development of mesenchymal and immune evasion phenotypes in KIRC cells. Mechanistically, STAMBPL1 elevated protein abundance and surface accumulation of TAM Receptor AXL through diminishing the TRIM21-mediated K63-linked ubiquitination and subsequent lysosomal degradation of AXL, thereby enhancing the expression of mesenchymal genes while suppressing chemokines CXCL9/10 and HLA/B/C. In addition, STAMBPL1 enhanced PD-L1 transcription via facilitating nuclear translocation of p65, and knockdown (KD) of STAMBPL1 augmented antitumor effects of PD-1 blockade. Furthermore, STAMBPL1 silencing and the tyrosine kinase inhibitor (TKI) sunitinib also exhibited a synergistic effect on the suppression of KIRC. Collectively, targeting the STAMBPL1/TRIM21/AXL axis can decrease mesenchymal phenotype and potentiate anti-tumor efficacy of cancer therapy.

The typical pathological feature of pancreatic ductal adenocarcinoma (PDAC) is a significant increase in stromal reaction, leading to a hypoxic and poorly vascularized tumor microenvironment. Tumor cells undergo metabolic reprogramming, such as the Warburg effect, yet the underlying mechanisms are not fully understood.

Interference and overexpression experiments were conducted to analyze the in vivo and in vitro effects of USP7 on the growth and glycolysis of tumor cells. Small-molecule inhibitors of USP7 and transgenic mouse models of PDAC were employed to assess the consequences of targeting USP7 in PDAC. The molecular mechanism underlying USP7-induced c-Myc stabilization was determined by RNA sequencing, co-IP and western blot analyses.

USP7 is abnormally overexpressed in PDAC and predicts a poor prognosis. Hypoxia and extracellular matrix stiffness can induce USP7 expression in PDAC cells. Genetic silencing of USP7 inhibits the glycolytic phenotypes in PDAC cells, while its overexpression has the opposite effect, as demonstrated by glucose uptake, lactate production, and extracellular acidification rate. Importantly, USP7 promotes PDAC tumor growth in a glycolysis-dependent manner. The small-molecule inhibitor P5091 targeting USP7 effectively suppresses the Warburg effect and cell growth in PDAC. In a transgenic mouse model of PDAC, named KPC, P5091 effectively blocks tumor progression. Mechanistically, USP7 interacts with c-Myc, enhancing its stability and expression, which in turn upregulates expression of glycolysis-related genes.

This study sheds light on the molecular mechanisms underlying the Warburg effect in PDAC and unveils USP7 as a potential therapeutic target for improving PDAC treatment.

Hypoxia Inducible transcription Factors (HIFs) are central to the metazoan oxygen-sensing response. Under low oxygen conditions (hypoxia), HIFs are stabilised and govern an adaptive transcriptional programme to cope with prolonged oxygen starvation. However, when oxygen is present, HIFs are continuously degraded by the proteasome in a process involving prolyl hydroxylation and subsequent ubiquitination by the Von Hippel Lindau (VHL) E3 ligase. The essential nature of VHL in the HIF response is well established but the role of other enzymes involved in ubiquitination is less clear. Deubiquitinating enzymes (DUBs) counteract ubiquitination and provide an important regulatory aspect to many signalling pathways involving ubiquitination. In this review, we look at the complex network of ubiquitination and deubiquitination in controlling HIF signalling in normal and low oxygen tensions. We discuss the relative importance of DUBs in opposing VHL, and explore roles of DUBs more broadly in hypoxia, in both VHL and HIF independent contexts. We also consider the catalytic and non-catalytic roles of DUBs, and elaborate on the potential benefits and challenges of inhibiting these enzymes for therapeutic use.

OTUD7B, a member of the ovarian tumor (OTU) protein superfamily, functions as a deubiquitinating enzyme and is associated with various biological processes and disease conditions, including tumors. In this study, we aimed to explore the expression patterns, prognostic significance, and the functional roles and underlying mechanisms of OTUD7B in gastric cancer (GC).

Using a blend of bioinformatics, clinical case reviews, and molecular experiments, we evaluated the expression of OTUD7B in GC at both mRNA and protein levels. We examined the relationship between OTUD7B expression and clinicopathological characteristics of GC patients. Additionally, in vitro assays were utilized to assess the effects of OTUD7B on the migratory and invasive capabilities of GC cells. RNA sequencing analysis was conducted to identify critical genes and pathways linked to OTUD7B in GC.

OTUD7B was found to be significantly overexpressed in GC, both at mRNA and protein levels. Higher levels of OTUD7B were positively associated with advanced tumor TNM stage, higher histological grade, and presence of lymph/vein invasion. These correlations were indicative of poorer overall survival (OS) and disease-free survival (DFS) in GC patients. In vitro assays revealed that genetic knockout of OTUD7B markedly reduced the migration and invasion of GC cells, while overexpression of OTUD7B led to enhanced cellular migration and invasion. Furthermore, RNA sequencing and bioinformatic analyses indicated that the absence of OTUD7B suppressed signaling pathways related to cancer progression, metastasis, and metabolism. Mechanistically, OTUD7B likely promotes GC metastasis through the WNT signaling pathway, specifically targeting β-catenin.

OTUD7B serves as a novel marker for poor prognosis in GC and actively promotes tumor metastasis. Our results shed light on the signaling pathways regulated by OTUD7B and highlight potential targets for therapeutic intervention.

Human health is endangered by the occurrence and progression of urological cancers, including renal cell carcinoma, prostate cancer and bladder cancer, which are usually associated with the activation of oncogenic factors and inhibition of cancer suppressors. The primary mechanism for protein breakdown in cells is the ubiquitin-proteasome system, whilst deubiquitinases contribute to the reversal of this process. However, both are important for protein homeostasis. Deubiquitination may also be involved in the control of the cell cycle, proliferation and apoptosis, and dysregulated deubiquitination is associated with the malignant transformation, invasion and metastasis of urologic malignancies. Therefore, a comprehensive summary of the mechanisms underlying deubiquitination in urological cancers may provide novel strategies and insights for diagnosis and treatment. The present review aimed to methodically clarify the role of deubiquitinating enzymes in urinary system cancers as well as their prospective application prospects for clinical treatment.

Metastasis is a major contributor to mortality in patients with esophageal squamous cell carcinoma (ESCC); effective treatment is currently lacking. Erianin, a bioactive ingredient of traditional Chinese medicine, Dendrobium chrysotoxum, has anti-tumor activity against multiple human tumors. However, the effect and associated underlying mechanism of Erianin on ESCC antimetastasis remain unclear.

To investigate the anti-metastatic properties of Erianin in ESCC both in vitro and in vivo and associated molecular mechanisms.

Wound healing assay, Transwell assay, CCK-8 assay, immunohistochemistry, and lung metastasis mouse model were carried out to examine ESCC cell migration and viability in vitro and in vivo. Drug affinity responsive target stability (DARTS), cellular thermal migration assay (CETSA), molecular docking, and Surface plasmon resonance (SPR) assay were used to confirm Erianin binding to ovarian tumor ubiquitin aldehyde-binding protein 1 (OTUB1) protein. Protein stability assay, cell transfection, and western blotting were used to confirm Erianin-mediated degradation of OTUB1 and Snail via the ubiquitin-proteasome pathway. qRT-PCR and western blotting were used to assess OTUB1expression in ESCC tissues.

Erianin suppressed the migration/invasion of ESCC cells without modulating cell viability in vitro and in vivo, bound to OTUB1 through DARTS, CETSA, and molecular docking, and SPR assay, and enhanced OTUB1 degradation via the ubiquitin-proteasome system. Moreover, Erianin inhibited the ESCC epithelial-mesenchymal transition by enhancing the ubiquitination and degradation of Snail via targeting OTUB1.

Erianin inhibited ESCC metastasis through ubiquitination and degradation of Snail via targeting OTUB1. Our findings suggest Erianin as a novel OTUB1 inhibitor for preventing ESCC metastasis.

Alzheimer's disease (AD) is a prevalent neurodegenerative disorder characterized by the accumulation of amyloid β protein (Aβ) and the hyper-phosphorylation of the microtubule-associated protein Tau. The ubiquitin-proteasome system (UPS) plays a pivotal role in determining the fate of proteins, and its dysregulation can contribute to the buildup of Aβ and Tau. Deubiquitinating enzymes (DUBs), working in conjunction with activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3), actively maintain the delicate balance of protein homeostasis. DUBs specifically remove ubiquitin tags from proteins marked for degradation, thereby averting their proteasomal breakdown. Several DUBs have demonstrated their capacity to regulate the levels of Aβ and Tau by modulating their degree of ubiquitination, underscoring their potential as therapeutic targets for AD. In this context, we present a comprehensive review of AD-associated DUBs and elucidate their physiological roles. Moreover, we delve into the current advancements in developing inhibitors targeting these DUBs, including the determination of cocrystal structures with their respective targets. Additionally, we assess the therapeutic efficacy of these inhibitors in AD, aiming to establish a theoretical foundation for future AD treatments.

Deubiquitylases (DUBs) are crucial in cell signalling and are often regulated by interactions within protein complexes. The BRCC36 isopeptidase complex (BRISC) regulates inflammatory signalling by cleaving K63-linked polyubiquitin chains on Type I interferon receptors (IFNAR1). As a Zn2+-dependent JAMM/MPN DUB, BRCC36 is challenging to target with selective inhibitors. We discovered first-in-class inhibitors, termed BRISC molecular glues (BLUEs), which stabilise a 16-subunit BRISC dimer in an autoinhibited conformation, blocking active sites and interactions with the targeting subunit SHMT2. This unique mode of action results in selective inhibition of BRISC over related complexes with the same catalytic subunit, splice variants and other JAMM/MPN DUBs. BLUE treatment reduced interferon-stimulated gene expression in cells containing wild type BRISC, and this effect was absent when using structure-guided, inhibitor-resistant BRISC mutants. Additionally, BLUEs increase IFNAR1 ubiquitylation and decrease IFNAR1 surface levels, offering a potential new strategy to mitigate Type I interferon-mediated diseases. Our approach also provides a template for designing selective inhibitors of large protein complexes by promoting, rather than blocking, protein-protein interactions.

Inhibition of ubiquitin-specific protease 7, USP7, has been proposed as a mechanism to affect many disease processes, primarily those implicated in oncology. The bound crystal structure of a published high-throughput screening hit with low-micromolar affinity for USP7 identified three regions of the compound for structure-guided optimization. Replacing one side of the compound with different aromatic moieties gave little improvement in affinity, and the central piperidine could not be improved. However, the binding site for the other side of the compound was poorly defined in the crystal structure, which suggested a wide variety of synthetically accessible options for optimization. These were assessed by screening reaction mixtures that introduced different substituents to this other side. Subsequent optimization led to a compound with low-nanomolar affinity for USP7, which showed target engagement in tumors, was tolerated in mice, and showed efficacy in xenograft models.