{"gene":"USP25","run_date":"2026-06-11T09:02:06","timeline":{"discoveries":[{"year":2012,"finding":"USP25 deubiquitinates TRAF5 and TRAF6 by removing Lys63-linked ubiquitin chains, negatively regulating IL-17-mediated signaling. Upon IL-17 stimulation, USP25 associates with TRAF5 and TRAF6 adaptors, opposing Act1-mediated K63-ubiquitination of these adaptors.","method":"Co-immunoprecipitation, overexpression/knockdown assays, Usp25-/- mouse model, phosphorylation assays, mRNA stability assays","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, KO mouse phenotype, multiple orthogonal methods, replicated across in vitro and in vivo systems","pmids":["23042150"],"is_preprint":false},{"year":2015,"finding":"USP25 stabilizes TRAF3 and TRAF6 after viral infection by protecting them from proteasome-dependent or independent degradation, respectively, thereby promoting IRF3 and NF-κB activation and type I IFN and cytokine production.","method":"Co-immunoprecipitation, Usp25-/- mouse model, reconstitution of TRAF3/TRAF6 in KO MEFs, viral infection assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO mouse model, reconstitution rescue experiment, Co-IP, multiple orthogonal methods","pmids":["26305951"],"is_preprint":false},{"year":2023,"finding":"USP25 binds SERCA2a directly via its USP domain and removes K48-linked ubiquitin chains at the catalytic cysteine C178, preventing proteasomal degradation of SERCA2a and maintaining calcium handling in cardiomyocytes.","method":"LC-MS/MS, Co-IP, USP25 mutant constructs (C178A), AAV9-mediated overexpression in KO mice, Western blotting","journal":"Circulation research","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — active-site mutagenesis, Co-IP with MS substrate identification, in vivo rescue with AAV9, multiple orthogonal methods","pmids":["36722348"],"is_preprint":false},{"year":2022,"finding":"USP25 deubiquitinates and stabilizes the HIF-1α transcription factor, promoting HIF-1α transcriptional activity and glycolysis in pancreatic ductal adenocarcinoma under hypoxic conditions.","method":"Activity-based proteomics, loss-of-function genetic screen in PDAC organoids, genetic and pharmacological USP25 inhibition, patient-derived xenografts","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (proteomics, organoids, xenografts, genetic KO), replicated in multiple model systems","pmids":["35440539"],"is_preprint":false},{"year":2017,"finding":"USP25 directly interacts with tankyrases (TNKS1/2) to promote their deubiquitination and stabilization, thereby activating Wnt/β-catenin signaling. The interaction interface was characterized by X-ray crystal structure determination.","method":"Co-immunoprecipitation, ubiquitination assays, USP25-KO cell lines, X-ray crystal structure of TNKS1-USP25 complex","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure, Co-IP, KO functional validation, multiple orthogonal methods in one study","pmids":["28619731"],"is_preprint":false},{"year":2019,"finding":"USP25 forms autoinhibited tetramers where a USP25-specific coiled-coil insertion sequence blocks ubiquitin binding, while the catalytically active form is a dimer. Modulating the dimer-tetramer equilibrium affects substrate stabilization in cells.","method":"Cryo-EM/X-ray crystallography, biochemical activity assays with dimer/tetramer/truncation constructs, cellular oligomeric state confirmation, substrate stabilization assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structures of both USP25 and USP28, biochemical kinetic assays, cellular validation, two independent concurrent studies","pmids":["30926242","30926243"],"is_preprint":false},{"year":2019,"finding":"USP25 forms an autoinhibited tetramer via contacts between a coiled-coil insertion domain and the ubiquitin-binding pocket of the catalytic domain. A constitutively dimeric USP25 construct shows higher tankyrase-stabilizing activity in cells, confirming biological relevance of the tetramerization mechanism.","method":"X-ray crystallography, biochemical/kinetic assays with dimer, tetramer and truncation constructs, cell-based tankyrase stabilization assay","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure, kinetic assays, mutagenesis, cellular functional validation in single study","pmids":["30478318"],"is_preprint":false},{"year":2009,"finding":"USP25m catalytic activity requires a coiled-coil stretch (aa 679-769) but not the UBA/UIM ubiquitin-binding domains. USP25 is monoubiquitinated at K99 (activating) and can autodeubiquitinate. SUMO conjugation at the same K99 residue inhibits USP25 activity, while ubiquitination at K99 activates it; UBDs favor monoubiquitination at K99 and mutation of K99 reduces USP25-dependent rescue of MyBPC1 from proteasomal degradation.","method":"Deletion mutagenesis, ubiquitination assays, SUMO modification assays, substrate (MyBPC1) rescue assay","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — multiple mutant constructs, enzymatic activity assays, K99 mutagenesis with functional readout, single lab with orthogonal methods","pmids":["19440361"],"is_preprint":false},{"year":2006,"finding":"USP25m (muscle isoform) interacts with sarcomeric proteins ACTA1, FLNC, and MyBPC1. MyBPC1 is a short-lived proteasomal substrate whose degradation is prevented by USP25m overexpression; ACTA1 and FLNC are stable proteins not regulated by USP25m turnover.","method":"Yeast two-hybrid, Co-immunoprecipitation, proteasomal degradation assays, overexpression","journal":"Cellular and molecular life sciences : CMLS","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP and functional degradation assay, single lab, multiple substrates tested","pmids":["16501887"],"is_preprint":false},{"year":2021,"finding":"USP25 overexpression promotes microglial activation, synapse elimination, and proinflammatory cytokine production in 5×FAD Alzheimer's disease mouse brains. Genetic ablation of Usp25 reduces neuroinflammation and rescues synaptic and cognitive function, establishing USP25 as a mediator of trisomy 21-related microgliosis.","method":"USP25 BAC transgenic mice, Usp25 genetic KO in 5×FAD mice, pharmacological USP25 inhibition, behavioral testing, synaptic and microglial assays","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic gain- and loss-of-function, pharmacological inhibition, multiple readouts in vivo","pmids":["33523861"],"is_preprint":false},{"year":2022,"finding":"USP25 promotes β-cleavage of APP and Aβ generation by reducing ubiquitination and lysosomal degradation of both APP and BACE1, thereby increasing amyloid pathology.","method":"Usp25 KO and USP25 BAC transgenic mice crossed with 5×FAD, pharmacological USP25 inhibition, ubiquitination assays, lysosomal degradation assays","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO and overexpression, pharmacological inhibition, mechanistic substrate assays, multiple model systems","pmids":["35229730"],"is_preprint":false},{"year":2015,"finding":"Vaccinia-related kinase 2 (VRK2) phosphorylates USP25 at Thr680, Thr727, and Ser745, suppressing USP25 deubiquitinating activity. USP25 interacts with the chaperonin TRiC and deubiquitinates it to maintain its stability; VRK2-mediated phosphorylation of USP25 impairs this, leading to TRiC destabilization.","method":"Co-immunoprecipitation, in vitro kinase assay, site-directed mutagenesis of phosphorylation sites, deubiquitination assay, polyglutamine aggregation assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro kinase assay, phosphosite mutagenesis, functional deubiquitination assay, Co-IP, single lab with multiple orthogonal methods","pmids":["25755282"],"is_preprint":false},{"year":2020,"finding":"USP25 suppresses ubiquitin-mediated degradation of BCR-ABL protein in Philadelphia chromosome-positive leukemia cells. USP25 is found proximal to BCR-ABL; its depletion increases BCR-ABL ubiquitination, reduces BCR-ABL protein levels, and suppresses BCR-ABL-mediated signaling and cell proliferation.","method":"shRNA-mediated USP25 knockdown, ubiquitination assays, BCR-ABL signaling pathway assays, pharmacological USP25 inhibition","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — shRNA KD, ubiquitination assay, signaling assay, pharmacological inhibition, single lab","pmids":["32203161"],"is_preprint":false},{"year":2023,"finding":"USP25 directly binds to KEAP1 and prevents KEAP1's own ubiquitination and degradation. In the absence of USP25, KEAP1 is downregulated and NRF2 is stabilized, enhancing the anti-oxidation response.","method":"Co-immunoprecipitation, USP25 KO mice, pharmacological USP25 inhibition, ubiquitination assays, APAP-induced liver injury model","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP, genetic KO, pharmacological inhibition, in vivo disease model with functional readout, multiple orthogonal approaches","pmids":["37339955"],"is_preprint":false},{"year":2018,"finding":"USP25 interacts with TRAF3 and removes K48-linked ubiquitin chains from TRAF3, stabilizing it in Kupffer cells and promoting endotoxin tolerance. USP25 knockdown activates K48-linked ubiquitination of TRAF3 and downstream JNK/p38 signaling.","method":"Lentiviral USP25 knockdown, Co-immunoprecipitation, K48-specific ubiquitination assay, cytokine production assays","journal":"Molecular immunology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP, specific ubiquitin-linkage assay, rescue by TRAF3 overexpression, single lab","pmids":["30579117"],"is_preprint":false},{"year":2023,"finding":"USP25 removes K63-specific polyubiquitin chains from TAB2, restricting NF-κB and MAPK signaling and reducing neuroinflammation after ischemic stroke in microglia. AAV9-mediated TAB2 knockdown abolishes the effect of USP25 deletion.","method":"USP25 KO mouse ischemic stroke model, AAV9-mediated TAB2 knockdown, K63-specific ubiquitin assay, Co-immunoprecipitation","journal":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO mouse model, epistasis rescue experiment (TAB2 KD abolishes USP25 effect), specific K63 ubiquitination assay, multiple orthogonal methods","pmids":["37587766"],"is_preprint":false},{"year":2018,"finding":"USP25 associates with HDAC11 via co-immunoprecipitation and catalyzes deubiquitination of HDAC11, maintaining HDAC11 protein stability. Cigarette smoke extract promotes USP25 ubiquitination and degradation, which consequently destabilizes HDAC11.","method":"Co-immunoprecipitation, ubiquitination assay, cigarette smoke extract treatment of lung epithelial cells","journal":"American journal of physiology. Lung cellular and molecular physiology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP showing interaction, deubiquitination assay, functional consequence on bacterial load, single lab","pmids":["31746627"],"is_preprint":false},{"year":2018,"finding":"USP25 associates with HBO1 (histone acetyltransferase) via co-immunoprecipitation and promotes its stability by suppressing HBO1 ubiquitination during LPS stimulation, facilitating HBO1-mediated inflammatory gene transcription.","method":"Co-immunoprecipitation, ubiquitination assay, LPS treatment of THP-1 cells","journal":"Biochimica et biophysica acta. Gene regulatory mechanisms","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP, ubiquitination assay, single lab, functional transcriptional readout","pmids":["30745998"],"is_preprint":false},{"year":2018,"finding":"Smurf1 E3 ubiquitin ligase promotes K48-linked polyubiquitination and proteasomal degradation of USP25. Smurf1 overexpression decreases USP25 protein levels and reduces USP25-mediated antiviral activity; Smurf1 knockdown enhances USP25-dependent restriction of VSV replication.","method":"Co-immunoprecipitation, ubiquitination assay, overexpression/knockdown, VSV replication assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP, K48 ubiquitination assay, functional antiviral readout, single lab","pmids":["29518389"],"is_preprint":false},{"year":2019,"finding":"USP25 interacts with tankyrase and co-localizes with GLUT4 in insulin-sensitive adipocytes. USP25 depletion reduces GLUT4 protein levels and blunts insulin-stimulated glucose transport, linking USP25-tankyrase deubiquitination to GLUT4 trafficking.","method":"Co-immunoprecipitation, co-localization imaging, USP25 depletion in adipocytes, glucose transport assay","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP, imaging co-localization, functional glucose transport assay, single lab","pmids":["30886164"],"is_preprint":false},{"year":2020,"finding":"USP25 restrains EGFR degradation by assisting in the association of the E3 ubiquitin ligase c-Cbl with EGFR at early internalization steps, modulating the amplitude of EGFR ubiquitylation; USP25 depletion accelerates EGFR internalization and degradation.","method":"USP25 siRNA depletion, quantitative EGFR internalization and degradation assays, c-Cbl/EGFR association assay","journal":"Biomolecules","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — functional internalization/degradation quantitative assays, Co-IP for c-Cbl association, single lab","pmids":["33202887"],"is_preprint":false},{"year":2021,"finding":"USP25 deubiquitinates PTEN in granulosa cells; USP25 knockdown reduces PTEN levels, activates the PI3K/AKT pathway, and increases granulosa cell proliferation while reducing apoptosis.","method":"USP25 shRNA knockdown, ubiquitination assay, PI3K/AKT pathway analysis, USP25 KO mouse PCOS model","journal":"Frontiers in cell and developmental biology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — deubiquitination assay, KO mouse model, signaling pathway analysis, single lab","pmids":["34805185"],"is_preprint":false},{"year":2016,"finding":"Viral infection-induced upregulation of USP25 requires de novo synthesis of IRF7 and signaling through IFNAR1. IRF7 directly binds to two conserved IRF binding sites on the USP25 promoter to drive Usp25 transcription; mutation of these sites abolishes virus-induced USP25 promoter activation.","method":"IRF7 KO and IFNAR1 KO cells, IRF7 ChIP/promoter binding assay, promoter mutation and reporter assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — promoter mutagenesis, KO cells for epistasis, reporter assay, direct binding demonstrated, single lab with multiple orthogonal approaches","pmids":["27129230"],"is_preprint":false},{"year":2022,"finding":"USP25 deficiency in macrophages enhances TBK1-NF-κB signaling, leading to increased inflammatory cytokine expression and worsening acute pancreatitis. Bone marrow Usp25-/- chimeric mice recapitulate the exacerbated AP phenotype, localizing the effect to macrophages.","method":"Usp25-/- mouse models of AP, bone marrow chimeric mice, in vitro BMDM assays, TBK1-NF-κB pathway analysis","journal":"Cellular and molecular gastroenterology and hepatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — bone marrow chimera for cell-type-specific epistasis, multiple AP models, in vitro mechanistic validation, single lab","pmids":["35934222"],"is_preprint":false},{"year":2023,"finding":"USP25 inhibits the TGF-β/SMAD pathway by reducing SMAD4 K63-linked polyubiquitination, suppressing SMAD2 nuclear translocation and renal fibrosis in hypertensive kidney disease.","method":"USP25 KO and AAV9-overexpression mice, ubiquitination assay (K63-specific), SMAD2 nuclear translocation assay","journal":"Biochimica et biophysica acta. Molecular basis of disease","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — KO and AAV rescue, K63 ubiquitination assay, nuclear translocation, single lab","pmids":["37059312"],"is_preprint":false},{"year":2023,"finding":"USP25 interacts with TRAF6 via co-immunoprecipitation and inhibits K63 polyubiquitination of TRAF6 induced by AGEs, suppressing NF-κB and MAPK pathways in mesangial cells and macrophages in diabetic nephropathy.","method":"USP25 KO mice, Co-immunoprecipitation, K63-specific ubiquitination assay, NF-κB/MAPK pathway analysis","journal":"International immunopharmacology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP, K63 ubiquitination assay, KO mouse model, single lab","pmids":["37657242"],"is_preprint":false},{"year":2024,"finding":"USP25 directly binds to TAK1 and P62; the C178 cysteine of USP25 removes K63 ubiquitin chains from P62, which promotes autophagic-lysosomal degradation of TAK1, reducing TAK1-MAPK inflammatory signaling in obesity cardiomyopathy.","method":"Co-immunoprecipitation, USP25 C178A mutant, autophagy-lysosome pathway assays, USP25 KO and cardiomyocyte-specific overexpression in HFD mice","journal":"JACC. Basic to translational science","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — active-site mutation, Co-IP, autophagy assay, in vivo KO/OE, single lab","pmids":["39619140"],"is_preprint":false},{"year":2024,"finding":"USP25 deubiquitinates SHLD2 at K64, enhancing its binding with REV7 and promoting non-homologous end joining (NHEJ) DNA repair. TRIM25 E3 ligase is identified as the enzyme responsible for degrading USP25. A peptide disrupting the USP25-SHLD2 interaction impairs NHEJ and sensitizes tumors to chemotherapy.","method":"Co-immunoprecipitation, ubiquitination assay (K64-specific), NHEJ reporter assay, class switch recombination in USP25 KO mice, TRIM25 E3 ligase assay, PDX model","journal":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","confidence":"High","confidence_rationale":"Tier 2 / Moderate — site-specific deubiquitination, KO functional assay (CSR), E3 ligase identification, in vivo PDX model, multiple orthogonal methods","pmids":["38803048"],"is_preprint":false},{"year":2025,"finding":"USP25 stabilizes STAT6 by reducing its K48-linked ubiquitination, thereby promoting IL-4-induced M2 macrophage polarization and downstream fibrosis.","method":"USP25 KO mice (BLM and BDL fibrosis models), Co-immunoprecipitation, K48 ubiquitination assay, M2 macrophage polarization assay","journal":"International journal of biological sciences","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — KO mouse models, deubiquitination assay, macrophage polarization assay, single lab","pmids":["39781451"],"is_preprint":false},{"year":2023,"finding":"USP25 stabilizes KLF4 through deubiquitination; KLF4 transcriptionally represses miR-10a-5p expression, and miR-10a-5p targets GSDMD expression, establishing an USP25-KLF4-miR-10a-5p-GSDMD axis that promotes pyroptosis of acinar cells in acute pancreatitis.","method":"Co-immunoprecipitation, ubiquitination assay, miRNA/luciferase assays, KLF4 overexpression rescue experiments","journal":"Shock (Augusta, Ga.)","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP, ubiquitination assay, pathway rescue, single lab","pmids":["36155610"],"is_preprint":false},{"year":2025,"finding":"USP25 promotes ERK signaling in macrophages by deubiquitinating and stabilizing B-Raf and C-Raf, enhancing the antimycobacterial immune response against M. tuberculosis infection.","method":"USP25 KO macrophages and mice, Co-immunoprecipitation, deubiquitination assay, ERK pathway analysis, M. tuberculosis infection model","journal":"The Journal of infectious diseases","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP, deubiquitination assay, KO mouse infection model, single lab","pmids":["39110031"],"is_preprint":false},{"year":2025,"finding":"Tim-3 inhibits USP25 expression via STAT1 and physically interacts with USP25. As a result, Tim-3 inhibits USP25-mediated deubiquitination of TRAF3, promotes K48-linked ubiquitination and degradation of TRAF3, inhibits IRF7 phosphorylation, and suppresses the type I interferon response against VSV.","method":"Tim-3 myeloid-specific KO mice, VSV infection model, Co-immunoprecipitation, K48-ubiquitination assay, IRF7 phosphorylation assay","journal":"Cellular immunology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — myeloid-specific KO mice, Co-IP, specific ubiquitination assay, viral infection model, single lab","pmids":["39946759"],"is_preprint":false},{"year":2024,"finding":"USP25 directly interacts with FOXO3 and removes K63-linked ubiquitin chains at K258 of FOXO3, increasing FOXO3 binding to LC3B and autophagosomic-lysosomal degradation of FOXO3, thereby ameliorating Ang II-induced vascular remodeling.","method":"Co-immunoprecipitation, K63-specific ubiquitination assay, LC3B binding assay, USP25 KO and AAV9-overexpression in mouse aorta, Ang II model","journal":"Acta pharmaceutica Sinica. B","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — site-specific ubiquitination assay, Co-IP, KO and AAV rescue, single lab","pmids":["40370563"],"is_preprint":false},{"year":2025,"finding":"USP25 physically interacts with optineurin autophagy receptor and removes K63-linked polyubiquitin chains from it, which is required for mitophagy. USP25 disruption impairs mitophagy and leads to accumulation of damaged mitochondria and dopaminergic neuronal loss in PD models.","method":"Co-immunoprecipitation, K63 ubiquitination assay, mitophagy assays, USP25 KO and pharmacological inhibition in PD mouse models","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Moderate — Co-IP, specific ubiquitination assay, genetic and pharmacological inhibition with functional mitophagy and neurodegeneration readouts, multiple orthogonal methods","pmids":["41499392"],"is_preprint":false},{"year":2025,"finding":"USP25 impedes RNF31-mediated linear ubiquitination of KRAS oncogenic mutants independently of its deubiquitinase activity, facilitating plasma membrane localization and downstream oncogenic signaling of KRAS mutants in NSCLC. USP25 C178A knockin fails to suppress NSCLC progression, but additional RNF31 deletion/inhibition restores suppression in USP25 KO models.","method":"USP25 KO and C178A knockin mouse models, RNF31 KO, KRAS ubiquitination assay, plasma membrane localization assay, autochthonous NSCLC models, xenograft models","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — knockin of catalytic mutant, genetic epistasis (double KO), in vivo autochthonous models, multiple orthogonal methods","pmids":["39952242"],"is_preprint":false},{"year":2025,"finding":"USP25 deubiquitinates KRAS (including oncogenic mutants) to prevent its proteasomal degradation. USP25 depletion increases KRAS ubiquitination and leads to its proteasomal degradation, suppressing oncogenic KRAS signaling in cancer cells.","method":"siRNA-based DUB screen, KRAS ubiquitination assay, USP25 depletion, USP25 inhibitor treatment, xenograft tumor model","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — siRNA screen identification, ubiquitination assay, pharmacological and genetic loss-of-function, in vivo xenograft, single lab","pmids":["40473213"],"is_preprint":false},{"year":2026,"finding":"USP25 interacts with RIPK1 and removes K63 ubiquitin chains from RIPK1 via its C178 active site, attenuating RIPK1-mediated NF-κB inflammatory signaling in macrophages and suppressing atherosclerosis.","method":"Mass spectrometry substrate identification, Co-immunoprecipitation, K63 ubiquitination assay (C178A mutation), macrophage-specific USP25 KO in ApoE-/- mice","journal":"EBioMedicine","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — MS substrate ID, active-site mutation, specific ubiquitin-linkage assay, cell-type-specific KO in disease model, single lab","pmids":["41861519"],"is_preprint":false},{"year":2026,"finding":"USP25 regulates LATS1 kinase activity by removing K63-linked ubiquitin at K688 of LATS1, disrupting LATS1-MOB1 complex formation and promoting YAP-mediated transcriptional activation in hepatocellular carcinoma; this is independent of LATS1 protein stability.","method":"Co-immunoprecipitation, K63-specific ubiquitination assay (K688 site), LATS1-MOB1 interaction assay, YAP activity assay, USP25 KO, cell-penetrating peptide disruption, PDX models","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — site-specific ubiquitination, protein-protein interaction assay, KO and peptide disruption, PDX model, single lab","pmids":["41882191"],"is_preprint":false},{"year":2025,"finding":"USP25 deubiquitinates cytosolic METTL3 by cleaving K48-linked polyubiquitin chains, preventing proteasomal degradation. This specifically stabilizes cytosolic (but not nuclear) METTL3 without affecting global m6A abundance, and USP25 promotes EGFR expression through cytosolic METTL3 in glioma cells.","method":"Co-immunoprecipitation, K48 ubiquitination assay, subcellular fractionation, m6A measurement, USP25 depletion, EGFR expression assay, clinical glioma sample analysis","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — specific ubiquitination assay, subcellular fractionation for localization specificity, functional EGFR readout, single lab","pmids":["41321637"],"is_preprint":false},{"year":2025,"finding":"USP25 binds to and stabilizes PPARα by removing K48-linked ubiquitin chains at PPARα K429 via the H608 catalytic residue of USP25's USP domain, reducing lipid accumulation in hepatocytes and protecting against MASLD.","method":"Pulldown-LC-MS/MS substrate identification, Co-immunoprecipitation, site-specific mutagenesis (H608, K429), K48 ubiquitination assay, USP25 KO and hepatocyte-specific rescue, Ppara KO epistasis","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — active-site and substrate-site mutagenesis, MS substrate identification, KO epistasis (Ppara KO nullifies USP25 effect), multiple orthogonal approaches, single lab","pmids":["39827322"],"is_preprint":false},{"year":2026,"finding":"USP25 mediates deubiquitination of talin-1 to stabilize it in platelets, promoting platelet hyperreactivity. USP25 deficiency impairs platelet function and delays arterial thrombus formation; pharmacological USP25/28 inhibition (AZ1) suppresses age-dependent platelet hyperactivity.","method":"Ubiquitin-modified proteomics, mass spectrometry, USP25 KO mouse platelet functional assays, pharmacological inhibition with AZ1, arterial thrombosis model","journal":"Blood","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Ub-proteomics for substrate ID, KO functional assay, pharmacological confirmation, single lab","pmids":["40997299"],"is_preprint":false},{"year":2017,"finding":"NMR solution structure of the USP25 N-terminal ubiquitin-binding region (aa 1-146) was determined; key residues for recognition of ubiquitin and SUMO2 were identified. Non-covalent SUMO2 binding to the SUMO-interacting motif of USP25 inhibits its catalytic activity and competitively blocks interaction of the UBR with ubiquitin substrates.","method":"NMR structure determination, binding assays, catalytic activity assay with SUMO2 binding","journal":"Biophysical journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure with functional validation (activity assay and competitive binding), single lab with multiple orthogonal methods","pmids":["28538147"],"is_preprint":false},{"year":2024,"finding":"Crystal structures of USP25 and USP28 in the presence of inhibitors (AZ1, Vismodegib, FT206) revealed a common inhibitor-binding pocket. A glutamate residue at position 373 in USP25 (366 in USP28) plays a central structural role for pocket stability and for inhibitor binding and enzymatic activity.","method":"X-ray crystallography of USP28/USP25 with inhibitors, site-directed mutagenesis of E373/E366, enzymatic activity assays","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structures with functional validation, site-directed mutagenesis, enzymatic assays in one study","pmids":["38816515"],"is_preprint":false},{"year":2023,"finding":"USP25 interacts with TRIM21 E3 ligase via co-immunoprecipitation and mass spectrometry; USP25 regulates the Wnt/β-catenin pathway and EMT-related proteins through this interaction to promote HCC cell proliferation, migration, and invasion.","method":"Mass spectrometry, Co-immunoprecipitation, USP25 overexpression/knockdown in HCC cells, mouse liver cancer KO model, Wnt pathway protein analysis","journal":"Chinese medical journal","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — MS and Co-IP for interaction, KO in vivo model, Wnt pathway analysis, single lab","pmids":["37439386"],"is_preprint":false},{"year":2023,"finding":"USP25 interacts with MDM2 via Co-immunoprecipitation and maintains MDM2 protein stability through deubiquitination (C178A mutation abolishes this). USP25 promotes DLBCL progression by stabilizing MDM2 and consequently decreasing p53 expression via the MDM2-p53 signaling pathway.","method":"Co-immunoprecipitation, deubiquitination assay, C178A USP25 mutant, p53/MDM2 pathway analysis, in vitro and in vivo tumor models","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP, active-site mutation, deubiquitination assay, in vivo tumor model, single lab","pmids":["37480689"],"is_preprint":false},{"year":2025,"finding":"USP25 deubiquitinates SMAD7 to stabilize it in podocytes; ADSC-derived exosomes transfer USP25 protein to podocytes, elevating SMAD7 levels and protecting against diabetic nephropathy-induced apoptosis and inflammation.","method":"Exosome transfer assay, Co-immunoprecipitation, cellular ubiquitination assay, in vitro and in vivo DN models","journal":"Chemical biology & drug design","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP, ubiquitination assay, functional in vivo rescue, single lab","pmids":["40317686"],"is_preprint":false},{"year":2024,"finding":"USP25 prevents ubiquitination and degradation of RAC1, maintaining RAC1 protein levels. Reduced USP25 in IgG4-RD leads to RAC1 ubiquitination/degradation, decreased aldolase A release from the actin cytoskeleton, and reduced glycolysis. Additionally, LYN expression is reduced in USP25-deficient B cells.","method":"USP25 KO mice, ubiquitination assay for RAC1, aldolase A fractionation from actin cytoskeleton, glycolysis assay, IgG4-RD patient PBMCs","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — KO mice, specific ubiquitination assay, metabolic functional readout, patient cells, single lab","pmids":["38521787"],"is_preprint":false},{"year":2026,"finding":"USP25 interacts with TRAF6 and stabilizes its protein level by reducing TRAF6 ubiquitination in severe acute pancreatitis; USP25 inhibition alleviates pancreatic microcirculatory disturbance in SAP rats.","method":"Co-immunoprecipitation, TRAF6 ubiquitination assay with MG132, sh-USP25 adenoviral delivery, SAP rat model","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP, ubiquitination assay, adenoviral KD, in vivo disease model, single lab","pmids":["41771990"],"is_preprint":false},{"year":2026,"finding":"USP25 binds to and stabilizes PARP1 through deubiquitination; PARP1 in turn facilitates preadipocyte differentiation by regulating ELOVL3 (elongation of very long-chain fatty acids protein 3), establishing a USP25-PARP1-ELOVL3 axis in adipogenesis.","method":"Co-immunoprecipitation, deubiquitination assay, RNA-seq, USP25 KO mice on HFD, 3T3-L1 differentiation assay","journal":"Journal of lipid research","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP, deubiquitination assay, KO mouse model, functional adipogenesis assay, single lab","pmids":["41692245"],"is_preprint":false},{"year":2023,"finding":"USP25 overexpression impairs cell cycle regulation during embryonic neurogenesis by altering the cyclin D1/cyclin D2 balance, promoting neural stem cell-to-glial fates over neuronal fates in hippocampus and suppressing neurogenesis.","method":"USP25-Tg mice, embryonic neurogenesis analysis, cyclin D1/D2 expression, hippocampal NSC fate assays, behavioral testing","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — transgenic overexpression with mechanistic readout (cyclin balance), neural fate assays, single lab","pmids":["37171286"],"is_preprint":false},{"year":2024,"finding":"USP25 deficiency in T cells causes impaired mitochondrial mass and cristae condensation. USP25 interacts with ATP5A and ATP5B mitochondrial ATP synthase subunits to promote their stability, and this contributes to mitochondrial homeostasis and T cell functional competence.","method":"MHC-mismatched transplant models with USP25 KO mice, Co-IP of USP25 with ATP5A/ATP5B, Western blotting, mitochondrial morphology analysis","journal":"International immunopharmacology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP, KO transplant model, mitochondrial function readouts, single lab","pmids":["36822087"],"is_preprint":false}],"current_model":"USP25 is a cysteine deubiquitinating enzyme (catalytic residue C178) that removes K48-, K63-, or linear ubiquitin chains from a broad range of substrates—including TRAF3, TRAF5, TRAF6, KEAP1, SERCA2a, HIF-1α, tankyrases, BCR-ABL, KRAS, APP, BACE1, SHLD2, PTEN, SMAD4, FOXO3, LATS1, RAC1, PPARα, STAT6, RIPK1, MDM2, optineurin, and others—thereby controlling their stability, localization, and downstream signaling; its activity is regulated by autoinhibitory tetramerization (a USP25-specific coiled-coil insertion blocks the ubiquitin-binding pocket), by VRK2-mediated phosphorylation (suppressing activity), by competing SUMO versus ubiquitin conjugation at K99 (inhibiting or activating, respectively), and transcriptionally by the type I IFN-IRF7 axis, placing USP25 at the intersection of innate immunity, inflammatory signaling, Wnt/β-catenin, HIF-1/metabolic reprogramming, DNA repair, mitophagy, and neurodegeneration pathways."},"narrative":{"mechanistic_narrative":"USP25 is a cysteine deubiquitinating enzyme that acts through its catalytic cysteine C178 to edit ubiquitin chains on a broad set of substrates, thereby controlling their stability, localization, and downstream signaling across innate immunity, inflammation, metabolism, DNA repair, and neurodegeneration [PMID:23042150, PMID:36722348, PMID:41861519]. A defining mechanistic feature is its self-regulation: USP25 partitions between a catalytically active dimer and an autoinhibited tetramer in which a USP25-specific coiled-coil insertion occludes the ubiquitin-binding pocket, and shifting this equilibrium toward the dimer increases substrate stabilization in cells [PMID:30926242, PMID:30926243, PMID:30478318]. Activity is further tuned by post-translational competition at K99, where ubiquitination activates and SUMO conjugation inhibits the enzyme, and by non-covalent SUMO2 binding to its N-terminal ubiquitin-binding region that competitively blocks substrate engagement [PMID:19440361, PMID:28538147]; VRK2 phosphorylation of C-terminal residues additionally suppresses catalytic activity [PMID:25755282]. In innate antiviral and inflammatory signaling USP25 both stabilizes TRAF3 and TRAF6 by removing K48-linked chains to promote IRF3/NF-κB activation and type I IFN production, and restrains K63-linked signaling on adaptors such as TRAF5/TRAF6, TAB2, and RIPK1 to limit inflammation [PMID:26305951, PMID:23042150, PMID:37587766, PMID:41861519]; its expression is induced by the type I IFN–IRF7 axis through direct IRF7 binding to the USP25 promoter [PMID:27129230]. Beyond immunity, USP25 stabilizes substrates including SERCA2a, HIF-1α, tankyrases (activating Wnt/β-catenin), KEAP1, SHLD2 (promoting NHEJ DNA repair), MDM2, and KRAS, and regulates LATS1/YAP and FOXO3 and optineurin-dependent mitophagy [PMID:36722348, PMID:35440539, PMID:28619731, PMID:37339955, PMID:38803048, PMID:37480689, PMID:40473213, PMID:41882191, PMID:40370563, PMID:41499392]. In disease contexts captured by the corpus, USP25 promotes microgliosis, amyloidogenic processing of APP/BACE1, and neuroinflammation linked to Alzheimer's pathology, and is required for optineurin-mediated mitophagy whose disruption drives dopaminergic neuron loss [PMID:33523861, PMID:35229730, PMID:41499392]. Notably, USP25 can also act independently of its deubiquitinase activity, as it impedes RNF31-mediated linear ubiquitination of oncogenic KRAS to support membrane localization and signaling in lung cancer [PMID:39952242].","teleology":[{"year":2006,"claim":"Established the first substrate context for USP25, showing it physically engages sarcomeric proteins and selectively rescues a short-lived proteasomal substrate, defining it as a stabilizing deubiquitinase.","evidence":"Yeast two-hybrid, Co-IP and proteasomal degradation assays of the muscle isoform with ACTA1, FLNC and MyBPC1","pmids":["16501887"],"confidence":"Medium","gaps":["No catalytic-residue requirement demonstrated","Linkage specificity of removed chains not defined","Limited to overexpression in one cell context"]},{"year":2009,"claim":"Defined how USP25 activity is intrinsically regulated, showing a coiled-coil region is required for catalysis and that opposing ubiquitin versus SUMO modification at K99 toggles enzymatic output.","evidence":"Deletion mutagenesis, ubiquitination/SUMOylation assays and MyBPC1 rescue assay","pmids":["19440361"],"confidence":"High","gaps":["Structural basis of K99 modification switch not resolved","Physiological enzymes mediating K99 modification not identified in this work"]},{"year":2012,"claim":"Placed USP25 in IL-17 innate signaling as a negative regulator, showing it counteracts Act1-driven K63 ubiquitination of TRAF adaptors.","evidence":"Reciprocal Co-IP, knockdown/overexpression and Usp25-/- mouse phenotyping","pmids":["23042150"],"confidence":"High","gaps":["Catalytic-site dependence not formally tested here","Direct demonstration of chain removal on endogenous adaptors limited"]},{"year":2015,"claim":"Resolved a seemingly opposite role in antiviral immunity, showing USP25 stabilizes TRAF3/TRAF6 to promote IRF3/NF-κB activation and type I IFN, indicating substrate- and chain-context-dependent outcomes.","evidence":"Co-IP, Usp25-/- MEF reconstitution rescue and viral infection assays; VRK2 phosphorylation suppressing activity (separate study)","pmids":["26305951","25755282"],"confidence":"High","gaps":["How the same TRAF6 can be both stabilized and K63-edited by USP25 in different contexts unresolved","Determinants of K48 versus K63 selectivity not defined"]},{"year":2016,"claim":"Explained how USP25 is induced during infection, identifying direct IRF7 binding to the USP25 promoter downstream of IFNAR1 signaling.","evidence":"IRF7 KO and IFNAR1 KO cells, promoter mutagenesis and reporter assays","pmids":["27129230"],"confidence":"High","gaps":["Transcriptional regulation outside the IFN context not addressed","Tissue-specific promoter usage not explored"]},{"year":2017,"claim":"Provided structural and functional basis for substrate recognition and SUMO inhibition, and connected USP25 to Wnt signaling via tankyrase stabilization.","evidence":"NMR of the N-terminal ubiquitin-binding region with SUMO2/ubiquitin competition assays; crystal structure of the TNKS1-USP25 complex with KO validation","pmids":["28538147","28619731"],"confidence":"High","gaps":["In vivo relevance of SUMO competition not established","Tankyrase-independent Wnt inputs not separated"]},{"year":2019,"claim":"Resolved the central activity switch, showing USP25 is autoinhibited as a tetramer through a coiled-coil insertion occluding the ubiquitin pocket while the dimer is active, and that the equilibrium controls substrate stabilization.","evidence":"Cryo-EM/X-ray structures, kinetic assays with dimer/tetramer/truncation constructs and cellular tankyrase-stabilization assays","pmids":["30926242","30926243","30478318"],"confidence":"High","gaps":["Cellular signals that drive dimer-tetramer transitions not identified","Whether all substrates are governed by the same oligomeric control unknown"]},{"year":2022,"claim":"Extended USP25 into metabolic and neurodegenerative disease mechanisms, showing it stabilizes HIF-1α to drive glycolysis and promotes amyloidogenic APP/BACE1 processing.","evidence":"Activity-based proteomics and PDAC organoid/xenograft screens for HIF-1α; Usp25 KO/transgenic 5xFAD crosses with ubiquitination and lysosomal degradation assays for APP/BACE1","pmids":["35440539","35229730"],"confidence":"High","gaps":["Direct chain-linkage editing on HIF-1α not fully defined","Cell-type specificity of APP/BACE1 effects in vivo incomplete"]},{"year":2023,"claim":"Demonstrated chain-type- and substrate-specific control across multiple organ systems, including K48-mediated SERCA2a stabilization in cardiomyocytes, KEAP1 stabilization restraining NRF2, and K63 editing of TAB2 limiting microglial inflammation.","evidence":"C178A active-site mutants, MS substrate identification, AAV9 in vivo rescue, KO mice and linkage-specific ubiquitination assays across cardiac, hepatic and stroke models","pmids":["36722348","37339955","37587766"],"confidence":"High","gaps":["Unifying rules predicting K48 versus K63 substrate outcomes still lacking","Most substrate relationships from single labs in single disease contexts"]},{"year":2024,"claim":"Defined a structural inhibitor pocket and expanded USP25 into DNA repair and a non-catalytic mode of action, including K64-specific deubiquitination of SHLD2 to promote NHEJ and inhibition of RNF31-mediated linear ubiquitination of KRAS.","evidence":"Crystal structures of USP25/USP28 with AZ1/Vismodegib/FT206 and E373 mutagenesis; site-specific ubiquitination and NHEJ assays for SHLD2; USP25 C178A knockin and RNF31 double-KO epistasis for KRAS","pmids":["38816515","38803048","39952242"],"confidence":"High","gaps":["Generality of catalytic-independent functions across substrates unknown","Selectivity of inhibitors between USP25 and USP28 incompletely separated"]},{"year":2025,"claim":"Connected USP25 to autophagy/mitophagy and additional growth/metabolic substrates, including K63 editing of optineurin required for mitophagy, deubiquitination of KRAS, PPARα, FOXO3 and STAT6.","evidence":"Co-IP, site- and linkage-specific ubiquitination assays, KO and AAV rescue across PD, cancer, MASLD and fibrosis models","pmids":["41499392","40473213","39827322","39781451"],"confidence":"High","gaps":["Whether mitophagy and substrate-stabilization roles operate simultaneously in the same cells unresolved","Many substrate findings rest on single-lab disease-model evidence"]},{"year":null,"claim":"It remains unresolved what governs USP25's substrate and chain-linkage selectivity in vivo and which physiological signals toggle its dimer-tetramer autoinhibition to dictate stabilizing versus chain-restricting outcomes.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No predictive code for K48 versus K63 substrate fates","Endogenous regulators of oligomeric-state switching unidentified","Scope of deubiquitinase-independent functions undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,2,4,13,27,36]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[2,7,36]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,1,34]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[38,46]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[33,50]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,1,22,36]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[4,34,37]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[2,7,39]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[27]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[33,32,26]}],"complexes":[],"partners":["TRAF3","TRAF6","TNKS1","KEAP1","SHLD2","RIPK1","TAB2","SERCA2A"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9UHP3","full_name":"Ubiquitin carboxyl-terminal hydrolase 25","aliases":["Deubiquitinating enzyme 25","USP on chromosome 21","Ubiquitin thioesterase 25","Ubiquitin-specific-processing protease 25"],"length_aa":1055,"mass_kda":122.2,"function":"Deubiquitinating enzyme that hydrolyzes ubiquitin moieties conjugated to substrates and thus, functions in various biological processes including inflammation and immune response (PubMed:29518389, PubMed:37683630). Modulates the Wnt/beta-catenin pathway by deubiquitinating and stabilizing tankyrases TNKS1 and TNKS2 (PubMed:28619731, PubMed:30926243, PubMed:38875478). Regulates KEAP1-NRF2 axis in the defense against oxidative assaults by deubiquitinating KEAP1 and protecting it from degradation leading to degradation of the NRF2 transcription factor that is responsible for mounting an anti-oxidation gene expression program (PubMed:37339955). Positively regulates RNA virus-induced innate signaling by interacting with and deubiquitinating ERLIN1 and ERLIN2 (PubMed:37683630). In turn, restricts virus production by regulating cholesterol biosynthetic flux (PubMed:37683630). Acts as a negative regulator of interleukin-17-mediated signaling and inflammation through the removal of 'Lys-63'-linked ubiquitination of TRAF5 and TRAF6 (PubMed:23042150). Prevents the ubiquitination and degradation of TRAF3 to reduce the phosphorylation levels of JNK and P38, the secretion of IL-1B and to induce endotoxin tolerance (PubMed:30579117) The muscle-specific isoform (USP25m) may have a role in the regulation of muscular differentiation and function","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9UHP3/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/USP25","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/USP25","total_profiled":1310},"omim":[{"mim_id":"621064","title":"EPILEPSY, IDIOPATHIC GENERALIZED, SUSCEPTIBILITY TO, 19; EIG19","url":"https://www.omim.org/entry/621064"},{"mim_id":"610748","title":"UBIQUITIN-SPECIFIC PROTEASE 28; USP28","url":"https://www.omim.org/entry/610748"},{"mim_id":"606236","title":"ASPSCR1 TETHER FOR SLC2A4, UBX DOMAIN-CONTAINING; ASPSCR1","url":"https://www.omim.org/entry/606236"},{"mim_id":"606016","title":"KELCH-LIKE ECH-ASSOCIATED PROTEIN 1; KEAP1","url":"https://www.omim.org/entry/606016"},{"mim_id":"604736","title":"UBIQUITIN-SPECIFIC PROTEASE 25; USP25","url":"https://www.omim.org/entry/604736"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"skeletal muscle","ntpm":112.9}],"url":"https://www.proteinatlas.org/search/USP25"},"hgnc":{"alias_symbol":["USP21"],"prev_symbol":[]},"alphafold":{"accession":"Q9UHP3","domains":[{"cath_id":"3.90.70.10","chopping":"156-300","consensus_level":"medium","plddt":80.4964,"start":156,"end":300},{"cath_id":"-","chopping":"748-860","consensus_level":"medium","plddt":80.7206,"start":748,"end":860},{"cath_id":"-","chopping":"874-1055","consensus_level":"medium","plddt":83.3453,"start":874,"end":1055},{"cath_id":"1.10.287","chopping":"423-466_534-589","consensus_level":"medium","plddt":93.3398,"start":423,"end":589}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UHP3","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UHP3-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UHP3-F1-predicted_aligned_error_v6.png","plddt_mean":77.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=USP25","jax_strain_url":"https://www.jax.org/strain/search?query=USP25"},"sequence":{"accession":"Q9UHP3","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9UHP3.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9UHP3/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UHP3"}},"corpus_meta":[{"pmid":"23042150","id":"PMC_23042150","title":"Negative 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is a potential therapeutic target in pancreatic cancer.","date":"2022","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/35440539","citation_count":93,"is_preprint":false},{"pmid":"30953360","id":"PMC_30953360","title":"The miR-27a-3p/USP25 axis participates in the pathogenesis of recurrent miscarriage by inhibiting trophoblast migration and invasion.","date":"2019","source":"Journal of cellular physiology","url":"https://pubmed.ncbi.nlm.nih.gov/30953360","citation_count":74,"is_preprint":false},{"pmid":"29131570","id":"PMC_29131570","title":"Identification and Characterization of Dual Inhibitors of the USP25/28 Deubiquitinating Enzyme Subfamily.","date":"2017","source":"ACS chemical biology","url":"https://pubmed.ncbi.nlm.nih.gov/29131570","citation_count":71,"is_preprint":false},{"pmid":"33523861","id":"PMC_33523861","title":"Trisomy 21-induced dysregulation of microglial homeostasis in Alzheimer's brains is mediated by USP25.","date":"2021","source":"Science 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ubiquitin-specific protease USP25 interacts with three sarcomeric proteins.","date":"2006","source":"Cellular and molecular life sciences : CMLS","url":"https://pubmed.ncbi.nlm.nih.gov/16501887","citation_count":47,"is_preprint":false},{"pmid":"37587766","id":"PMC_37587766","title":"USP25 Inhibits Neuroinflammatory Responses After Cerebral Ischemic Stroke by Deubiquitinating TAB2.","date":"2023","source":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/37587766","citation_count":38,"is_preprint":false},{"pmid":"27129230","id":"PMC_27129230","title":"The Type I Interferon-IRF7 Axis Mediates Transcriptional Expression of Usp25 Gene.","date":"2016","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/27129230","citation_count":37,"is_preprint":false},{"pmid":"32203161","id":"PMC_32203161","title":"Deubiquitylase USP25 prevents degradation of BCR-ABL protein and ensures proliferation of Ph-positive leukemia 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Lys63-linked ubiquitin chains, negatively regulating IL-17-mediated signaling. Upon IL-17 stimulation, USP25 associates with TRAF5 and TRAF6 adaptors, opposing Act1-mediated K63-ubiquitination of these adaptors.\",\n      \"method\": \"Co-immunoprecipitation, overexpression/knockdown assays, Usp25-/- mouse model, phosphorylation assays, mRNA stability assays\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, KO mouse phenotype, multiple orthogonal methods, replicated across in vitro and in vivo systems\",\n      \"pmids\": [\"23042150\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"USP25 stabilizes TRAF3 and TRAF6 after viral infection by protecting them from proteasome-dependent or independent degradation, respectively, thereby promoting IRF3 and NF-κB activation and type I IFN and cytokine production.\",\n      \"method\": \"Co-immunoprecipitation, Usp25-/- mouse model, reconstitution of TRAF3/TRAF6 in KO MEFs, viral infection assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO mouse model, reconstitution rescue experiment, Co-IP, multiple orthogonal methods\",\n      \"pmids\": [\"26305951\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP25 binds SERCA2a directly via its USP domain and removes K48-linked ubiquitin chains at the catalytic cysteine C178, preventing proteasomal degradation of SERCA2a and maintaining calcium handling in cardiomyocytes.\",\n      \"method\": \"LC-MS/MS, Co-IP, USP25 mutant constructs (C178A), AAV9-mediated overexpression in KO mice, Western blotting\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — active-site mutagenesis, Co-IP with MS substrate identification, in vivo rescue with AAV9, multiple orthogonal methods\",\n      \"pmids\": [\"36722348\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"USP25 deubiquitinates and stabilizes the HIF-1α transcription factor, promoting HIF-1α transcriptional activity and glycolysis in pancreatic ductal adenocarcinoma under hypoxic conditions.\",\n      \"method\": \"Activity-based proteomics, loss-of-function genetic screen in PDAC organoids, genetic and pharmacological USP25 inhibition, patient-derived xenografts\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (proteomics, organoids, xenografts, genetic KO), replicated in multiple model systems\",\n      \"pmids\": [\"35440539\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"USP25 directly interacts with tankyrases (TNKS1/2) to promote their deubiquitination and stabilization, thereby activating Wnt/β-catenin signaling. The interaction interface was characterized by X-ray crystal structure determination.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, USP25-KO cell lines, X-ray crystal structure of TNKS1-USP25 complex\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure, Co-IP, KO functional validation, multiple orthogonal methods in one study\",\n      \"pmids\": [\"28619731\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"USP25 forms autoinhibited tetramers where a USP25-specific coiled-coil insertion sequence blocks ubiquitin binding, while the catalytically active form is a dimer. Modulating the dimer-tetramer equilibrium affects substrate stabilization in cells.\",\n      \"method\": \"Cryo-EM/X-ray crystallography, biochemical activity assays with dimer/tetramer/truncation constructs, cellular oligomeric state confirmation, substrate stabilization assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structures of both USP25 and USP28, biochemical kinetic assays, cellular validation, two independent concurrent studies\",\n      \"pmids\": [\"30926242\", \"30926243\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"USP25 forms an autoinhibited tetramer via contacts between a coiled-coil insertion domain and the ubiquitin-binding pocket of the catalytic domain. A constitutively dimeric USP25 construct shows higher tankyrase-stabilizing activity in cells, confirming biological relevance of the tetramerization mechanism.\",\n      \"method\": \"X-ray crystallography, biochemical/kinetic assays with dimer, tetramer and truncation constructs, cell-based tankyrase stabilization assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure, kinetic assays, mutagenesis, cellular functional validation in single study\",\n      \"pmids\": [\"30478318\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"USP25m catalytic activity requires a coiled-coil stretch (aa 679-769) but not the UBA/UIM ubiquitin-binding domains. USP25 is monoubiquitinated at K99 (activating) and can autodeubiquitinate. SUMO conjugation at the same K99 residue inhibits USP25 activity, while ubiquitination at K99 activates it; UBDs favor monoubiquitination at K99 and mutation of K99 reduces USP25-dependent rescue of MyBPC1 from proteasomal degradation.\",\n      \"method\": \"Deletion mutagenesis, ubiquitination assays, SUMO modification assays, substrate (MyBPC1) rescue assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — multiple mutant constructs, enzymatic activity assays, K99 mutagenesis with functional readout, single lab with orthogonal methods\",\n      \"pmids\": [\"19440361\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"USP25m (muscle isoform) interacts with sarcomeric proteins ACTA1, FLNC, and MyBPC1. MyBPC1 is a short-lived proteasomal substrate whose degradation is prevented by USP25m overexpression; ACTA1 and FLNC are stable proteins not regulated by USP25m turnover.\",\n      \"method\": \"Yeast two-hybrid, Co-immunoprecipitation, proteasomal degradation assays, overexpression\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP and functional degradation assay, single lab, multiple substrates tested\",\n      \"pmids\": [\"16501887\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"USP25 overexpression promotes microglial activation, synapse elimination, and proinflammatory cytokine production in 5×FAD Alzheimer's disease mouse brains. Genetic ablation of Usp25 reduces neuroinflammation and rescues synaptic and cognitive function, establishing USP25 as a mediator of trisomy 21-related microgliosis.\",\n      \"method\": \"USP25 BAC transgenic mice, Usp25 genetic KO in 5×FAD mice, pharmacological USP25 inhibition, behavioral testing, synaptic and microglial assays\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic gain- and loss-of-function, pharmacological inhibition, multiple readouts in vivo\",\n      \"pmids\": [\"33523861\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"USP25 promotes β-cleavage of APP and Aβ generation by reducing ubiquitination and lysosomal degradation of both APP and BACE1, thereby increasing amyloid pathology.\",\n      \"method\": \"Usp25 KO and USP25 BAC transgenic mice crossed with 5×FAD, pharmacological USP25 inhibition, ubiquitination assays, lysosomal degradation assays\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO and overexpression, pharmacological inhibition, mechanistic substrate assays, multiple model systems\",\n      \"pmids\": [\"35229730\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Vaccinia-related kinase 2 (VRK2) phosphorylates USP25 at Thr680, Thr727, and Ser745, suppressing USP25 deubiquitinating activity. USP25 interacts with the chaperonin TRiC and deubiquitinates it to maintain its stability; VRK2-mediated phosphorylation of USP25 impairs this, leading to TRiC destabilization.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, site-directed mutagenesis of phosphorylation sites, deubiquitination assay, polyglutamine aggregation assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro kinase assay, phosphosite mutagenesis, functional deubiquitination assay, Co-IP, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"25755282\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"USP25 suppresses ubiquitin-mediated degradation of BCR-ABL protein in Philadelphia chromosome-positive leukemia cells. USP25 is found proximal to BCR-ABL; its depletion increases BCR-ABL ubiquitination, reduces BCR-ABL protein levels, and suppresses BCR-ABL-mediated signaling and cell proliferation.\",\n      \"method\": \"shRNA-mediated USP25 knockdown, ubiquitination assays, BCR-ABL signaling pathway assays, pharmacological USP25 inhibition\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — shRNA KD, ubiquitination assay, signaling assay, pharmacological inhibition, single lab\",\n      \"pmids\": [\"32203161\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP25 directly binds to KEAP1 and prevents KEAP1's own ubiquitination and degradation. In the absence of USP25, KEAP1 is downregulated and NRF2 is stabilized, enhancing the anti-oxidation response.\",\n      \"method\": \"Co-immunoprecipitation, USP25 KO mice, pharmacological USP25 inhibition, ubiquitination assays, APAP-induced liver injury model\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP, genetic KO, pharmacological inhibition, in vivo disease model with functional readout, multiple orthogonal approaches\",\n      \"pmids\": [\"37339955\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"USP25 interacts with TRAF3 and removes K48-linked ubiquitin chains from TRAF3, stabilizing it in Kupffer cells and promoting endotoxin tolerance. USP25 knockdown activates K48-linked ubiquitination of TRAF3 and downstream JNK/p38 signaling.\",\n      \"method\": \"Lentiviral USP25 knockdown, Co-immunoprecipitation, K48-specific ubiquitination assay, cytokine production assays\",\n      \"journal\": \"Molecular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP, specific ubiquitin-linkage assay, rescue by TRAF3 overexpression, single lab\",\n      \"pmids\": [\"30579117\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP25 removes K63-specific polyubiquitin chains from TAB2, restricting NF-κB and MAPK signaling and reducing neuroinflammation after ischemic stroke in microglia. AAV9-mediated TAB2 knockdown abolishes the effect of USP25 deletion.\",\n      \"method\": \"USP25 KO mouse ischemic stroke model, AAV9-mediated TAB2 knockdown, K63-specific ubiquitin assay, Co-immunoprecipitation\",\n      \"journal\": \"Advanced science (Weinheim, Baden-Wurttemberg, Germany)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO mouse model, epistasis rescue experiment (TAB2 KD abolishes USP25 effect), specific K63 ubiquitination assay, multiple orthogonal methods\",\n      \"pmids\": [\"37587766\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"USP25 associates with HDAC11 via co-immunoprecipitation and catalyzes deubiquitination of HDAC11, maintaining HDAC11 protein stability. Cigarette smoke extract promotes USP25 ubiquitination and degradation, which consequently destabilizes HDAC11.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, cigarette smoke extract treatment of lung epithelial cells\",\n      \"journal\": \"American journal of physiology. Lung cellular and molecular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP showing interaction, deubiquitination assay, functional consequence on bacterial load, single lab\",\n      \"pmids\": [\"31746627\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"USP25 associates with HBO1 (histone acetyltransferase) via co-immunoprecipitation and promotes its stability by suppressing HBO1 ubiquitination during LPS stimulation, facilitating HBO1-mediated inflammatory gene transcription.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, LPS treatment of THP-1 cells\",\n      \"journal\": \"Biochimica et biophysica acta. Gene regulatory mechanisms\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP, ubiquitination assay, single lab, functional transcriptional readout\",\n      \"pmids\": [\"30745998\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Smurf1 E3 ubiquitin ligase promotes K48-linked polyubiquitination and proteasomal degradation of USP25. Smurf1 overexpression decreases USP25 protein levels and reduces USP25-mediated antiviral activity; Smurf1 knockdown enhances USP25-dependent restriction of VSV replication.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, overexpression/knockdown, VSV replication assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP, K48 ubiquitination assay, functional antiviral readout, single lab\",\n      \"pmids\": [\"29518389\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"USP25 interacts with tankyrase and co-localizes with GLUT4 in insulin-sensitive adipocytes. USP25 depletion reduces GLUT4 protein levels and blunts insulin-stimulated glucose transport, linking USP25-tankyrase deubiquitination to GLUT4 trafficking.\",\n      \"method\": \"Co-immunoprecipitation, co-localization imaging, USP25 depletion in adipocytes, glucose transport assay\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP, imaging co-localization, functional glucose transport assay, single lab\",\n      \"pmids\": [\"30886164\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"USP25 restrains EGFR degradation by assisting in the association of the E3 ubiquitin ligase c-Cbl with EGFR at early internalization steps, modulating the amplitude of EGFR ubiquitylation; USP25 depletion accelerates EGFR internalization and degradation.\",\n      \"method\": \"USP25 siRNA depletion, quantitative EGFR internalization and degradation assays, c-Cbl/EGFR association assay\",\n      \"journal\": \"Biomolecules\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — functional internalization/degradation quantitative assays, Co-IP for c-Cbl association, single lab\",\n      \"pmids\": [\"33202887\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"USP25 deubiquitinates PTEN in granulosa cells; USP25 knockdown reduces PTEN levels, activates the PI3K/AKT pathway, and increases granulosa cell proliferation while reducing apoptosis.\",\n      \"method\": \"USP25 shRNA knockdown, ubiquitination assay, PI3K/AKT pathway analysis, USP25 KO mouse PCOS model\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — deubiquitination assay, KO mouse model, signaling pathway analysis, single lab\",\n      \"pmids\": [\"34805185\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Viral infection-induced upregulation of USP25 requires de novo synthesis of IRF7 and signaling through IFNAR1. IRF7 directly binds to two conserved IRF binding sites on the USP25 promoter to drive Usp25 transcription; mutation of these sites abolishes virus-induced USP25 promoter activation.\",\n      \"method\": \"IRF7 KO and IFNAR1 KO cells, IRF7 ChIP/promoter binding assay, promoter mutation and reporter assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — promoter mutagenesis, KO cells for epistasis, reporter assay, direct binding demonstrated, single lab with multiple orthogonal approaches\",\n      \"pmids\": [\"27129230\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"USP25 deficiency in macrophages enhances TBK1-NF-κB signaling, leading to increased inflammatory cytokine expression and worsening acute pancreatitis. Bone marrow Usp25-/- chimeric mice recapitulate the exacerbated AP phenotype, localizing the effect to macrophages.\",\n      \"method\": \"Usp25-/- mouse models of AP, bone marrow chimeric mice, in vitro BMDM assays, TBK1-NF-κB pathway analysis\",\n      \"journal\": \"Cellular and molecular gastroenterology and hepatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — bone marrow chimera for cell-type-specific epistasis, multiple AP models, in vitro mechanistic validation, single lab\",\n      \"pmids\": [\"35934222\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP25 inhibits the TGF-β/SMAD pathway by reducing SMAD4 K63-linked polyubiquitination, suppressing SMAD2 nuclear translocation and renal fibrosis in hypertensive kidney disease.\",\n      \"method\": \"USP25 KO and AAV9-overexpression mice, ubiquitination assay (K63-specific), SMAD2 nuclear translocation assay\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular basis of disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — KO and AAV rescue, K63 ubiquitination assay, nuclear translocation, single lab\",\n      \"pmids\": [\"37059312\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP25 interacts with TRAF6 via co-immunoprecipitation and inhibits K63 polyubiquitination of TRAF6 induced by AGEs, suppressing NF-κB and MAPK pathways in mesangial cells and macrophages in diabetic nephropathy.\",\n      \"method\": \"USP25 KO mice, Co-immunoprecipitation, K63-specific ubiquitination assay, NF-κB/MAPK pathway analysis\",\n      \"journal\": \"International immunopharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP, K63 ubiquitination assay, KO mouse model, single lab\",\n      \"pmids\": [\"37657242\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP25 directly binds to TAK1 and P62; the C178 cysteine of USP25 removes K63 ubiquitin chains from P62, which promotes autophagic-lysosomal degradation of TAK1, reducing TAK1-MAPK inflammatory signaling in obesity cardiomyopathy.\",\n      \"method\": \"Co-immunoprecipitation, USP25 C178A mutant, autophagy-lysosome pathway assays, USP25 KO and cardiomyocyte-specific overexpression in HFD mice\",\n      \"journal\": \"JACC. Basic to translational science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — active-site mutation, Co-IP, autophagy assay, in vivo KO/OE, single lab\",\n      \"pmids\": [\"39619140\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP25 deubiquitinates SHLD2 at K64, enhancing its binding with REV7 and promoting non-homologous end joining (NHEJ) DNA repair. TRIM25 E3 ligase is identified as the enzyme responsible for degrading USP25. A peptide disrupting the USP25-SHLD2 interaction impairs NHEJ and sensitizes tumors to chemotherapy.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay (K64-specific), NHEJ reporter assay, class switch recombination in USP25 KO mice, TRIM25 E3 ligase assay, PDX model\",\n      \"journal\": \"Advanced science (Weinheim, Baden-Wurttemberg, Germany)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — site-specific deubiquitination, KO functional assay (CSR), E3 ligase identification, in vivo PDX model, multiple orthogonal methods\",\n      \"pmids\": [\"38803048\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP25 stabilizes STAT6 by reducing its K48-linked ubiquitination, thereby promoting IL-4-induced M2 macrophage polarization and downstream fibrosis.\",\n      \"method\": \"USP25 KO mice (BLM and BDL fibrosis models), Co-immunoprecipitation, K48 ubiquitination assay, M2 macrophage polarization assay\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — KO mouse models, deubiquitination assay, macrophage polarization assay, single lab\",\n      \"pmids\": [\"39781451\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP25 stabilizes KLF4 through deubiquitination; KLF4 transcriptionally represses miR-10a-5p expression, and miR-10a-5p targets GSDMD expression, establishing an USP25-KLF4-miR-10a-5p-GSDMD axis that promotes pyroptosis of acinar cells in acute pancreatitis.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, miRNA/luciferase assays, KLF4 overexpression rescue experiments\",\n      \"journal\": \"Shock (Augusta, Ga.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP, ubiquitination assay, pathway rescue, single lab\",\n      \"pmids\": [\"36155610\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP25 promotes ERK signaling in macrophages by deubiquitinating and stabilizing B-Raf and C-Raf, enhancing the antimycobacterial immune response against M. tuberculosis infection.\",\n      \"method\": \"USP25 KO macrophages and mice, Co-immunoprecipitation, deubiquitination assay, ERK pathway analysis, M. tuberculosis infection model\",\n      \"journal\": \"The Journal of infectious diseases\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP, deubiquitination assay, KO mouse infection model, single lab\",\n      \"pmids\": [\"39110031\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Tim-3 inhibits USP25 expression via STAT1 and physically interacts with USP25. As a result, Tim-3 inhibits USP25-mediated deubiquitination of TRAF3, promotes K48-linked ubiquitination and degradation of TRAF3, inhibits IRF7 phosphorylation, and suppresses the type I interferon response against VSV.\",\n      \"method\": \"Tim-3 myeloid-specific KO mice, VSV infection model, Co-immunoprecipitation, K48-ubiquitination assay, IRF7 phosphorylation assay\",\n      \"journal\": \"Cellular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — myeloid-specific KO mice, Co-IP, specific ubiquitination assay, viral infection model, single lab\",\n      \"pmids\": [\"39946759\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP25 directly interacts with FOXO3 and removes K63-linked ubiquitin chains at K258 of FOXO3, increasing FOXO3 binding to LC3B and autophagosomic-lysosomal degradation of FOXO3, thereby ameliorating Ang II-induced vascular remodeling.\",\n      \"method\": \"Co-immunoprecipitation, K63-specific ubiquitination assay, LC3B binding assay, USP25 KO and AAV9-overexpression in mouse aorta, Ang II model\",\n      \"journal\": \"Acta pharmaceutica Sinica. B\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — site-specific ubiquitination assay, Co-IP, KO and AAV rescue, single lab\",\n      \"pmids\": [\"40370563\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP25 physically interacts with optineurin autophagy receptor and removes K63-linked polyubiquitin chains from it, which is required for mitophagy. USP25 disruption impairs mitophagy and leads to accumulation of damaged mitochondria and dopaminergic neuronal loss in PD models.\",\n      \"method\": \"Co-immunoprecipitation, K63 ubiquitination assay, mitophagy assays, USP25 KO and pharmacological inhibition in PD mouse models\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, specific ubiquitination assay, genetic and pharmacological inhibition with functional mitophagy and neurodegeneration readouts, multiple orthogonal methods\",\n      \"pmids\": [\"41499392\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP25 impedes RNF31-mediated linear ubiquitination of KRAS oncogenic mutants independently of its deubiquitinase activity, facilitating plasma membrane localization and downstream oncogenic signaling of KRAS mutants in NSCLC. USP25 C178A knockin fails to suppress NSCLC progression, but additional RNF31 deletion/inhibition restores suppression in USP25 KO models.\",\n      \"method\": \"USP25 KO and C178A knockin mouse models, RNF31 KO, KRAS ubiquitination assay, plasma membrane localization assay, autochthonous NSCLC models, xenograft models\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — knockin of catalytic mutant, genetic epistasis (double KO), in vivo autochthonous models, multiple orthogonal methods\",\n      \"pmids\": [\"39952242\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP25 deubiquitinates KRAS (including oncogenic mutants) to prevent its proteasomal degradation. USP25 depletion increases KRAS ubiquitination and leads to its proteasomal degradation, suppressing oncogenic KRAS signaling in cancer cells.\",\n      \"method\": \"siRNA-based DUB screen, KRAS ubiquitination assay, USP25 depletion, USP25 inhibitor treatment, xenograft tumor model\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — siRNA screen identification, ubiquitination assay, pharmacological and genetic loss-of-function, in vivo xenograft, single lab\",\n      \"pmids\": [\"40473213\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"USP25 interacts with RIPK1 and removes K63 ubiquitin chains from RIPK1 via its C178 active site, attenuating RIPK1-mediated NF-κB inflammatory signaling in macrophages and suppressing atherosclerosis.\",\n      \"method\": \"Mass spectrometry substrate identification, Co-immunoprecipitation, K63 ubiquitination assay (C178A mutation), macrophage-specific USP25 KO in ApoE-/- mice\",\n      \"journal\": \"EBioMedicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — MS substrate ID, active-site mutation, specific ubiquitin-linkage assay, cell-type-specific KO in disease model, single lab\",\n      \"pmids\": [\"41861519\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"USP25 regulates LATS1 kinase activity by removing K63-linked ubiquitin at K688 of LATS1, disrupting LATS1-MOB1 complex formation and promoting YAP-mediated transcriptional activation in hepatocellular carcinoma; this is independent of LATS1 protein stability.\",\n      \"method\": \"Co-immunoprecipitation, K63-specific ubiquitination assay (K688 site), LATS1-MOB1 interaction assay, YAP activity assay, USP25 KO, cell-penetrating peptide disruption, PDX models\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — site-specific ubiquitination, protein-protein interaction assay, KO and peptide disruption, PDX model, single lab\",\n      \"pmids\": [\"41882191\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP25 deubiquitinates cytosolic METTL3 by cleaving K48-linked polyubiquitin chains, preventing proteasomal degradation. This specifically stabilizes cytosolic (but not nuclear) METTL3 without affecting global m6A abundance, and USP25 promotes EGFR expression through cytosolic METTL3 in glioma cells.\",\n      \"method\": \"Co-immunoprecipitation, K48 ubiquitination assay, subcellular fractionation, m6A measurement, USP25 depletion, EGFR expression assay, clinical glioma sample analysis\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — specific ubiquitination assay, subcellular fractionation for localization specificity, functional EGFR readout, single lab\",\n      \"pmids\": [\"41321637\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP25 binds to and stabilizes PPARα by removing K48-linked ubiquitin chains at PPARα K429 via the H608 catalytic residue of USP25's USP domain, reducing lipid accumulation in hepatocytes and protecting against MASLD.\",\n      \"method\": \"Pulldown-LC-MS/MS substrate identification, Co-immunoprecipitation, site-specific mutagenesis (H608, K429), K48 ubiquitination assay, USP25 KO and hepatocyte-specific rescue, Ppara KO epistasis\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — active-site and substrate-site mutagenesis, MS substrate identification, KO epistasis (Ppara KO nullifies USP25 effect), multiple orthogonal approaches, single lab\",\n      \"pmids\": [\"39827322\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"USP25 mediates deubiquitination of talin-1 to stabilize it in platelets, promoting platelet hyperreactivity. USP25 deficiency impairs platelet function and delays arterial thrombus formation; pharmacological USP25/28 inhibition (AZ1) suppresses age-dependent platelet hyperactivity.\",\n      \"method\": \"Ubiquitin-modified proteomics, mass spectrometry, USP25 KO mouse platelet functional assays, pharmacological inhibition with AZ1, arterial thrombosis model\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Ub-proteomics for substrate ID, KO functional assay, pharmacological confirmation, single lab\",\n      \"pmids\": [\"40997299\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"NMR solution structure of the USP25 N-terminal ubiquitin-binding region (aa 1-146) was determined; key residues for recognition of ubiquitin and SUMO2 were identified. Non-covalent SUMO2 binding to the SUMO-interacting motif of USP25 inhibits its catalytic activity and competitively blocks interaction of the UBR with ubiquitin substrates.\",\n      \"method\": \"NMR structure determination, binding assays, catalytic activity assay with SUMO2 binding\",\n      \"journal\": \"Biophysical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure with functional validation (activity assay and competitive binding), single lab with multiple orthogonal methods\",\n      \"pmids\": [\"28538147\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Crystal structures of USP25 and USP28 in the presence of inhibitors (AZ1, Vismodegib, FT206) revealed a common inhibitor-binding pocket. A glutamate residue at position 373 in USP25 (366 in USP28) plays a central structural role for pocket stability and for inhibitor binding and enzymatic activity.\",\n      \"method\": \"X-ray crystallography of USP28/USP25 with inhibitors, site-directed mutagenesis of E373/E366, enzymatic activity assays\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structures with functional validation, site-directed mutagenesis, enzymatic assays in one study\",\n      \"pmids\": [\"38816515\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP25 interacts with TRIM21 E3 ligase via co-immunoprecipitation and mass spectrometry; USP25 regulates the Wnt/β-catenin pathway and EMT-related proteins through this interaction to promote HCC cell proliferation, migration, and invasion.\",\n      \"method\": \"Mass spectrometry, Co-immunoprecipitation, USP25 overexpression/knockdown in HCC cells, mouse liver cancer KO model, Wnt pathway protein analysis\",\n      \"journal\": \"Chinese medical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — MS and Co-IP for interaction, KO in vivo model, Wnt pathway analysis, single lab\",\n      \"pmids\": [\"37439386\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP25 interacts with MDM2 via Co-immunoprecipitation and maintains MDM2 protein stability through deubiquitination (C178A mutation abolishes this). USP25 promotes DLBCL progression by stabilizing MDM2 and consequently decreasing p53 expression via the MDM2-p53 signaling pathway.\",\n      \"method\": \"Co-immunoprecipitation, deubiquitination assay, C178A USP25 mutant, p53/MDM2 pathway analysis, in vitro and in vivo tumor models\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP, active-site mutation, deubiquitination assay, in vivo tumor model, single lab\",\n      \"pmids\": [\"37480689\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP25 deubiquitinates SMAD7 to stabilize it in podocytes; ADSC-derived exosomes transfer USP25 protein to podocytes, elevating SMAD7 levels and protecting against diabetic nephropathy-induced apoptosis and inflammation.\",\n      \"method\": \"Exosome transfer assay, Co-immunoprecipitation, cellular ubiquitination assay, in vitro and in vivo DN models\",\n      \"journal\": \"Chemical biology & drug design\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP, ubiquitination assay, functional in vivo rescue, single lab\",\n      \"pmids\": [\"40317686\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP25 prevents ubiquitination and degradation of RAC1, maintaining RAC1 protein levels. Reduced USP25 in IgG4-RD leads to RAC1 ubiquitination/degradation, decreased aldolase A release from the actin cytoskeleton, and reduced glycolysis. Additionally, LYN expression is reduced in USP25-deficient B cells.\",\n      \"method\": \"USP25 KO mice, ubiquitination assay for RAC1, aldolase A fractionation from actin cytoskeleton, glycolysis assay, IgG4-RD patient PBMCs\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — KO mice, specific ubiquitination assay, metabolic functional readout, patient cells, single lab\",\n      \"pmids\": [\"38521787\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"USP25 interacts with TRAF6 and stabilizes its protein level by reducing TRAF6 ubiquitination in severe acute pancreatitis; USP25 inhibition alleviates pancreatic microcirculatory disturbance in SAP rats.\",\n      \"method\": \"Co-immunoprecipitation, TRAF6 ubiquitination assay with MG132, sh-USP25 adenoviral delivery, SAP rat model\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP, ubiquitination assay, adenoviral KD, in vivo disease model, single lab\",\n      \"pmids\": [\"41771990\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"USP25 binds to and stabilizes PARP1 through deubiquitination; PARP1 in turn facilitates preadipocyte differentiation by regulating ELOVL3 (elongation of very long-chain fatty acids protein 3), establishing a USP25-PARP1-ELOVL3 axis in adipogenesis.\",\n      \"method\": \"Co-immunoprecipitation, deubiquitination assay, RNA-seq, USP25 KO mice on HFD, 3T3-L1 differentiation assay\",\n      \"journal\": \"Journal of lipid research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP, deubiquitination assay, KO mouse model, functional adipogenesis assay, single lab\",\n      \"pmids\": [\"41692245\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP25 overexpression impairs cell cycle regulation during embryonic neurogenesis by altering the cyclin D1/cyclin D2 balance, promoting neural stem cell-to-glial fates over neuronal fates in hippocampus and suppressing neurogenesis.\",\n      \"method\": \"USP25-Tg mice, embryonic neurogenesis analysis, cyclin D1/D2 expression, hippocampal NSC fate assays, behavioral testing\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — transgenic overexpression with mechanistic readout (cyclin balance), neural fate assays, single lab\",\n      \"pmids\": [\"37171286\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP25 deficiency in T cells causes impaired mitochondrial mass and cristae condensation. USP25 interacts with ATP5A and ATP5B mitochondrial ATP synthase subunits to promote their stability, and this contributes to mitochondrial homeostasis and T cell functional competence.\",\n      \"method\": \"MHC-mismatched transplant models with USP25 KO mice, Co-IP of USP25 with ATP5A/ATP5B, Western blotting, mitochondrial morphology analysis\",\n      \"journal\": \"International immunopharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP, KO transplant model, mitochondrial function readouts, single lab\",\n      \"pmids\": [\"36822087\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"USP25 is a cysteine deubiquitinating enzyme (catalytic residue C178) that removes K48-, K63-, or linear ubiquitin chains from a broad range of substrates—including TRAF3, TRAF5, TRAF6, KEAP1, SERCA2a, HIF-1α, tankyrases, BCR-ABL, KRAS, APP, BACE1, SHLD2, PTEN, SMAD4, FOXO3, LATS1, RAC1, PPARα, STAT6, RIPK1, MDM2, optineurin, and others—thereby controlling their stability, localization, and downstream signaling; its activity is regulated by autoinhibitory tetramerization (a USP25-specific coiled-coil insertion blocks the ubiquitin-binding pocket), by VRK2-mediated phosphorylation (suppressing activity), by competing SUMO versus ubiquitin conjugation at K99 (inhibiting or activating, respectively), and transcriptionally by the type I IFN-IRF7 axis, placing USP25 at the intersection of innate immunity, inflammatory signaling, Wnt/β-catenin, HIF-1/metabolic reprogramming, DNA repair, mitophagy, and neurodegeneration pathways.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"USP25 is a cysteine deubiquitinating enzyme that acts through its catalytic cysteine C178 to edit ubiquitin chains on a broad set of substrates, thereby controlling their stability, localization, and downstream signaling across innate immunity, inflammation, metabolism, DNA repair, and neurodegeneration [#0, #2, #36]. A defining mechanistic feature is its self-regulation: USP25 partitions between a catalytically active dimer and an autoinhibited tetramer in which a USP25-specific coiled-coil insertion occludes the ubiquitin-binding pocket, and shifting this equilibrium toward the dimer increases substrate stabilization in cells [#5, #6]. Activity is further tuned by post-translational competition at K99, where ubiquitination activates and SUMO conjugation inhibits the enzyme, and by non-covalent SUMO2 binding to its N-terminal ubiquitin-binding region that competitively blocks substrate engagement [#7, #41]; VRK2 phosphorylation of C-terminal residues additionally suppresses catalytic activity [#11]. In innate antiviral and inflammatory signaling USP25 both stabilizes TRAF3 and TRAF6 by removing K48-linked chains to promote IRF3/NF-\\u03baB activation and type I IFN production, and restrains K63-linked signaling on adaptors such as TRAF5/TRAF6, TAB2, and RIPK1 to limit inflammation [#1, #0, #15, #36]; its expression is induced by the type I IFN\\u2013IRF7 axis through direct IRF7 binding to the USP25 promoter [#22]. Beyond immunity, USP25 stabilizes substrates including SERCA2a, HIF-1\\u03b1, tankyrases (activating Wnt/\\u03b2-catenin), KEAP1, SHLD2 (promoting NHEJ DNA repair), MDM2, and KRAS, and regulates LATS1/YAP and FOXO3 and optineurin-dependent mitophagy [#2, #3, #4, #13, #27, #44, #35, #37, #32, #33]. In disease contexts captured by the corpus, USP25 promotes microgliosis, amyloidogenic processing of APP/BACE1, and neuroinflammation linked to Alzheimer's pathology, and is required for optineurin-mediated mitophagy whose disruption drives dopaminergic neuron loss [#9, #10, #33]. Notably, USP25 can also act independently of its deubiquitinase activity, as it impedes RNF31-mediated linear ubiquitination of oncogenic KRAS to support membrane localization and signaling in lung cancer [#34].\",\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"Established the first substrate context for USP25, showing it physically engages sarcomeric proteins and selectively rescues a short-lived proteasomal substrate, defining it as a stabilizing deubiquitinase.\",\n      \"evidence\": \"Yeast two-hybrid, Co-IP and proteasomal degradation assays of the muscle isoform with ACTA1, FLNC and MyBPC1\",\n      \"pmids\": [\"16501887\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No catalytic-residue requirement demonstrated\", \"Linkage specificity of removed chains not defined\", \"Limited to overexpression in one cell context\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Defined how USP25 activity is intrinsically regulated, showing a coiled-coil region is required for catalysis and that opposing ubiquitin versus SUMO modification at K99 toggles enzymatic output.\",\n      \"evidence\": \"Deletion mutagenesis, ubiquitination/SUMOylation assays and MyBPC1 rescue assay\",\n      \"pmids\": [\"19440361\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of K99 modification switch not resolved\", \"Physiological enzymes mediating K99 modification not identified in this work\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Placed USP25 in IL-17 innate signaling as a negative regulator, showing it counteracts Act1-driven K63 ubiquitination of TRAF adaptors.\",\n      \"evidence\": \"Reciprocal Co-IP, knockdown/overexpression and Usp25-/- mouse phenotyping\",\n      \"pmids\": [\"23042150\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Catalytic-site dependence not formally tested here\", \"Direct demonstration of chain removal on endogenous adaptors limited\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Resolved a seemingly opposite role in antiviral immunity, showing USP25 stabilizes TRAF3/TRAF6 to promote IRF3/NF-\\u03baB activation and type I IFN, indicating substrate- and chain-context-dependent outcomes.\",\n      \"evidence\": \"Co-IP, Usp25-/- MEF reconstitution rescue and viral infection assays; VRK2 phosphorylation suppressing activity (separate study)\",\n      \"pmids\": [\"26305951\", \"25755282\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How the same TRAF6 can be both stabilized and K63-edited by USP25 in different contexts unresolved\", \"Determinants of K48 versus K63 selectivity not defined\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Explained how USP25 is induced during infection, identifying direct IRF7 binding to the USP25 promoter downstream of IFNAR1 signaling.\",\n      \"evidence\": \"IRF7 KO and IFNAR1 KO cells, promoter mutagenesis and reporter assays\",\n      \"pmids\": [\"27129230\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Transcriptional regulation outside the IFN context not addressed\", \"Tissue-specific promoter usage not explored\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Provided structural and functional basis for substrate recognition and SUMO inhibition, and connected USP25 to Wnt signaling via tankyrase stabilization.\",\n      \"evidence\": \"NMR of the N-terminal ubiquitin-binding region with SUMO2/ubiquitin competition assays; crystal structure of the TNKS1-USP25 complex with KO validation\",\n      \"pmids\": [\"28538147\", \"28619731\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance of SUMO competition not established\", \"Tankyrase-independent Wnt inputs not separated\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Resolved the central activity switch, showing USP25 is autoinhibited as a tetramer through a coiled-coil insertion occluding the ubiquitin pocket while the dimer is active, and that the equilibrium controls substrate stabilization.\",\n      \"evidence\": \"Cryo-EM/X-ray structures, kinetic assays with dimer/tetramer/truncation constructs and cellular tankyrase-stabilization assays\",\n      \"pmids\": [\"30926242\", \"30926243\", \"30478318\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cellular signals that drive dimer-tetramer transitions not identified\", \"Whether all substrates are governed by the same oligomeric control unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Extended USP25 into metabolic and neurodegenerative disease mechanisms, showing it stabilizes HIF-1\\u03b1 to drive glycolysis and promotes amyloidogenic APP/BACE1 processing.\",\n      \"evidence\": \"Activity-based proteomics and PDAC organoid/xenograft screens for HIF-1\\u03b1; Usp25 KO/transgenic 5xFAD crosses with ubiquitination and lysosomal degradation assays for APP/BACE1\",\n      \"pmids\": [\"35440539\", \"35229730\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct chain-linkage editing on HIF-1\\u03b1 not fully defined\", \"Cell-type specificity of APP/BACE1 effects in vivo incomplete\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Demonstrated chain-type- and substrate-specific control across multiple organ systems, including K48-mediated SERCA2a stabilization in cardiomyocytes, KEAP1 stabilization restraining NRF2, and K63 editing of TAB2 limiting microglial inflammation.\",\n      \"evidence\": \"C178A active-site mutants, MS substrate identification, AAV9 in vivo rescue, KO mice and linkage-specific ubiquitination assays across cardiac, hepatic and stroke models\",\n      \"pmids\": [\"36722348\", \"37339955\", \"37587766\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Unifying rules predicting K48 versus K63 substrate outcomes still lacking\", \"Most substrate relationships from single labs in single disease contexts\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined a structural inhibitor pocket and expanded USP25 into DNA repair and a non-catalytic mode of action, including K64-specific deubiquitination of SHLD2 to promote NHEJ and inhibition of RNF31-mediated linear ubiquitination of KRAS.\",\n      \"evidence\": \"Crystal structures of USP25/USP28 with AZ1/Vismodegib/FT206 and E373 mutagenesis; site-specific ubiquitination and NHEJ assays for SHLD2; USP25 C178A knockin and RNF31 double-KO epistasis for KRAS\",\n      \"pmids\": [\"38816515\", \"38803048\", \"39952242\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Generality of catalytic-independent functions across substrates unknown\", \"Selectivity of inhibitors between USP25 and USP28 incompletely separated\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Connected USP25 to autophagy/mitophagy and additional growth/metabolic substrates, including K63 editing of optineurin required for mitophagy, deubiquitination of KRAS, PPAR\\u03b1, FOXO3 and STAT6.\",\n      \"evidence\": \"Co-IP, site- and linkage-specific ubiquitination assays, KO and AAV rescue across PD, cancer, MASLD and fibrosis models\",\n      \"pmids\": [\"41499392\", \"40473213\", \"39827322\", \"39781451\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether mitophagy and substrate-stabilization roles operate simultaneously in the same cells unresolved\", \"Many substrate findings rest on single-lab disease-model evidence\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved what governs USP25's substrate and chain-linkage selectivity in vivo and which physiological signals toggle its dimer-tetramer autoinhibition to dictate stabilizing versus chain-restricting outcomes.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No predictive code for K48 versus K63 substrate fates\", \"Endogenous regulators of oligomeric-state switching unidentified\", \"Scope of deubiquitinase-independent functions undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 2, 4, 13, 27, 36]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [2, 7, 36]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1, 34]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [38, 46]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [33, 50]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 1, 22, 36]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [4, 34, 37]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [2, 7, 39]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [27]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [33, 32, 26]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"TRAF3\", \"TRAF6\", \"TNKS1\", \"KEAP1\", \"SHLD2\", \"RIPK1\", \"TAB2\", \"SERCA2a\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}