{"gene":"OTUD4","run_date":"2026-04-29T11:37:57","timeline":{"discoveries":[{"year":2015,"finding":"OTUD4 acts as a scaffold (in a catalytic-activity-independent manner) to recruit the deubiquitinases USP7 and USP9X, which in turn directly deubiquitinate and stabilize the DNA demethylases ALKBH2 and ALKBH3, thereby promoting resistance to DNA alkylation damage.","method":"Co-IP/pulldown, catalytic-dead mutant analysis, loss-of-function (knockdown of OTUD4, USP7, USP9X) with alkylating-agent sensitivity readout","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, catalytic-dead mutant ruling out direct DUB activity, multiple deubiquitinase knockdowns with defined cellular phenotype; moderate evidence from single lab with orthogonal methods","pmids":["25944111"],"is_preprint":false},{"year":2018,"finding":"OTUD4 is phosphorylated near its catalytic domain, which converts its nominal K48-linkage specificity into K63-linkage-specific deubiquitinase activity; this phospho-activated K63 activity also requires an adjacent ubiquitin-interacting motif (UIM) that increases affinity for K63-linked chains. The K63 deubiquitinase targets MyD88, thereby negatively regulating TLR-mediated NF-κB activation.","method":"In vitro DUB linkage-specificity assay, phospho-mutant analysis, UIM deletion, macrophage KO (Otud4-/- mice) with TLR stimulation and NF-κB readout, mass spectrometry phosphoproteomics","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro biochemical linkage-specificity assays, phospho-mutant and UIM mutant dissection, genetic KO with defined inflammatory phenotype in primary macrophages","pmids":["29395066"],"is_preprint":false},{"year":2018,"finding":"Viral infection induces IRF3/7-dependent upregulation of OTUD4, which then interacts with MAVS and removes K48-linked polyubiquitin chains from MAVS, thereby stabilizing MAVS and promoting IRF3/NF-κB-dependent antiviral type I interferon signaling.","method":"Co-IP, ubiquitination assay, OTUD4 KO/knockdown (conditional Otud4fl/fl mice and cell lines), VSV infection with IFN/cytokine readout, MAVS reconstitution rescue experiment","journal":"Cell research","confidence":"High","confidence_rationale":"Tier 2 — Co-IP, ubiquitin linkage assay, conditional KO mice with in vivo viral challenge, and reconstitution rescue; single lab but multiple orthogonal methods","pmids":["30410068"],"is_preprint":false},{"year":2019,"finding":"OTUD4 is a component of neuronal RNA transport granules under physiological conditions and is recruited to cytoplasmic stress granules upon cellular stress; it interacts with a network of RNA-binding proteins including FMRP, SMN1, G3BP1, and TIA1 in an RNA-dependent manner, and is itself an RNA-binding protein. Knockdown of OTUD4 impairs stress granule formation and leads to apoptotic cell death.","method":"Pulldown + mass spectrometry on mouse brain lysates, RNA-dependence assay, live-cell imaging of neuronal RNA granules, stress granule immunofluorescence, siRNA knockdown with apoptosis readout","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2–3 — pulldown/MS for interactome, direct RNA-binding shown, localization by imaging with functional consequence (stress granule defect, apoptosis); single lab","pmids":["31138677"],"is_preprint":false},{"year":2020,"finding":"OTUD4 interacts with TRAF6 and removes K63-linked auto-polyubiquitin chains from TRAF6, thereby suppressing NF-κB activation and reducing inflammatory responses in hepatic ischemia-reperfusion injury.","method":"Co-IP, ubiquitination assay (K63 linkage), OTUD4 overexpression in hypoxia/reoxygenation model and in vivo hepatic IR model with NF-κB and cytokine readouts","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2–3 — Co-IP, K63-specific ubiquitination assay, in vivo overexpression model; single lab, moderate methods","pmids":["31964525"],"is_preprint":false},{"year":2020,"finding":"OTUD4 enhances TGFβ signalling through both catalytically dependent and independent mechanisms: it promotes membrane retention of TGFβ receptor I and inactivates the TGFβ negative regulator SMURF2; OTUD4 is itself a transcriptional target of TGFβ, forming a positive feedback loop.","method":"Catalytic-dead mutant analysis, receptor surface assay, Co-IP, loss-of-function with TGFβ pathway readouts (SMAD phosphorylation)","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2–3 — catalytic-dead mutant dissection, Co-IP, functional TGFβ signaling readout; single lab with multiple approaches","pmids":["32973272"],"is_preprint":false},{"year":2022,"finding":"OTUD4 directly deubiquitinates GSDME, removing ubiquitin chains and preventing its proteasomal degradation, thereby stabilizing GSDME and enhancing radiation-induced pyroptosis (caspase-3-mediated GSDME cleavage) and radiosensitivity in nasopharyngeal carcinoma.","method":"Co-IP, mass spectrometry, ubiquitination assay, in vitro and in vivo functional assays with OTUD4/GSDME modulation, live-cell imaging of pyroptosis","journal":"Journal of experimental & clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 2–3 — Co-IP, MS identification, ubiquitination assay, in vivo tumor model; single lab","pmids":["36411454"],"is_preprint":false},{"year":2023,"finding":"OTUD4 interacts with and deubiquitinates PFKFB3, a master activator of glycolysis, preventing its degradation and thereby promoting TGFβ1-driven cardiac fibroblast activation and cardiac fibrosis after myocardial infarction.","method":"Co-IP, ubiquitination assay, OTUD4 knockdown/overexpression in cardiac fibroblasts, in vivo post-MI mouse model with fibrosis readout","journal":"Journal of molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2–3 — Co-IP, deubiquitination assay, in vivo model; single lab","pmids":["37162556"],"is_preprint":false},{"year":2023,"finding":"In Paneth cells, OTUD4 deubiquitinates MyD88 (removing K63-linked chains), thereby dampening NF-κB and MAPK activation and restricting antimicrobial peptide expression; knockout of OTUD4 leads to hyper-K63-ubiquitination of MyD88, increased AMP production, and resistance to DSS colitis and Salmonella infection.","method":"Conditional KO mice (Vil-Cre and Def-Cre Otud4fl/fl), intestinal organoids, ubiquitination assay, LPS/PGN stimulation with NF-κB/MAPK readouts","journal":"Cell insight","confidence":"High","confidence_rationale":"Tier 2 — cell-type-specific conditional KO mice, ubiquitination assay, defined mechanistic node (MyD88 K63-deubiquitination), in vivo infection models; single lab but multiple orthogonal approaches","pmids":["37193092"],"is_preprint":false},{"year":2023,"finding":"IRTKS recruits OTUD4 to SETDB1, where OTUD4 removes K48-linked polyubiquitin chains at K182/K1050 of SETDB1, preventing proteasomal degradation of SETDB1, increasing H3K9me3 at the CDH1 locus, and suppressing E-cadherin expression to drive EMT and tumor metastasis.","method":"Co-IP, ubiquitination assay (K48 linkage, specific sites identified), chromatin accessibility assay, E-cadherin transcription readout, in vitro invasion and in vivo metastasis assays","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2–3 — Co-IP, site-specific ubiquitination assay, downstream epigenetic and transcriptional readout; single lab","pmids":["37739210"],"is_preprint":false},{"year":2023,"finding":"OTUD4 directly interacts with and deubiquitinates Snail1, preventing its degradation and thereby promoting epithelial-mesenchymal transition and metastasis in triple-negative breast cancer.","method":"Co-IP, ubiquitination assay, OTUD4 knockdown with migration/invasion/metastasis readouts, reconstitution of Snail1 in OTUD4-deficient cells","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2–3 — Co-IP, deubiquitination assay, rescue experiment with Snail1 reconstitution; single lab","pmids":["38040050"],"is_preprint":false},{"year":2024,"finding":"OTUD4 directly interacts with CDK1 and removes K11-, K29-, and K33-linked polyubiquitin chains to stabilize CDK1; it also binds FGFR1 and reduces K6- and K27-linked polyubiquitination of FGFR1, thereby indirectly further stabilizing CDK1 and activating the downstream MAPK signaling pathway in glioblastoma.","method":"Co-IP, ubiquitination assay (linkage-specific), OTUD4 knockdown/overexpression with proliferation/invasion readouts, in vivo xenograft","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2–3 — Co-IP, linkage-specific ubiquitination assay, in vivo xenograft; single lab","pmids":["38429268"],"is_preprint":false},{"year":2024,"finding":"OTUD4 deubiquitinates and stabilizes CD73, counteracting TRIM21-mediated ubiquitination of CD73, within a TGF-β-orchestrated signaling axis; this OTUD4/CD73 proteolytic axis suppresses antitumor immune responses in triple-negative breast cancer.","method":"Multiomic analysis, Co-IP, ubiquitination assay (TRIM21 as E3 ligase), pharmacological inhibitor (ST80) disrupting OTUD4-CD73 interaction, in vivo TNBC models with immune readout","journal":"The Journal of clinical investigation","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP, defined E3/DUB pair, specific inhibitor validation, in vivo immunological readout; single lab but multiple orthogonal approaches","pmids":["38530357"],"is_preprint":false},{"year":2024,"finding":"OTUD4 acts as a non-catalytic adaptor to recruit USP7 to the KSHV replication and transcription activator (K-RTA), facilitating K-RTA deubiquitination by USP7 and thereby stabilizing K-RTA to promote KSHV lytic reactivation.","method":"Co-IP, ubiquitination assay, OTUD4 catalytic-dead mutant (showing DUB activity dispensable), USP7 recruitment assay, viral reactivation readout","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP, catalytic-dead mutant excluding direct DUB activity, USP7 recruitment shown, viral reactivation phenotype; single lab with multiple orthogonal methods","pmids":["38215174"],"is_preprint":false},{"year":2024,"finding":"OTUD4 directly interacts with and deubiquitinates RBM47, preventing its proteasomal degradation; stabilized RBM47 promotes ATF3 mRNA stability and ATF3-mediated ferroptosis, suppressing ccRCC progression.","method":"Co-IP, ubiquitination assay, OTUD4 overexpression/knockdown, RBM47 interference rescue experiment, ferroptosis assay","journal":"Apoptosis","confidence":"Medium","confidence_rationale":"Tier 2–3 — Co-IP, deubiquitination assay, rescue experiment; single lab","pmids":["38553613"],"is_preprint":false},{"year":2025,"finding":"OTUD4 interacts with core components of the TAK1 signalosome (TAK1, TAB1, TAB3) and removes K63-linked polyubiquitin chains from TAK1 and TAB3, suppressing TNF-induced NF-κB activation. A histidine-centered loop (His loop) in the catalytic domain confers K63-linkage specificity; a tumor-associated H148Y variant in this loop retains TAK1 binding but abolishes DUB activity, leading to sustained NF-κB activation.","method":"Co-IP, in vitro K63-specific DUB assay, His-loop mutant and H148Y variant analysis, TNF stimulation with NF-κB readout","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1–2 — in vitro DUB assay, active-site mutagenesis (H148Y), Co-IP with signalosome components; single lab","pmids":["41062071"],"is_preprint":false},{"year":2025,"finding":"OTUD4 directly deubiquitinates GPX4, removing ubiquitin chains to prevent its proteasomal degradation; OTUD4 also impedes RHEB-mediated autophagic degradation of GPX4, collectively suppressing ferroptosis and promoting tumor cell survival.","method":"CRISPR-Cas9 KO cell lines, ferroptosis inducer sensitivity assay, Co-IP, ubiquitination assay, RHEB-autophagy axis analysis, in vivo tumor models","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 — CRISPR KO, Co-IP, ubiquitination assay, in vivo tumor growth; single lab with multiple orthogonal methods","pmids":["40338740"],"is_preprint":false},{"year":2025,"finding":"OTUD4 interacts with UBAC1 and maintains UBAC1 protein stability through deubiquitination; OTUD4 deficiency decreases UBAC1 expression, thereby impairing autophagic flux in cardiomyocytes and modulating myocardial ischemia-reperfusion injury.","method":"Co-IP, ubiquitination assay, OTUD4 knockdown/overexpression in OGD-R cell model and LAD-ligation MIRI mouse model, autophagic flux markers (LC3, p62, LAMP2), AKT/mTOR pathway readout","journal":"Journal of molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2–3 — Co-IP, deubiquitination assay, in vivo cardiac model; single lab","pmids":["41107601"],"is_preprint":false},{"year":2025,"finding":"SPARC competitively binds OTUD4 in conjunction with MyD88, disrupting OTUD4's deubiquitinase regulation of MyD88 and facilitating p65 nuclear translocation, thereby activating the p65-MLCK/MLC2 pathway and compromising intestinal barrier integrity in Crohn's disease.","method":"Co-IP, competitive binding assay, p65 nuclear translocation imaging, SPARC KO mouse colitis model, barrier permeability readouts","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 2–3 — Co-IP showing competitive binding, in vivo KO model, signaling pathway readouts; single lab","pmids":["39888301"],"is_preprint":false},{"year":2025,"finding":"OTUD4 directly interacts with and deubiquitinates p53, stabilizing it and enhancing its transcriptional activity to suppress colorectal cancer progression; p53 knockdown abrogates OTUD4-mediated tumor suppression.","method":"Co-IP, ubiquitination assay, OTUD4 knockdown/overexpression, p53 knockdown rescue experiment, proliferation/migration/invasion readouts","journal":"Discover oncology","confidence":"Medium","confidence_rationale":"Tier 2–3 — Co-IP, deubiquitination assay, genetic rescue; single lab","pmids":["41266709"],"is_preprint":false},{"year":2025,"finding":"TRIM21 promotes K48-linked ubiquitination of the small hepatitis B surface antigen (SHBs) at K122 leading to proteasomal degradation, while OTUD4 counteracts TRIM21-induced ubiquitination to stabilize SHBs; OTUD4 interacts with SHBs via its N-terminal 1–180 amino acids.","method":"Co-IP, GST pulldown (in vitro), K48-ubiquitination assay (site-directed at K122), TRIM21/OTUD4 overexpression with SHBs stability readout","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 1–2 — GST pulldown (in vitro), Co-IP, site-specific ubiquitination assay; single lab with multiple orthogonal methods","pmids":["40277358"],"is_preprint":false},{"year":2025,"finding":"OTUD4 directly deubiquitinates and stabilizes ZMYND8, promoting assembly of the DDX3X-CK1ε complex that activates WNT/β-catenin signaling, upregulates CSF1, and drives M2 macrophage polarization to foster an immunosuppressive niche in TNBC spinal metastasis.","method":"Co-IP, ubiquitination assay, OTUD4 KO/overexpression, in vivo spinal metastasis model, macrophage co-culture readouts","journal":"Neoplasia","confidence":"Medium","confidence_rationale":"Tier 2–3 — Co-IP, deubiquitination assay, in vivo model; single lab","pmids":["41297414"],"is_preprint":false},{"year":2025,"finding":"WSB1 promotes interaction between ISOC2 and OTUD4, leading to OTUD4-mediated deubiquitination and stabilization of ISOC2, which suppresses P16INK4a expression and drives prostate cancer progression.","method":"Mass spectrometry, Co-IP, ubiquitination assay, WSB1/ISOC2/OTUD4 knockdown with proliferation/migration readouts, in vivo xenograft","journal":"American journal of cancer research","confidence":"Medium","confidence_rationale":"Tier 2–3 — MS, Co-IP, deubiquitination assay, in vivo xenograft; single lab","pmids":["41244113"],"is_preprint":false},{"year":2025,"finding":"OTUD4 directly deubiquitinates MYH9, preventing its proteasomal degradation, thereby stabilizing MYH9 which suppresses prostate cancer progression through interactions with cell adhesion molecules.","method":"Co-IP, mass spectrometry, ubiquitination assay, OTUD4 KO/overexpression, in vivo xenograft","journal":"Oncology research","confidence":"Medium","confidence_rationale":"Tier 2–3 — Co-IP, MS, deubiquitination assay, in vivo model; single lab","pmids":["41930143"],"is_preprint":false},{"year":2026,"finding":"OTUD4 stabilizes EGFR by directly interacting with it (via OTUD4 aa 568–1114 and EGFR aa 958–1210) and cleaving K48-linked polyubiquitin chains; OTUD4 is additionally recruited by NRP1 to further deubiquitinate and stabilize EGFR, activating the PI3K/AKT pathway in TNBC.","method":"Co-IP (domain-mapping), ubiquitination assay (K48 linkage), OTUD4 knockdown, NRP1 co-recruitment assay, PI3K/AKT pathway readout","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2–3 — domain-mapped Co-IP, K48 ubiquitination assay, NRP1 recruitment; single lab","pmids":["41730840"],"is_preprint":false}],"current_model":"OTUD4 is an OTU-family deubiquitinase with dual K48 and (phospho-activated, UIM-assisted) K63 deubiquitinase activities that targets a broad range of substrates—including MAVS, MyD88, TRAF6, TAK1/TAB3, CDK1, FGFR1, GSDME, GPX4, Snail1, SETDB1, CD73, p53, RBM47, PFKFB3, ZMYND8, EGFR, and others—to regulate innate immune signaling, TGFβ signaling, NF-κB/MAPK pathways, ferroptosis, DNA repair, stress granule assembly, and tumor progression; it can also act as a catalysis-independent scaffold to recruit USP7 and USP9X to specific substrates (ALKBH2/3, K-RTA)."},"narrative":{"teleology":[{"year":2015,"claim":"Establishing that OTUD4 can function independently of its own catalytic activity as a scaffold to recruit USP7 and USP9X answered how alkylation-repair demethylases ALKBH2/3 are stabilized and revealed an unexpected non-enzymatic mode of action for an OTU-family protein.","evidence":"Co-IP, catalytic-dead mutant analysis, and knockdown of OTUD4/USP7/USP9X with alkylating-agent sensitivity readout in human cells","pmids":["25944111"],"confidence":"High","gaps":["Whether the scaffold function extends to other substrates beyond ALKBH2/3","Structural basis of OTUD4–USP7/USP9X interaction unknown"]},{"year":2018,"claim":"Demonstrating that phosphorylation near the catalytic domain switches OTUD4 from K48 to K63 specificity—dependent on an adjacent UIM—resolved how a single DUB achieves dual-linkage specificity and identified MyD88 as a physiological K63-deubiquitination target that restrains TLR-driven NF-κB activation.","evidence":"In vitro linkage-specificity assays, phospho- and UIM-mutant analysis, Otud4-KO macrophages with TLR stimulation","pmids":["29395066"],"confidence":"High","gaps":["Identity of the kinase(s) responsible for activating phosphorylation","Whether phospho-switching is reversible and dynamically regulated in vivo"]},{"year":2018,"claim":"Showing that OTUD4 removes K48-linked ubiquitin from MAVS to stabilize it during viral infection established OTUD4 as a positive regulator of antiviral innate immunity, complementing its negative role in TLR/NF-κB signaling.","evidence":"Conditional Otud4-KO mice challenged with VSV, Co-IP, K48-ubiquitination assay, MAVS reconstitution rescue","pmids":["30410068"],"confidence":"High","gaps":["E3 ligase whose K48-ubiquitination of MAVS is countered by OTUD4 not identified","Whether OTUD4 acts on other RLR-pathway components"]},{"year":2019,"claim":"Identifying OTUD4 as an RNA-binding protein that localizes to neuronal RNA transport granules and is required for stress granule assembly expanded its functional repertoire beyond ubiquitin biology into RNA metabolism and stress adaptation.","evidence":"Pulldown/MS from mouse brain, RNA-dependence assay, stress granule imaging, siRNA knockdown with apoptosis readout","pmids":["31138677"],"confidence":"Medium","gaps":["RNA targets of OTUD4 not identified","Whether DUB activity is required for RNA granule function","Independent replication in non-neuronal cell types needed"]},{"year":2020,"claim":"Demonstrating OTUD4-mediated K63-deubiquitination of TRAF6 and enhancement of TGFβ signaling (via TGFβRI retention and SMURF2 inactivation) broadened the set of immune and developmental pathways regulated by OTUD4.","evidence":"Co-IP, K63 ubiquitination assay in hepatic IR model; catalytic-dead mutant and receptor-surface assay for TGFβ axis","pmids":["31964525","32973272"],"confidence":"Medium","gaps":["How OTUD4 inactivates SMURF2 mechanistically unclear","TGFβ feedback loop not validated in vivo"]},{"year":2023,"claim":"Identifying OTUD4 as a deubiquitinase for multiple pro-tumorigenic and metabolic substrates—GSDME (pyroptosis), PFKFB3 (glycolysis), SETDB1 (epigenetics/EMT), Snail1 (EMT)—and confirming MyD88 K63-deubiquitination in Paneth cells in vivo consolidated OTUD4 as a versatile regulator of cell fate and inflammation across tissues.","evidence":"Co-IP and ubiquitination assays for each substrate; conditional intestinal KO mice (Paneth-cell-specific) with infection challenge; in vivo tumor and cardiac fibrosis models","pmids":["36411454","37162556","37739210","38040050","37193092"],"confidence":"Medium","gaps":["How substrate selectivity is determined among many targets is unknown","Whether OTUD4 activity on these substrates is phospho-regulated as for MyD88 is untested"]},{"year":2024,"claim":"Revealing that OTUD4 cleaves atypical ubiquitin chains (K11, K29, K33 on CDK1; K6, K27 on FGFR1) and stabilizes GPX4 to suppress ferroptosis while also stabilizing CD73 to enable immune evasion extended its chain-type repertoire and linked it to ferroptosis regulation and tumor immunology.","evidence":"Linkage-specific ubiquitination assays, CRISPR-KO ferroptosis sensitivity, TRIM21-counteracting ubiquitination of CD73, in vivo TNBC immune readout, pharmacological inhibitor ST80","pmids":["38429268","40338740","38530357"],"confidence":"Medium","gaps":["How OTUD4 achieves specificity for such diverse chain types is structurally unresolved","ST80 inhibitor mechanism and selectivity not fully characterized"]},{"year":2025,"claim":"Structural and functional dissection of OTUD4's His-loop conferring K63 specificity, identification of a tumor-associated H148Y loss-of-function variant, and expansion of the substrate repertoire to include p53, ZMYND8, MYH9, ISOC2, UBAC1, EGFR, RBM47, and viral SHBs established OTUD4 as a broadly acting deubiquitinase with clinical relevance, and confirmed the scaffold function for USP7 in KSHV reactivation.","evidence":"His-loop mutagenesis and in vitro DUB assay; domain-mapped Co-IPs; Co-IP/deubiquitination assays with rescue experiments; in vivo models (spinal metastasis, xenograft, cardiac IR, colitis); competitive binding assay (SPARC–OTUD4–MyD88)","pmids":["41062071","41266709","41297414","41930143","41244113","41107601","41730840","38215174","39888301","40277358","38553613"],"confidence":"Medium","gaps":["No crystal structure of OTUD4 catalytic domain available","Relative contribution of catalytic vs. scaffold activities in physiological settings unknown","Most substrate studies from single laboratories awaiting independent replication"]},{"year":null,"claim":"Key open questions include the structural basis of OTUD4's unusually broad chain-type specificity and phospho-switching mechanism, the identity of kinase(s) regulating the K48-to-K63 switch, how substrate selectivity is achieved among dozens of targets, and whether the scaffold and DUB activities are coordinated or context-exclusive.","evidence":"","pmids":[],"confidence":"Low","gaps":["No crystal or cryo-EM structure of OTUD4","Kinase(s) for activating phosphorylation unidentified","Systematic substrate-prioritization under physiological conditions not performed"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[1,2,4,6,8,9,10,11,14,15,16,17,19,20,21,22,23,24]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,2,4,6,8,9,10,11,14,15,16,17,19,20,21,22,23,24]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,13]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[3]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[3,8]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[9]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[1,2,4,8,15,18]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[5,7,11,12,21,24]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,6,9,10,16,17,19,20,22,23]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[0]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[6,14,16]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[9,10,11,12,22,24]}],"complexes":[],"partners":["USP7","USP9X","MYD88","MAVS","TRAF6","TAK1","GPX4","EGFR"],"other_free_text":[]},"mechanistic_narrative":"OTUD4 is an OTU-family deubiquitinase that functions as both a catalytically active enzyme with broad ubiquitin-chain specificity and a non-catalytic scaffold for recruiting other deubiquitinases, thereby regulating innate immune signaling, stress responses, DNA repair, and tumor progression. Its intrinsic K48-directed deubiquitinase activity stabilizes diverse substrates—including MAVS, GSDME, GPX4, SETDB1, CD73, p53, Snail1, EGFR, and others—by preventing their proteasomal degradation [PMID:30410068, PMID:36411454, PMID:40338740, PMID:37739210, PMID:38530357, PMID:41266709, PMID:41730840], while phosphorylation near its catalytic domain, together with an adjacent ubiquitin-interacting motif, switches its specificity toward K63-linked chains, enabling it to deubiquitinate MyD88, TRAF6, and TAK1/TAB3 to restrain NF-κB and MAPK signaling [PMID:29395066, PMID:31964525, PMID:41062071, PMID:37193092]. Independent of catalytic activity, OTUD4 scaffolds USP7 and USP9X to stabilize the DNA alkylation-repair demethylases ALKBH2/3 and the viral activator K-RTA [PMID:25944111, PMID:38215174]. OTUD4 additionally localizes to neuronal RNA transport granules and stress granules, binds RNA, and is required for stress granule assembly and cell survival under stress [PMID:31138677]."},"prefetch_data":{"uniprot":{"accession":"Q01804","full_name":"OTU domain-containing protein 4","aliases":["HIV-1-induced protein HIN-1"],"length_aa":1114,"mass_kda":124.0,"function":"Deubiquitinase which hydrolyzes the isopeptide bond between the ubiquitin C-terminus and the lysine epsilon-amino group of the target protein (PubMed:23827681, PubMed:25944111, PubMed:29395066). May negatively regulate inflammatory and pathogen recognition signaling in innate immune response. Upon phosphorylation at Ser-202 and Ser-204 residues, via IL-1 receptor and Toll-like receptor signaling pathway, specifically deubiquitinates 'Lys-63'-polyubiquitinated MYD88 adapter protein triggering down-regulation of NF-kappa-B-dependent transcription of inflammatory mediators (PubMed:29395066). Independently of the catalytic activity, acts as a scaffold for alternative deubiquitinases to assemble specific deubiquitinase-substrate complexes. Associates with USP7 and USP9X deubiquitinases to stabilize alkylation repair enzyme ALKBH3, thereby promoting the repair of alkylated DNA lesions (PubMed:25944111)","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q01804/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/OTUD4","classification":"Not Classified","n_dependent_lines":11,"n_total_lines":1208,"dependency_fraction":0.009105960264900662},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/OTUD4","total_profiled":1310},"omim":[{"mim_id":"611744","title":"OTU DOMAIN-CONTAINING PROTEIN 4; OTUD4","url":"https://www.omim.org/entry/611744"},{"mim_id":"609948","title":"RING FINGER PROTEIN 216; RNF216","url":"https://www.omim.org/entry/609948"},{"mim_id":"212840","title":"GORDON HOLMES SYNDROME; GDHS","url":"https://www.omim.org/entry/212840"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/OTUD4"},"hgnc":{"alias_symbol":["HSHIN1","KIAA1046","DUBA6"],"prev_symbol":[]},"alphafold":{"accession":"Q01804","domains":[{"cath_id":"3.90.70.80","chopping":"25-192_244-274","consensus_level":"medium","plddt":91.9933,"start":25,"end":274},{"cath_id":"2.30.30,2.30.30","chopping":"285-333","consensus_level":"high","plddt":58.0578,"start":285,"end":333},{"cath_id":"-","chopping":"660-735","consensus_level":"high","plddt":66.4125,"start":660,"end":735}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q01804","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q01804-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q01804-F1-predicted_aligned_error_v6.png","plddt_mean":48.91},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=OTUD4","jax_strain_url":"https://www.jax.org/strain/search?query=OTUD4"},"sequence":{"accession":"Q01804","fasta_url":"https://rest.uniprot.org/uniprotkb/Q01804.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q01804/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q01804"}},"corpus_meta":[{"pmid":"30410068","id":"PMC_30410068","title":"Induction 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Metastases of Triple-Negative Breast Cancer.","date":"2025","source":"Neoplasia (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/41297414","citation_count":0,"is_preprint":false},{"pmid":"41687501","id":"PMC_41687501","title":"OTUD4 regulates pancreatic cancer progression via Hippo/YAP axis.","date":"2026","source":"Neoplasia (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/41687501","citation_count":0,"is_preprint":false},{"pmid":"41266709","id":"PMC_41266709","title":"OTUD4 suppresses colorectal cancer progression through deubiquitinating p53.","date":"2025","source":"Discover oncology","url":"https://pubmed.ncbi.nlm.nih.gov/41266709","citation_count":0,"is_preprint":false},{"pmid":"41652905","id":"PMC_41652905","title":"Fenofibrate potentiates the therapeutic efficacy of EZH2 inhibitors on melanoma via TRIM21- and OTUD4-mediated EZH2 ubiquitination.","date":"2026","source":"British journal of 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pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2015,\n      \"finding\": \"OTUD4 acts as a scaffold (in a catalytic-activity-independent manner) to recruit the deubiquitinases USP7 and USP9X, which in turn directly deubiquitinate and stabilize the DNA demethylases ALKBH2 and ALKBH3, thereby promoting resistance to DNA alkylation damage.\",\n      \"method\": \"Co-IP/pulldown, catalytic-dead mutant analysis, loss-of-function (knockdown of OTUD4, USP7, USP9X) with alkylating-agent sensitivity readout\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, catalytic-dead mutant ruling out direct DUB activity, multiple deubiquitinase knockdowns with defined cellular phenotype; moderate evidence from single lab with orthogonal methods\",\n      \"pmids\": [\"25944111\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"OTUD4 is phosphorylated near its catalytic domain, which converts its nominal K48-linkage specificity into K63-linkage-specific deubiquitinase activity; this phospho-activated K63 activity also requires an adjacent ubiquitin-interacting motif (UIM) that increases affinity for K63-linked chains. The K63 deubiquitinase targets MyD88, thereby negatively regulating TLR-mediated NF-κB activation.\",\n      \"method\": \"In vitro DUB linkage-specificity assay, phospho-mutant analysis, UIM deletion, macrophage KO (Otud4-/- mice) with TLR stimulation and NF-κB readout, mass spectrometry phosphoproteomics\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro biochemical linkage-specificity assays, phospho-mutant and UIM mutant dissection, genetic KO with defined inflammatory phenotype in primary macrophages\",\n      \"pmids\": [\"29395066\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Viral infection induces IRF3/7-dependent upregulation of OTUD4, which then interacts with MAVS and removes K48-linked polyubiquitin chains from MAVS, thereby stabilizing MAVS and promoting IRF3/NF-κB-dependent antiviral type I interferon signaling.\",\n      \"method\": \"Co-IP, ubiquitination assay, OTUD4 KO/knockdown (conditional Otud4fl/fl mice and cell lines), VSV infection with IFN/cytokine readout, MAVS reconstitution rescue experiment\",\n      \"journal\": \"Cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP, ubiquitin linkage assay, conditional KO mice with in vivo viral challenge, and reconstitution rescue; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"30410068\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"OTUD4 is a component of neuronal RNA transport granules under physiological conditions and is recruited to cytoplasmic stress granules upon cellular stress; it interacts with a network of RNA-binding proteins including FMRP, SMN1, G3BP1, and TIA1 in an RNA-dependent manner, and is itself an RNA-binding protein. Knockdown of OTUD4 impairs stress granule formation and leads to apoptotic cell death.\",\n      \"method\": \"Pulldown + mass spectrometry on mouse brain lysates, RNA-dependence assay, live-cell imaging of neuronal RNA granules, stress granule immunofluorescence, siRNA knockdown with apoptosis readout\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — pulldown/MS for interactome, direct RNA-binding shown, localization by imaging with functional consequence (stress granule defect, apoptosis); single lab\",\n      \"pmids\": [\"31138677\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"OTUD4 interacts with TRAF6 and removes K63-linked auto-polyubiquitin chains from TRAF6, thereby suppressing NF-κB activation and reducing inflammatory responses in hepatic ischemia-reperfusion injury.\",\n      \"method\": \"Co-IP, ubiquitination assay (K63 linkage), OTUD4 overexpression in hypoxia/reoxygenation model and in vivo hepatic IR model with NF-κB and cytokine readouts\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — Co-IP, K63-specific ubiquitination assay, in vivo overexpression model; single lab, moderate methods\",\n      \"pmids\": [\"31964525\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"OTUD4 enhances TGFβ signalling through both catalytically dependent and independent mechanisms: it promotes membrane retention of TGFβ receptor I and inactivates the TGFβ negative regulator SMURF2; OTUD4 is itself a transcriptional target of TGFβ, forming a positive feedback loop.\",\n      \"method\": \"Catalytic-dead mutant analysis, receptor surface assay, Co-IP, loss-of-function with TGFβ pathway readouts (SMAD phosphorylation)\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — catalytic-dead mutant dissection, Co-IP, functional TGFβ signaling readout; single lab with multiple approaches\",\n      \"pmids\": [\"32973272\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"OTUD4 directly deubiquitinates GSDME, removing ubiquitin chains and preventing its proteasomal degradation, thereby stabilizing GSDME and enhancing radiation-induced pyroptosis (caspase-3-mediated GSDME cleavage) and radiosensitivity in nasopharyngeal carcinoma.\",\n      \"method\": \"Co-IP, mass spectrometry, ubiquitination assay, in vitro and in vivo functional assays with OTUD4/GSDME modulation, live-cell imaging of pyroptosis\",\n      \"journal\": \"Journal of experimental & clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — Co-IP, MS identification, ubiquitination assay, in vivo tumor model; single lab\",\n      \"pmids\": [\"36411454\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"OTUD4 interacts with and deubiquitinates PFKFB3, a master activator of glycolysis, preventing its degradation and thereby promoting TGFβ1-driven cardiac fibroblast activation and cardiac fibrosis after myocardial infarction.\",\n      \"method\": \"Co-IP, ubiquitination assay, OTUD4 knockdown/overexpression in cardiac fibroblasts, in vivo post-MI mouse model with fibrosis readout\",\n      \"journal\": \"Journal of molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — Co-IP, deubiquitination assay, in vivo model; single lab\",\n      \"pmids\": [\"37162556\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In Paneth cells, OTUD4 deubiquitinates MyD88 (removing K63-linked chains), thereby dampening NF-κB and MAPK activation and restricting antimicrobial peptide expression; knockout of OTUD4 leads to hyper-K63-ubiquitination of MyD88, increased AMP production, and resistance to DSS colitis and Salmonella infection.\",\n      \"method\": \"Conditional KO mice (Vil-Cre and Def-Cre Otud4fl/fl), intestinal organoids, ubiquitination assay, LPS/PGN stimulation with NF-κB/MAPK readouts\",\n      \"journal\": \"Cell insight\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — cell-type-specific conditional KO mice, ubiquitination assay, defined mechanistic node (MyD88 K63-deubiquitination), in vivo infection models; single lab but multiple orthogonal approaches\",\n      \"pmids\": [\"37193092\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"IRTKS recruits OTUD4 to SETDB1, where OTUD4 removes K48-linked polyubiquitin chains at K182/K1050 of SETDB1, preventing proteasomal degradation of SETDB1, increasing H3K9me3 at the CDH1 locus, and suppressing E-cadherin expression to drive EMT and tumor metastasis.\",\n      \"method\": \"Co-IP, ubiquitination assay (K48 linkage, specific sites identified), chromatin accessibility assay, E-cadherin transcription readout, in vitro invasion and in vivo metastasis assays\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — Co-IP, site-specific ubiquitination assay, downstream epigenetic and transcriptional readout; single lab\",\n      \"pmids\": [\"37739210\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"OTUD4 directly interacts with and deubiquitinates Snail1, preventing its degradation and thereby promoting epithelial-mesenchymal transition and metastasis in triple-negative breast cancer.\",\n      \"method\": \"Co-IP, ubiquitination assay, OTUD4 knockdown with migration/invasion/metastasis readouts, reconstitution of Snail1 in OTUD4-deficient cells\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — Co-IP, deubiquitination assay, rescue experiment with Snail1 reconstitution; single lab\",\n      \"pmids\": [\"38040050\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"OTUD4 directly interacts with CDK1 and removes K11-, K29-, and K33-linked polyubiquitin chains to stabilize CDK1; it also binds FGFR1 and reduces K6- and K27-linked polyubiquitination of FGFR1, thereby indirectly further stabilizing CDK1 and activating the downstream MAPK signaling pathway in glioblastoma.\",\n      \"method\": \"Co-IP, ubiquitination assay (linkage-specific), OTUD4 knockdown/overexpression with proliferation/invasion readouts, in vivo xenograft\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — Co-IP, linkage-specific ubiquitination assay, in vivo xenograft; single lab\",\n      \"pmids\": [\"38429268\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"OTUD4 deubiquitinates and stabilizes CD73, counteracting TRIM21-mediated ubiquitination of CD73, within a TGF-β-orchestrated signaling axis; this OTUD4/CD73 proteolytic axis suppresses antitumor immune responses in triple-negative breast cancer.\",\n      \"method\": \"Multiomic analysis, Co-IP, ubiquitination assay (TRIM21 as E3 ligase), pharmacological inhibitor (ST80) disrupting OTUD4-CD73 interaction, in vivo TNBC models with immune readout\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP, defined E3/DUB pair, specific inhibitor validation, in vivo immunological readout; single lab but multiple orthogonal approaches\",\n      \"pmids\": [\"38530357\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"OTUD4 acts as a non-catalytic adaptor to recruit USP7 to the KSHV replication and transcription activator (K-RTA), facilitating K-RTA deubiquitination by USP7 and thereby stabilizing K-RTA to promote KSHV lytic reactivation.\",\n      \"method\": \"Co-IP, ubiquitination assay, OTUD4 catalytic-dead mutant (showing DUB activity dispensable), USP7 recruitment assay, viral reactivation readout\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP, catalytic-dead mutant excluding direct DUB activity, USP7 recruitment shown, viral reactivation phenotype; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"38215174\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"OTUD4 directly interacts with and deubiquitinates RBM47, preventing its proteasomal degradation; stabilized RBM47 promotes ATF3 mRNA stability and ATF3-mediated ferroptosis, suppressing ccRCC progression.\",\n      \"method\": \"Co-IP, ubiquitination assay, OTUD4 overexpression/knockdown, RBM47 interference rescue experiment, ferroptosis assay\",\n      \"journal\": \"Apoptosis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — Co-IP, deubiquitination assay, rescue experiment; single lab\",\n      \"pmids\": [\"38553613\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"OTUD4 interacts with core components of the TAK1 signalosome (TAK1, TAB1, TAB3) and removes K63-linked polyubiquitin chains from TAK1 and TAB3, suppressing TNF-induced NF-κB activation. A histidine-centered loop (His loop) in the catalytic domain confers K63-linkage specificity; a tumor-associated H148Y variant in this loop retains TAK1 binding but abolishes DUB activity, leading to sustained NF-κB activation.\",\n      \"method\": \"Co-IP, in vitro K63-specific DUB assay, His-loop mutant and H148Y variant analysis, TNF stimulation with NF-κB readout\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro DUB assay, active-site mutagenesis (H148Y), Co-IP with signalosome components; single lab\",\n      \"pmids\": [\"41062071\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"OTUD4 directly deubiquitinates GPX4, removing ubiquitin chains to prevent its proteasomal degradation; OTUD4 also impedes RHEB-mediated autophagic degradation of GPX4, collectively suppressing ferroptosis and promoting tumor cell survival.\",\n      \"method\": \"CRISPR-Cas9 KO cell lines, ferroptosis inducer sensitivity assay, Co-IP, ubiquitination assay, RHEB-autophagy axis analysis, in vivo tumor models\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — CRISPR KO, Co-IP, ubiquitination assay, in vivo tumor growth; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"40338740\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"OTUD4 interacts with UBAC1 and maintains UBAC1 protein stability through deubiquitination; OTUD4 deficiency decreases UBAC1 expression, thereby impairing autophagic flux in cardiomyocytes and modulating myocardial ischemia-reperfusion injury.\",\n      \"method\": \"Co-IP, ubiquitination assay, OTUD4 knockdown/overexpression in OGD-R cell model and LAD-ligation MIRI mouse model, autophagic flux markers (LC3, p62, LAMP2), AKT/mTOR pathway readout\",\n      \"journal\": \"Journal of molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — Co-IP, deubiquitination assay, in vivo cardiac model; single lab\",\n      \"pmids\": [\"41107601\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SPARC competitively binds OTUD4 in conjunction with MyD88, disrupting OTUD4's deubiquitinase regulation of MyD88 and facilitating p65 nuclear translocation, thereby activating the p65-MLCK/MLC2 pathway and compromising intestinal barrier integrity in Crohn's disease.\",\n      \"method\": \"Co-IP, competitive binding assay, p65 nuclear translocation imaging, SPARC KO mouse colitis model, barrier permeability readouts\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — Co-IP showing competitive binding, in vivo KO model, signaling pathway readouts; single lab\",\n      \"pmids\": [\"39888301\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"OTUD4 directly interacts with and deubiquitinates p53, stabilizing it and enhancing its transcriptional activity to suppress colorectal cancer progression; p53 knockdown abrogates OTUD4-mediated tumor suppression.\",\n      \"method\": \"Co-IP, ubiquitination assay, OTUD4 knockdown/overexpression, p53 knockdown rescue experiment, proliferation/migration/invasion readouts\",\n      \"journal\": \"Discover oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — Co-IP, deubiquitination assay, genetic rescue; single lab\",\n      \"pmids\": [\"41266709\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TRIM21 promotes K48-linked ubiquitination of the small hepatitis B surface antigen (SHBs) at K122 leading to proteasomal degradation, while OTUD4 counteracts TRIM21-induced ubiquitination to stabilize SHBs; OTUD4 interacts with SHBs via its N-terminal 1–180 amino acids.\",\n      \"method\": \"Co-IP, GST pulldown (in vitro), K48-ubiquitination assay (site-directed at K122), TRIM21/OTUD4 overexpression with SHBs stability readout\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 — GST pulldown (in vitro), Co-IP, site-specific ubiquitination assay; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"40277358\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"OTUD4 directly deubiquitinates and stabilizes ZMYND8, promoting assembly of the DDX3X-CK1ε complex that activates WNT/β-catenin signaling, upregulates CSF1, and drives M2 macrophage polarization to foster an immunosuppressive niche in TNBC spinal metastasis.\",\n      \"method\": \"Co-IP, ubiquitination assay, OTUD4 KO/overexpression, in vivo spinal metastasis model, macrophage co-culture readouts\",\n      \"journal\": \"Neoplasia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — Co-IP, deubiquitination assay, in vivo model; single lab\",\n      \"pmids\": [\"41297414\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"WSB1 promotes interaction between ISOC2 and OTUD4, leading to OTUD4-mediated deubiquitination and stabilization of ISOC2, which suppresses P16INK4a expression and drives prostate cancer progression.\",\n      \"method\": \"Mass spectrometry, Co-IP, ubiquitination assay, WSB1/ISOC2/OTUD4 knockdown with proliferation/migration readouts, in vivo xenograft\",\n      \"journal\": \"American journal of cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — MS, Co-IP, deubiquitination assay, in vivo xenograft; single lab\",\n      \"pmids\": [\"41244113\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"OTUD4 directly deubiquitinates MYH9, preventing its proteasomal degradation, thereby stabilizing MYH9 which suppresses prostate cancer progression through interactions with cell adhesion molecules.\",\n      \"method\": \"Co-IP, mass spectrometry, ubiquitination assay, OTUD4 KO/overexpression, in vivo xenograft\",\n      \"journal\": \"Oncology research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — Co-IP, MS, deubiquitination assay, in vivo model; single lab\",\n      \"pmids\": [\"41930143\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"OTUD4 stabilizes EGFR by directly interacting with it (via OTUD4 aa 568–1114 and EGFR aa 958–1210) and cleaving K48-linked polyubiquitin chains; OTUD4 is additionally recruited by NRP1 to further deubiquitinate and stabilize EGFR, activating the PI3K/AKT pathway in TNBC.\",\n      \"method\": \"Co-IP (domain-mapping), ubiquitination assay (K48 linkage), OTUD4 knockdown, NRP1 co-recruitment assay, PI3K/AKT pathway readout\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — domain-mapped Co-IP, K48 ubiquitination assay, NRP1 recruitment; single lab\",\n      \"pmids\": [\"41730840\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"OTUD4 is an OTU-family deubiquitinase with dual K48 and (phospho-activated, UIM-assisted) K63 deubiquitinase activities that targets a broad range of substrates—including MAVS, MyD88, TRAF6, TAK1/TAB3, CDK1, FGFR1, GSDME, GPX4, Snail1, SETDB1, CD73, p53, RBM47, PFKFB3, ZMYND8, EGFR, and others—to regulate innate immune signaling, TGFβ signaling, NF-κB/MAPK pathways, ferroptosis, DNA repair, stress granule assembly, and tumor progression; it can also act as a catalysis-independent scaffold to recruit USP7 and USP9X to specific substrates (ALKBH2/3, K-RTA).\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"OTUD4 is an OTU-family deubiquitinase that functions as both a catalytically active enzyme with broad ubiquitin-chain specificity and a non-catalytic scaffold for recruiting other deubiquitinases, thereby regulating innate immune signaling, stress responses, DNA repair, and tumor progression. Its intrinsic K48-directed deubiquitinase activity stabilizes diverse substrates—including MAVS, GSDME, GPX4, SETDB1, CD73, p53, Snail1, EGFR, and others—by preventing their proteasomal degradation [PMID:30410068, PMID:36411454, PMID:40338740, PMID:37739210, PMID:38530357, PMID:41266709, PMID:41730840], while phosphorylation near its catalytic domain, together with an adjacent ubiquitin-interacting motif, switches its specificity toward K63-linked chains, enabling it to deubiquitinate MyD88, TRAF6, and TAK1/TAB3 to restrain NF-κB and MAPK signaling [PMID:29395066, PMID:31964525, PMID:41062071, PMID:37193092]. Independent of catalytic activity, OTUD4 scaffolds USP7 and USP9X to stabilize the DNA alkylation-repair demethylases ALKBH2/3 and the viral activator K-RTA [PMID:25944111, PMID:38215174]. OTUD4 additionally localizes to neuronal RNA transport granules and stress granules, binds RNA, and is required for stress granule assembly and cell survival under stress [PMID:31138677].\",\n  \"teleology\": [\n    {\n      \"year\": 2015,\n      \"claim\": \"Establishing that OTUD4 can function independently of its own catalytic activity as a scaffold to recruit USP7 and USP9X answered how alkylation-repair demethylases ALKBH2/3 are stabilized and revealed an unexpected non-enzymatic mode of action for an OTU-family protein.\",\n      \"evidence\": \"Co-IP, catalytic-dead mutant analysis, and knockdown of OTUD4/USP7/USP9X with alkylating-agent sensitivity readout in human cells\",\n      \"pmids\": [\"25944111\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the scaffold function extends to other substrates beyond ALKBH2/3\", \"Structural basis of OTUD4–USP7/USP9X interaction unknown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Demonstrating that phosphorylation near the catalytic domain switches OTUD4 from K48 to K63 specificity—dependent on an adjacent UIM—resolved how a single DUB achieves dual-linkage specificity and identified MyD88 as a physiological K63-deubiquitination target that restrains TLR-driven NF-κB activation.\",\n      \"evidence\": \"In vitro linkage-specificity assays, phospho- and UIM-mutant analysis, Otud4-KO macrophages with TLR stimulation\",\n      \"pmids\": [\"29395066\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the kinase(s) responsible for activating phosphorylation\", \"Whether phospho-switching is reversible and dynamically regulated in vivo\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Showing that OTUD4 removes K48-linked ubiquitin from MAVS to stabilize it during viral infection established OTUD4 as a positive regulator of antiviral innate immunity, complementing its negative role in TLR/NF-κB signaling.\",\n      \"evidence\": \"Conditional Otud4-KO mice challenged with VSV, Co-IP, K48-ubiquitination assay, MAVS reconstitution rescue\",\n      \"pmids\": [\"30410068\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"E3 ligase whose K48-ubiquitination of MAVS is countered by OTUD4 not identified\", \"Whether OTUD4 acts on other RLR-pathway components\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identifying OTUD4 as an RNA-binding protein that localizes to neuronal RNA transport granules and is required for stress granule assembly expanded its functional repertoire beyond ubiquitin biology into RNA metabolism and stress adaptation.\",\n      \"evidence\": \"Pulldown/MS from mouse brain, RNA-dependence assay, stress granule imaging, siRNA knockdown with apoptosis readout\",\n      \"pmids\": [\"31138677\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"RNA targets of OTUD4 not identified\", \"Whether DUB activity is required for RNA granule function\", \"Independent replication in non-neuronal cell types needed\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Demonstrating OTUD4-mediated K63-deubiquitination of TRAF6 and enhancement of TGFβ signaling (via TGFβRI retention and SMURF2 inactivation) broadened the set of immune and developmental pathways regulated by OTUD4.\",\n      \"evidence\": \"Co-IP, K63 ubiquitination assay in hepatic IR model; catalytic-dead mutant and receptor-surface assay for TGFβ axis\",\n      \"pmids\": [\"31964525\", \"32973272\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How OTUD4 inactivates SMURF2 mechanistically unclear\", \"TGFβ feedback loop not validated in vivo\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identifying OTUD4 as a deubiquitinase for multiple pro-tumorigenic and metabolic substrates—GSDME (pyroptosis), PFKFB3 (glycolysis), SETDB1 (epigenetics/EMT), Snail1 (EMT)—and confirming MyD88 K63-deubiquitination in Paneth cells in vivo consolidated OTUD4 as a versatile regulator of cell fate and inflammation across tissues.\",\n      \"evidence\": \"Co-IP and ubiquitination assays for each substrate; conditional intestinal KO mice (Paneth-cell-specific) with infection challenge; in vivo tumor and cardiac fibrosis models\",\n      \"pmids\": [\"36411454\", \"37162556\", \"37739210\", \"38040050\", \"37193092\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How substrate selectivity is determined among many targets is unknown\", \"Whether OTUD4 activity on these substrates is phospho-regulated as for MyD88 is untested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Revealing that OTUD4 cleaves atypical ubiquitin chains (K11, K29, K33 on CDK1; K6, K27 on FGFR1) and stabilizes GPX4 to suppress ferroptosis while also stabilizing CD73 to enable immune evasion extended its chain-type repertoire and linked it to ferroptosis regulation and tumor immunology.\",\n      \"evidence\": \"Linkage-specific ubiquitination assays, CRISPR-KO ferroptosis sensitivity, TRIM21-counteracting ubiquitination of CD73, in vivo TNBC immune readout, pharmacological inhibitor ST80\",\n      \"pmids\": [\"38429268\", \"40338740\", \"38530357\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How OTUD4 achieves specificity for such diverse chain types is structurally unresolved\", \"ST80 inhibitor mechanism and selectivity not fully characterized\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Structural and functional dissection of OTUD4's His-loop conferring K63 specificity, identification of a tumor-associated H148Y loss-of-function variant, and expansion of the substrate repertoire to include p53, ZMYND8, MYH9, ISOC2, UBAC1, EGFR, RBM47, and viral SHBs established OTUD4 as a broadly acting deubiquitinase with clinical relevance, and confirmed the scaffold function for USP7 in KSHV reactivation.\",\n      \"evidence\": \"His-loop mutagenesis and in vitro DUB assay; domain-mapped Co-IPs; Co-IP/deubiquitination assays with rescue experiments; in vivo models (spinal metastasis, xenograft, cardiac IR, colitis); competitive binding assay (SPARC–OTUD4–MyD88)\",\n      \"pmids\": [\"41062071\", \"41266709\", \"41297414\", \"41930143\", \"41244113\", \"41107601\", \"41730840\", \"38215174\", \"39888301\", \"40277358\", \"38553613\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No crystal structure of OTUD4 catalytic domain available\", \"Relative contribution of catalytic vs. scaffold activities in physiological settings unknown\", \"Most substrate studies from single laboratories awaiting independent replication\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key open questions include the structural basis of OTUD4's unusually broad chain-type specificity and phospho-switching mechanism, the identity of kinase(s) regulating the K48-to-K63 switch, how substrate selectivity is achieved among dozens of targets, and whether the scaffold and DUB activities are coordinated or context-exclusive.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No crystal or cryo-EM structure of OTUD4\", \"Kinase(s) for activating phosphorylation unidentified\", \"Systematic substrate-prioritization under physiological conditions not performed\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [1, 2, 4, 6, 8, 9, 10, 11, 14, 15, 16, 17, 19, 20, 21, 22, 23, 24]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 2, 4, 6, 8, 9, 10, 11, 14, 15, 16, 17, 19, 20, 21, 22, 23, 24]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 13]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [3, 8]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [9]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [1, 2, 4, 8, 15, 18]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [5, 7, 11, 12, 21, 24]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 6, 9, 10, 16, 17, 19, 20, 22, 23]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [6, 14, 16]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [9, 10, 11, 12, 22, 24]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"USP7\",\n      \"USP9X\",\n      \"MyD88\",\n      \"MAVS\",\n      \"TRAF6\",\n      \"TAK1\",\n      \"GPX4\",\n      \"EGFR\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}