{"gene":"OTUD1","run_date":"2026-04-29T11:37:57","timeline":{"discoveries":[{"year":2018,"finding":"OTUD1 is a deubiquitinase that removes K63-linked polyubiquitin chains from YAP, reversing SCFSKP2-mediated non-proteolytic K63-linked ubiquitination at K321 and K497. This ubiquitination promotes YAP interaction with TEAD, nuclear localization, and transcriptional activity independently of Hippo signaling; OTUD1 overexpression retains YAP in the cytoplasm and inhibits its activity.","method":"DUB library screen, co-immunoprecipitation, ubiquitination assays, site-directed mutagenesis, overexpression/knockdown with localization readouts","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, ubiquitin linkage mapping, mutagenesis, functional readout; replicated in companion commentary","pmids":["29891922","31225491"],"is_preprint":false},{"year":2017,"finding":"OTUD1 directly deubiquitinates SMAD7, cleaving K33-linked polyubiquitin chains at SMAD7 K220, which prevents SMAD7 proteasomal degradation and exposes the SMAD7 PY motif for SMURF2 binding, leading to TβRI turnover at the cell surface and suppression of TGF-β pro-metastatic signaling.","method":"Loss-of-function screen in mice, co-immunoprecipitation, in vitro deubiquitination assay, ubiquitin linkage-specific analysis, site-directed mutagenesis, xenograft and orthotopic transplantation models","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro deubiquitination, mutagenesis at SMAD7 K220, multiple in vivo models, mechanistic pathway placement","pmids":["29235476"],"is_preprint":false},{"year":2021,"finding":"OTUD1 deubiquitinates and stabilizes IREB2 (iron-responsive element-binding protein 2), which in turn promotes TFRC-mediated iron transport, increases ROS generation, and induces ferroptosis; OTUD1 loss reduces iron import and dampens tumor-reactive T-cell accumulation.","method":"Co-immunoprecipitation, ubiquitination assay, genetic knockdown/knockout with ferroptosis and immune cell readouts","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 2 — Co-IP plus functional rescue, multiple cellular and in vivo readouts","pmids":["33393230"],"is_preprint":false},{"year":2021,"finding":"OTUD1 physically interacts with RIPK1 and selectively cleaves K63-linked polyubiquitin chains from RIPK1, thereby inhibiting recruitment of NEMO and suppressing NF-κB activation; a UC-associated OTUD1 G430V mutation abolishes this activity.","method":"Co-immunoprecipitation, ubiquitin linkage-specific assay, knockout mouse model (DSS colitis), bone marrow transplantation, patient mutation functional analysis","journal":"Cellular & molecular immunology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, linkage-specific deubiquitination, KO mouse with defined phenotype, disease-variant validation","pmids":["34876703"],"is_preprint":false},{"year":2018,"finding":"RNA virus infection induces OTUD1 expression via NF-κB-dependent mechanisms; OTUD1 then deubiquitinates and stabilizes the E3 ligase Smurf1, which promotes ubiquitination-dependent degradation of MAVS, TRAF3, and TRAF6, thereby inhibiting type I IFN production as a negative feedback loop.","method":"Co-immunoprecipitation, ubiquitination assay, OTUD1-deficient mice with viral infection challenge, overexpression/knockdown","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 — mechanistic pathway reconstituted in cells and validated in KO mice with viral challenge","pmids":["29734366"],"is_preprint":false},{"year":2020,"finding":"OTUD1 deubiquitinates IRF3 by cleaving K6-linked polyubiquitin chains at IRF3 K98; K6-linked ubiquitination is required for IRF3 DNA-binding capacity at IFN gene promoters without affecting IRF3 stability, dimerization, or nuclear translocation. OTUD1 removal of this modification dissociates IRF3 from target gene promoters.","method":"Co-immunoprecipitation, linkage-specific ubiquitination assay, chromatin immunoprecipitation, Otud1-/- cells and mice with viral/LPS challenge","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 — linkage-specific deubiquitination, ChIP validation, KO mouse phenotype, multiple orthogonal methods","pmids":["32075857"],"is_preprint":false},{"year":2018,"finding":"OTUD1 directly interacts with IRF3 and removes K63-linked polyubiquitin chains at IRF3 K98, inhibiting IRF3 nuclear translocation and transcriptional activity; loss-of-function OTUD1 mutations found in autoimmune patients impair this deubiquitinase activity or IRF3 association. FOXO3 signaling is required for OTUD1 induction upon antigenic stimulation.","method":"Co-immunoprecipitation, ubiquitination assay, patient mutation functional analysis, OTUD1-deficient cellular models","journal":"Journal of autoimmunity","confidence":"High","confidence_rationale":"Tier 2 — patient variants tested functionally, Co-IP, ubiquitination assay, pathway placement","pmids":["30100102"],"is_preprint":false},{"year":2022,"finding":"OTUD1 suppresses canonical NF-κB activation by hydrolyzing K63-linked ubiquitin chains from NF-κB signaling factors including LUBAC substrates. OTUD1 also binds KEAP1 through an ETGE motif in its N-terminal intrinsically disordered region, regulating the antioxidant response and ROS-induced cell death (oxeiptosis). In Otud1-/- mice, inflammation and oxidative damage are enhanced in IBD, acute hepatitis, and sepsis models.","method":"DUB screen (88 human DUBs), mass spectrometry interactome, ubiquitination assay, Otud1-/- mouse models, domain deletion analysis","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 2 — systematic screen, MS interactome, domain mapping, multiple KO mouse disease models","pmids":["35941131"],"is_preprint":false},{"year":2021,"finding":"OTUD1 deubiquitinates AIF at K244 (disrupting mitochondrial structure and oxidative phosphorylation) and at K255 (enhancing AIF DNA-binding ability to promote caspase-independent parthanatos). OTUD1 also stabilizes DCAF10 and recruits the CUL4A-DDB1 complex to promote MCL1 degradation, activating caspase-dependent apoptosis.","method":"Co-immunoprecipitation, site-directed mutagenesis at AIF K244 and K255, ubiquitination assays, functional apoptosis readouts","journal":"Advanced science","confidence":"High","confidence_rationale":"Tier 1–2 — mutagenesis at specific ubiquitination sites, reconstituted complex, dual apoptotic pathway readouts","pmids":["33898171"],"is_preprint":false},{"year":2021,"finding":"OTUD1 directly interacts with CARD9 and cleaves polyubiquitin chains from CARD9, which is required for CARD9-mediated NF-κB and MAPK activation in antifungal innate immunity; OTUD1-deficient mice are more susceptible to fungal infection.","method":"Co-immunoprecipitation, ubiquitination assay, Otud1-/- mouse fungal infection model","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 — Co-IP, deubiquitination assay, KO mouse in vivo phenotype","pmids":["33789983"],"is_preprint":false},{"year":2023,"finding":"OTUD1 binds the SH2 domain of STAT3 and deubiquitinates STAT3 K63-linked chains via its catalytic C320 residue, promoting STAT3 phosphorylation and nuclear translocation, thereby inducing inflammatory, fibrotic, and hypertrophic responses in cardiomyocytes; OTUD1 KO mice are protected from angiotensin II- and TAC-induced cardiac remodeling.","method":"LC-MS/MS combined with Co-IP, site-directed mutagenesis (C320), AAV9 overexpression, OTUD1 KO mouse models","journal":"Theranostics","confidence":"High","confidence_rationale":"Tier 2 — MS-based substrate identification, mutagenesis, gain/loss-of-function mouse models with defined cardiac phenotype","pmids":["37153745"],"is_preprint":false},{"year":2022,"finding":"OTUD1's N-terminal intrinsically disordered region contains a short peptide (OUN-36) that binds the Akt PH domain at residues required for PtdIns(3,4,5)P3 recognition, inhibiting Akt membrane localization and phosphorylation independently of OTUD1 deubiquitinase activity.","method":"Co-immunoprecipitation, domain deletion analysis, peptide binding assay, cell membrane localization studies, Akt phosphorylation assays","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 — mechanistic domain dissection, PH domain binding identified, deubiquitinase-independent mechanism established","pmids":["36640312"],"is_preprint":false},{"year":2023,"finding":"OTUD1 interacts with CARD9 and removes K33-linked ubiquitin from CARD9, promoting assembly of the CARD9-BCL10-MALT1 (CBM) complex and NF-κB activation in macrophages during isoproterenol-induced cardiac inflammation; myeloid-specific OTUD1 deletion attenuates cardiac remodeling.","method":"Co-immunoprecipitation, linkage-specific ubiquitination assay, Card9-/- and Otud1-/- bone marrow reconstitution, primary macrophage mechanistic studies","journal":"Clinical and translational medicine","confidence":"High","confidence_rationale":"Tier 2 — linkage-specific deubiquitination, myeloid-specific KO reconstitution, CBM complex assembly validated","pmids":["39118286"],"is_preprint":false},{"year":2024,"finding":"OTUD1 binds directly to GAF1 and PDEase domains of PDE5A and reverses K48-linked ubiquitination of PDE5A via its C320 catalytic residue, preventing proteasomal degradation of PDE5A, which inactivates the cGMP-PKG-SERCA2a signaling axis and dysregulates calcium handling in cardiomyocytes, promoting heart failure.","method":"LC-MS/MS combined with Co-IP, site-directed mutagenesis (C320), OTUD1 KO mouse (ISO and MI models), domain binding mapping","journal":"Biochimica et biophysica acta. Molecular basis of disease","confidence":"High","confidence_rationale":"Tier 2 — MS-identified interaction, catalytic site mutagenesis, domain mapping, KO mouse with cardiac phenotype","pmids":["38185350"],"is_preprint":false},{"year":2023,"finding":"OTUD1 interacts with Notch2-ICD (NICD) and cleaves ubiquitin at NICD K1770, leading to NICD protein accumulation in T cells, which promotes Th1/Th17 differentiation and amplifies graft-versus-host disease; dapagliflozin was identified as an inhibitor of the OTUD1/NICD axis.","method":"Co-immunoprecipitation, site-specific ubiquitination assay (K1770), FDA-approved drug screen, aGVHD mouse model","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 — site-specific deubiquitination, Co-IP, in vivo aGVHD model validation","pmids":["36574342"],"is_preprint":false},{"year":2022,"finding":"OTUD1 interacts with and deubiquitinates PTEN, stabilizing PTEN protein levels to suppress PI3K/AKT and TNF-α/NF-κB signaling in renal cancer cells.","method":"Co-immunoprecipitation, ubiquitination assay, RNA-seq pathway analysis, knockdown/overexpression","journal":"International journal of biological sciences","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP and deubiquitination assay, single lab, no site-specific mutagenesis","pmids":["35280681"],"is_preprint":false},{"year":2021,"finding":"OTUD1 interacts with MCL1 and promotes its deubiquitination in an enzymatic-activity-dependent manner, stabilizing MCL1 protein and antagonizing BH3-mimetic inhibitor-induced cell death.","method":"DUB library screen, co-immunoprecipitation, ubiquitination assay, cell viability assay","journal":"Cancer cell international","confidence":"Medium","confidence_rationale":"Tier 3 — single Co-IP/deubiquitination assay, DUB screen, single lab","pmids":["31467488"],"is_preprint":false},{"year":2021,"finding":"OTUD1 deubiquitinates and stabilizes Bim at lysine 3, preventing its proteasomal degradation; melatonin-induced Sp1 activation drives OTUD1 transcription, leading to Bim stabilization and apoptosis in cancer cells.","method":"Co-immunoprecipitation, site-directed mutagenesis (Bim K3), ubiquitination assay, siRNA knockdown, xenograft model","journal":"Journal of pineal research","confidence":"Medium","confidence_rationale":"Tier 2 — mutagenesis at K3, Co-IP, in vivo xenograft; single lab","pmids":["34826170"],"is_preprint":false},{"year":2022,"finding":"OTUD1 catalytic activity resolves ribosome stalling on polyA sequences by inhibiting ZNF598-mediated RPS10 ubiquitination, and promotes polysome formation; OTUD1 also associates with ribosome subunits and elongation factors and regulates stability of rare-codon-rich mRNAs by antagonizing ZNF598.","method":"Proximity labeling-based interactome (BioID), co-immunoprecipitation with ribosome subunits, polysome profiling, ubiquitination assay for RPS10, gene expression analysis","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 — proximity interactome plus biochemical validation, but single lab and novel function","pmids":["36445135"],"is_preprint":false},{"year":2023,"finding":"OTUD1 cleaves K63-linked polyubiquitin chains from SEC23B at K81, negatively regulating the COPII secretory machinery and limiting ER-to-Golgi protein trafficking, thereby restricting surface expression of CD9 and CD47 and suppressing secretory neutrophil polarization.","method":"Co-immunoprecipitation, linkage-specific ubiquitination assay (K81), neutrophil-specific OTUD1 depletion, Brefeldin A rescue experiment","journal":"Advanced science","confidence":"High","confidence_rationale":"Tier 2 — site-specific K63 deubiquitination, functional rescue with transport inhibitor, neutrophil-specific KO phenotype","pmids":["37639212"],"is_preprint":false},{"year":2023,"finding":"OTUD1 deubiquitinates and stabilizes NRF2 through its C320 catalytic residue and ETGE motif, attenuating hepatic ischemia/reperfusion-induced oxidative stress; an ETGE-containing peptide mimics this protective effect in vivo.","method":"Co-immunoprecipitation, deubiquitination assay, site-directed mutagenesis (C320), OTUD1 KO mouse (I/R model), ETGE peptide in vivo treatment","journal":"Redox biology","confidence":"High","confidence_rationale":"Tier 2 — catalytic site mutagenesis, ETGE motif functional validation, KO mouse with disease phenotype","pmids":["39079388"],"is_preprint":false},{"year":2023,"finding":"OTUD1 interacts with RIP2 and cleaves K63-linked polyubiquitin chains from RIP2, inhibiting NF-κB activation; OTUD1 deficiency exacerbates cerebral ischemic injury by allowing excess RIP2 ubiquitination and NF-κB-driven inflammation.","method":"Co-immunoprecipitation, confocal microscopy co-localization, immunofluorescence, dual-luciferase NF-κB assay, OTUD1-/- mouse (MCAO model)","journal":"Journal of neuroinflammation","confidence":"High","confidence_rationale":"Tier 2 — Co-IP, linkage-specific deubiquitination, KO mouse in vivo with defined ischemia phenotype","pmids":["38012669"],"is_preprint":false},{"year":2023,"finding":"OTUD1 deubiquitinates CDK9 via K63-linkage at its C320 catalytic residue, promoting CDK9 phosphorylation and activation to induce inflammatory responses and fibrosis in kidney epithelial cells; OTUD1 KO mice are protected from angiotensin II-induced hypertensive renal disease.","method":"Co-immunoprecipitation, site-directed mutagenesis (C320), OTUD1 KO mouse (Ang II model), CDK9 inhibitor rescue","journal":"Acta pharmacologica Sinica","confidence":"High","confidence_rationale":"Tier 2 — catalytic site mutagenesis, KO mouse disease model, pharmacological rescue","pmids":["38110583"],"is_preprint":false},{"year":2024,"finding":"OTUD1 directly binds AMPKα2, deubiquitinates it at K60/K379, and inhibits AMPKT172 phosphorylation by impeding the AMPKα2-CAMKK2 interaction, resulting in mitochondrial dysfunction in cardiomyocytes and diabetic cardiomyopathy; cardiomyocyte-specific OTUD1 KO restores AMPK activity.","method":"Co-immunoprecipitation, site-directed mutagenesis (K60/K379), cardiomyocyte-specific KO mouse (T1DM and T2DM models), single-cell RNA-seq, CAMKK2 interaction assay","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1–2 — site-specific mutagenesis, cardiomyocyte-specific KO, upstream kinase interaction assay, replicated in two diabetes models","pmids":["40683882"],"is_preprint":false},{"year":2025,"finding":"OTUD1 promotes HK2 dissociation from mitochondria via selective K63-linked deubiquitination of HK2, activating the NLRP3 inflammasome and triggering microglia pyroptosis, leading to neuroinflammation and cognitive deficits in sepsis-associated encephalopathy.","method":"Co-immunoprecipitation, molecular docking, 3D confocal microscopy, K63-specific ubiquitination assay, OTUD1 KO mouse (CLP model), single-cell RNA-seq","journal":"Journal of neuroinflammation","confidence":"High","confidence_rationale":"Tier 2 — linkage-specific deubiquitination, domain binding mapped, KO mouse in vivo, multiple validation methods","pmids":["40500776"],"is_preprint":false},{"year":2024,"finding":"OTUD1 deubiquitinates and stabilizes PGAM5 by reversing its ubiquitination, leading to robust activation of the ASK1-p38/JNK signaling pathway to accelerate cardiac hypertrophy; m6A modification by METTL3 of OTUD1 mRNA promotes its stability and elevated expression.","method":"RNA immunoprecipitation (RIP) for m6A, IP-MS for substrate identification, domain deletion constructs, cardiac-specific KO mice (TAC model), ASK1 knockdown rescue","journal":"International journal of biological sciences","confidence":"High","confidence_rationale":"Tier 2 — IP-MS substrate identification, KO mouse model, upstream m6A regulation validated, pathway rescue by ASK1 KD","pmids":["39309432"],"is_preprint":false},{"year":2023,"finding":"OTUD1 contains an N-terminal ETGE motif within an alanine/proline/glycine-rich intrinsically disordered region that mediates binding to KEAP1, placing OTUD1 in the KEAP1-NRF2 antioxidant axis; the OTU catalytic domain preferentially hydrolyzes K63-linked ubiquitin chains, with the UIM enhancing this preference.","method":"Domain analysis, in vitro ubiquitin chain hydrolysis assay, KEAP1 binding assay, UIM deletion analysis","journal":"Antioxidants","confidence":"Medium","confidence_rationale":"Tier 2 — review synthesizing prior biochemical data, domain-function relationships established","pmids":["36829909"],"is_preprint":false},{"year":2023,"finding":"An ubiquitin variant inhibitor (UbVOD.1) was developed that binds OTUD1 with nanomolar affinity bridging the catalytic OTU domain and UIM, inhibiting OTUD1 activity toward mono-Ub, K63-linked di-Ub, and poly-Ub chains of other linkages in vitro, and inhibiting OTUD1-mediated deubiquitination of RIPK1 in cells as a di-UbV.","method":"Phage display UbV library selection, in vitro deubiquitination IC50 assay, in vivo di-UbV expression with RIPK1 ubiquitination readout","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstituted inhibition with IC50 quantification, cellular validation with defined substrate","pmids":["37589489"],"is_preprint":false},{"year":2022,"finding":"OTUD1 deubiquitinates and stabilizes KLF4 in non-small cell lung cancer cells, with OTUD1 overexpression inhibiting NSCLC cell progression and KLF4 knockdown reversing this effect.","method":"Co-immunoprecipitation, cycloheximide chase, deubiquitination assay, siRNA knockdown rescue","journal":"Thoracic cancer","confidence":"Medium","confidence_rationale":"Tier 3 — Co-IP and deubiquitination assay, single lab, no site-specific mutagenesis","pmids":["35098684"],"is_preprint":false},{"year":2023,"finding":"OTUD1 deubiquitinates and stabilizes FHL1 in lung adenocarcinoma cells, inhibiting tumor proliferation and migration in vitro and in vivo.","method":"IP-MS, co-immunoprecipitation, ubiquitination assay, xenograft and urethane-induced lung cancer models","journal":"Cellular oncology","confidence":"Medium","confidence_rationale":"Tier 2 — IP-MS plus Co-IP, in vivo validation; single lab","pmids":["36929488"],"is_preprint":false},{"year":2025,"finding":"OTUD1 interacts with BST-2, reducing its K48- and K63-linked ubiquitination and increasing BST-2 protein stability, thereby inhibiting HIV-1 particle release.","method":"siRNA DUB screen, co-immunoprecipitation, ubiquitin linkage-specific assay, HIV-1 release assay","journal":"Viruses","confidence":"Medium","confidence_rationale":"Tier 2 — DUB screen followed by Co-IP and linkage-specific assay, functional viral release readout; single lab","pmids":["40007014"],"is_preprint":false},{"year":2025,"finding":"OTUD1 directly binds C/EBPβ and removes K48-linked ubiquitin chains at C/EBPβ K253, stabilizing C/EBPβ and activating C/EBPβ-NF-κB-mediated inflammatory responses in microglia, promoting Alzheimer's disease pathology.","method":"LC-MS/MS combined with Co-IP, site-directed mutagenesis (K253), OTUD1 knockdown in microglia, AD mouse model cognitive readout","journal":"Acta pharmacologica Sinica","confidence":"High","confidence_rationale":"Tier 2 — MS-based substrate identification, site-specific mutagenesis, in vivo AD model with cognitive phenotype","pmids":["40335710"],"is_preprint":false},{"year":2025,"finding":"OTUD1 deubiquitinates TIPE2, and this interaction suppresses TAK1-mediated MAPK and NF-κB signaling to protect against sepsis-induced lung injury.","method":"Co-immunoprecipitation, immunofluorescence co-localization, western blotting for deubiquitination, in vivo sepsis model","journal":"Biochemical pharmacology","confidence":"Medium","confidence_rationale":"Tier 3 — Co-IP and deubiquitination assay, single lab, pathway placement supported by prior TIPE2 literature","pmids":["38996928"],"is_preprint":false},{"year":2025,"finding":"OTUD1 directly interacts with β-catenin and reduces K63-linked ubiquitination at β-catenin K496, K508, and K625 via its C320 catalytic residue, facilitating β-catenin phosphorylation and restricting its nuclear translocation, thereby downregulating angiogenesis-related factors and impairing wound healing in diabetic mice.","method":"IP-MS, co-immunoprecipitation, site-directed mutagenesis (C320 and K496/K508/K625), endothelial-specific AAV knockdown, diabetic mouse wound healing model","journal":"Journal of advanced research","confidence":"High","confidence_rationale":"Tier 2 — IP-MS, site-specific mutagenesis at both catalytic and substrate ubiquitination sites, endothelial-specific in vivo KD","pmids":["40300668"],"is_preprint":false},{"year":2025,"finding":"OTUD1 deubiquitinates PRDX1 by reversing K48-linked ubiquitination, maintaining PRDX1 stability, mitigating mitochondrial dysfunction, and suppressing osteoclast differentiation; OTUD1 KO leads to enhanced osteoclastogenesis and reduced bone mass.","method":"LC-MS/MS combined with Co-IP, ubiquitin linkage assay, whole-body and myeloid-specific KO mice, micro-CT, histomorphometry","journal":"Theranostics","confidence":"High","confidence_rationale":"Tier 2 — MS-based substrate identification, linkage-specific assay, myeloid-specific KO with quantitative bone phenotype","pmids":["40585986"],"is_preprint":false},{"year":2025,"finding":"OTUD1 deubiquitinates p53 via K48-dependent deubiquitination through its 300–481 region, stabilizing p53 and promoting a procoagulant gene expression program in endothelial cells; an L357 point mutation of OTUD1 rescues endothelial homeostasis.","method":"Co-immunoprecipitation, domain deletion/point mutagenesis (L357), K48-specific ubiquitination assay, ROS manipulation, in vivo thrombosis model","journal":"Environment international","confidence":"Medium","confidence_rationale":"Tier 2 — domain and point mutagenesis, K48-specific assay, in vivo thrombus model; single lab","pmids":["41967174"],"is_preprint":false},{"year":2024,"finding":"OTUD1 binds RACK1 and cleaves K63-linked polyubiquitin chains from RACK1, enhancing RACK1 phosphorylation and modulating downstream MAPKs and NF-κB signaling to promote cardiomyocyte apoptosis after ischemia/reperfusion; RACK1 silencing reverses OTUD1-promoted H/R injury.","method":"IP-MS, co-immunoprecipitation, K63-specific deubiquitination assay, RACK1 siRNA rescue, OTUD1 KO mouse (I/R model)","journal":"Acta pharmacologica Sinica","confidence":"High","confidence_rationale":"Tier 2 — IP-MS substrate identification, linkage-specific assay, KO mouse model, epistasis by RACK1 KD","pmids":["40394237"],"is_preprint":false},{"year":2025,"finding":"OTUD1 deubiquitinates SMAD7/MADH7 in endometriosis stromal cells, inhibiting fibrosis-related protein expression and reducing endometriosis lesion growth in vivo.","method":"Co-immunoprecipitation, ubiquitination assay, in vitro stromal cell model, C57BL/6N mouse in vivo lesion model","journal":"Molecular human reproduction","confidence":"Medium","confidence_rationale":"Tier 3 — Co-IP and deubiquitination, consistent with SMAD7 mechanism described for breast cancer but single lab in new context","pmids":["40279273"],"is_preprint":false}],"current_model":"OTUD1 is an OTU-family deubiquitinase with a disordered N-terminal APGR/ETGE region, a catalytic OTU domain (active-site C320), and a UIM that enhances K63-chain preference; it removes K63-, K48-, K33-, and K6-linked ubiquitin chains from a broad substrate repertoire—including SMAD7, IRF3, RIPK1, CARD9, STAT3, AIF, MCL1, IREB2, NRF2, YAP, AKT, PTEN, AMPKα2, PDE5A, CDK9, PGAM5, SEC23B, HK2, and β-catenin—to control TGF-β/metastasis, innate antiviral/antifungal immunity, NF-κB inflammation, Hippo-independent YAP activity, ferroptosis, apoptosis, ribosome quality control, and cardiac/renal/bone pathophysiology, while its ETGE motif additionally engages KEAP1 to regulate oxidative stress responses."},"narrative":{"teleology":[{"year":2017,"claim":"Establishing OTUD1 as a deubiquitinase that suppresses TGF-β-driven metastasis revealed its first defined substrate (SMAD7) and demonstrated it cleaves an atypical K33 linkage at a specific lysine, coupling deubiquitination to E3-ligase recruitment and receptor turnover.","evidence":"Loss-of-function screen in mice, in vitro DUB assay with K33-linkage specificity, SMAD7 K220 mutagenesis, orthotopic transplantation","pmids":["29235476"],"confidence":"High","gaps":["Whether K33-linked ubiquitination of SMAD7 is catalyzed by a specific E3 ligase was not identified","Structural basis for K33 selectivity unresolved"]},{"year":2018,"claim":"Two contemporaneous studies established OTUD1 as a negative regulator of innate antiviral immunity through two distinct mechanisms—stabilizing Smurf1 to degrade MAVS/TRAF3/TRAF6, and removing K63-linked ubiquitin from IRF3 K98 to block its nuclear function—revealing OTUD1 as a multi-substrate feedback inhibitor in the IFN pathway.","evidence":"OTUD1 KO mice with viral challenge for Smurf1 axis; Co-IP and ubiquitination assays with patient mutation validation for IRF3 axis","pmids":["29734366","30100102"],"confidence":"High","gaps":["Whether Smurf1 stabilization and IRF3 deubiquitination operate in the same cell type simultaneously","Relative contribution of each axis to net IFN output in vivo"]},{"year":2018,"claim":"Identification of YAP as a K63-deubiquitination target showed OTUD1 opposes SCF-SKP2-mediated non-proteolytic ubiquitination to control YAP nuclear localization independently of canonical Hippo kinase signaling, broadening OTUD1's role beyond immune regulation.","evidence":"DUB library screen, K321/K497 mutagenesis, localization assays","pmids":["29891922"],"confidence":"High","gaps":["Physiological contexts where OTUD1-YAP axis dominates over Hippo pathway unclear","No in vivo phenotype shown for this axis"]},{"year":2020,"claim":"Demonstrating that OTUD1 cleaves a K6-linked ubiquitin chain from IRF3 that specifically controls DNA-binding capacity—without affecting stability, dimerization, or nuclear entry—revealed an unprecedented non-degradative, non-K63 regulatory mechanism for transcription factor activity.","evidence":"Linkage-specific assay, ChIP showing IRF3 dissociation from promoters, Otud1−/− mice with viral/LPS challenge","pmids":["32075857"],"confidence":"High","gaps":["The E3 ligase installing K6-linked ubiquitin on IRF3 was not identified","Whether K6 and K63 modifications on IRF3 K98 are mutually exclusive or sequential"]},{"year":2021,"claim":"Multiple 2021 studies expanded OTUD1's substrate repertoire to ferroptosis (IREB2), NF-κB (RIPK1), antifungal immunity (CARD9), and apoptosis (AIF, MCL1), establishing it as a pleiotropic deubiquitinase whose net cellular outcome depends on cell type and stimulus context.","evidence":"RIPK1 K63-deubiquitination with UC patient G430V variant in DSS colitis KO mice; IREB2 stabilization with ferroptosis/immune readouts; CARD9 deubiquitination with fungal KO mouse model; AIF site-specific mutagenesis (K244/K255) and MCL1 degradation via DCAF10-CUL4A complex","pmids":["34876703","33393230","33789983","33898171"],"confidence":"High","gaps":["How substrate selectivity is achieved among many competing targets in the same cell","Whether the G430V UC mutation affects substrates beyond RIPK1"]},{"year":2022,"claim":"Discovery that OTUD1 possesses deubiquitinase-independent functions—an N-terminal OUN-36 peptide blocking Akt PH-domain membrane recruitment, and an ETGE motif engaging KEAP1—revealed that OTUD1's disordered N-terminus operates as an autonomous signaling module.","evidence":"PH domain binding assay and Akt phosphorylation studies for OUN-36; DUB screen, MS interactome, ETGE domain deletion with KO mouse inflammatory models for KEAP1 binding","pmids":["36640312","35941131"],"confidence":"High","gaps":["Structural basis of OUN-36/PH domain interaction not solved","Whether ETGE-KEAP1 and catalytic functions are coordinated or independent in vivo"]},{"year":2022,"claim":"Identification of OTUD1 as an antagonist of ZNF598-mediated ribosome ubiquitination during translational stalling linked this deubiquitinase to ribosome quality control, a function distinct from its signaling roles.","evidence":"BioID proximity labeling, polysome profiling, RPS10 ubiquitination assay, rare-codon mRNA stability analysis","pmids":["36445135"],"confidence":"Medium","gaps":["Whether OTUD1 acts on stalled ribosomes in vivo under physiological stress","No structural or reconstituted in vitro ribosome-OTUD1 complex","Independent replication needed"]},{"year":2023,"claim":"Studies in 2023 established OTUD1 as a regulator of cardiac pathophysiology through STAT3 K63-deubiquitination (promoting inflammatory cardiac remodeling), SEC23B K63-deubiquitination (restricting COPII secretory transport), NRF2 stabilization (protecting against oxidative injury), and CARD9 K33-deubiquitination (promoting CBM complex assembly in macrophages), revealing tissue-specific and linkage-specific substrate programs.","evidence":"LC-MS/MS with Co-IP and C320 mutagenesis for STAT3 in KO mice; K81 site-specific assay and neutrophil KO for SEC23B; ETGE peptide treatment and KO mouse I/R model for NRF2; myeloid-specific KO and CBM reconstitution for CARD9","pmids":["37153745","37639212","39079388","39118286"],"confidence":"High","gaps":["How OTUD1 is directed to K33 vs K63 chains on different substrates in the same cell type","Whether SEC23B regulation is specific to neutrophils or generalizable"]},{"year":2023,"claim":"Development of UbVOD.1, a ubiquitin variant inhibitor bridging the OTU domain and UIM, provided the first tool-grade inhibitor of OTUD1 and confirmed that the UIM contributes to catalytic engagement with ubiquitin chains.","evidence":"Phage display selection, in vitro IC50 quantification, cellular RIPK1 ubiquitination rescue with di-UbV","pmids":["37589489"],"confidence":"High","gaps":["No in vivo application of UbVOD.1 demonstrated","Selectivity across the full OTU DUB family not comprehensively tested"]},{"year":2024,"claim":"OTUD1 was shown to regulate cardiac calcium handling by stabilizing PDE5A through K48-deubiquitination, inactivating the cGMP-PKG-SERCA2a axis, and to impair AMPK activation by deubiquitinating AMPKα2 at K60/K379 and blocking CAMKK2 binding, establishing OTUD1 as a driver of heart failure and diabetic cardiomyopathy.","evidence":"LC-MS/MS, C320 mutagenesis, domain mapping for PDE5A in ISO/MI KO mice; AMPKα2 K60/K379 mutagenesis, cardiomyocyte-specific KO in T1DM/T2DM models with scRNA-seq","pmids":["38185350","40683882"],"confidence":"High","gaps":["Whether PDE5A and AMPKα2 deubiquitination interact in the same cardiac pathology","Therapeutic targeting of OTUD1 in heart failure not yet demonstrated"]},{"year":2025,"claim":"Recent work further expanded OTUD1 substrates to β-catenin (controlling angiogenesis in diabetic wound healing), C/EBPβ (driving Alzheimer's neuroinflammation), PRDX1 (suppressing osteoclastogenesis), HK2 (promoting NLRP3-driven pyroptosis), and p53 (promoting endothelial procoagulant programs), demonstrating context-dependent pro- or anti-inflammatory outcomes.","evidence":"IP-MS with site-specific mutagenesis (β-catenin K496/K508/K625, C/EBPβ K253), myeloid-specific KO with micro-CT for PRDX1, OTUD1 KO in CLP sepsis model for HK2, L357 mutagenesis with thrombosis model for p53","pmids":["40300668","40335710","40585986","40500776","41967174"],"confidence":"High","gaps":["How OTUD1 substrate selection is regulated post-translationally in different tissues","No unifying structural model of OTUD1 engaged with different chain types","Contribution of disordered N-terminus to substrate recruitment largely unmapped"]},{"year":null,"claim":"Key unresolved questions include how OTUD1 achieves substrate selectivity among its many targets in a given cell, whether its catalytic and non-catalytic (ETGE, OUN-36) functions are coordinately regulated, and whether a full-length structural model can explain its multi-linkage specificity.","evidence":"","pmids":[],"confidence":"High","gaps":["No full-length crystal or cryo-EM structure","No systematic selectivity profiling across chain types with endogenous substrates","Upstream signals governing OTUD1 protein turnover and activity remain fragmentary"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0,1,2,3,4,5,6,7,8,9,10,12,13,14,19,20,21,22,23,24,25,27,31,33,34,35,36]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,2,3,5,8,10,13,19,22,23,24,31,33,34,36]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[11,18]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,11,18]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,5]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[3,4,5,6,7,9,12,21,24,30,31,32]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,10,11,15,22,23,25,33,36]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[2,8,17,24,36]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1,3,5,13,18,27]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[18]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[19]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[7,20]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[3,10,13,22,23,25]}],"complexes":[],"partners":["SMAD7","IRF3","RIPK1","CARD9","KEAP1","STAT3","PRKAA2","PDE5A"],"other_free_text":[]},"mechanistic_narrative":"OTUD1 is an OTU-family deubiquitinase that functions as a broad-specificity regulator of ubiquitin-dependent signaling, cleaving K63-, K48-, K33-, and K6-linked polyubiquitin chains from diverse substrates to control inflammation, innate immunity, cell death, antioxidant defense, and protein trafficking. Its catalytic OTU domain (active-site C320) preferentially hydrolyzes K63-linked chains—enhanced by a C-terminal UIM—to modulate NF-κB signaling (via RIPK1, RIP2, CARD9, CDK9), antiviral IFN responses (via IRF3, Smurf1/MAVS), Hippo-independent YAP activity, β-catenin nuclear translocation, COPII-mediated ER-to-Golgi transport (via SEC23B), ribosome quality control (via ZNF598/RPS10), and AMPK activation, while K48-chain cleavage stabilizes substrates including PTEN, NRF2, p53, PRDX1, and C/EBPβ [PMID:29891922, PMID:34876703, PMID:32075857, PMID:29734366, PMID:37639212, PMID:36445135, PMID:40300668, PMID:39079388, PMID:40683882, PMID:35280681]. Beyond its catalytic function, OTUD1 possesses an N-terminal intrinsically disordered region containing an ETGE motif that binds KEAP1 to regulate the NRF2 antioxidant response, and a short peptide (OUN-36) that directly inhibits Akt membrane recruitment independently of deubiquitinase activity [PMID:35941131, PMID:36640312]. OTUD1 loss-of-function exacerbates inflammatory bowel disease, cardiac hypertrophy, sepsis, and fungal susceptibility in mouse models, while a UC-associated G430V mutation abolishes RIPK1 deubiquitination, linking OTUD1 dysfunction to human inflammatory disease [PMID:34876703, PMID:37153745, PMID:33789983]."},"prefetch_data":{"uniprot":{"accession":"Q5VV17","full_name":"OTU domain-containing protein 1","aliases":["DUBA-7"],"length_aa":481,"mass_kda":51.1,"function":"Deubiquitinating enzyme that specifically hydrolyzes 'Lys-63'-linked polyubiquitin to monoubiquitin (PubMed:23827681). Required for the stability and translation of a subset mRNAs with a high abundance of rare codons by mediating deubiquitination of 40S ribosomal protein RPS10/eS10, thereby antagonizing ZNF598-mediated 40S ubiquitination (PubMed:36445135). The abundance of rare codons in mRNAs can limit the translation rate and can lead to ribosome collisions that trigger activation of ribosome quality control (RQC) pathway by ZNF598 (PubMed:36445135). OTUD1-mediated deubiquitination prevents activation of the RQC and subsequent dissociation of ribosomes and stimulates formation of polysomes and translation (PubMed:36445135)","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q5VV17/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/OTUD1","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/OTUD1","total_profiled":1310},"omim":[{"mim_id":"620295","title":"DDB1- AND CUL4-ASSOCIATED FACTOR 10; DCAF10","url":"https://www.omim.org/entry/620295"},{"mim_id":"612022","title":"OTU DOMAIN-CONTAINING PROTEIN 1; OTUD1","url":"https://www.omim.org/entry/612022"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Cytosol","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"bone marrow","ntpm":57.0}],"url":"https://www.proteinatlas.org/search/OTUD1"},"hgnc":{"alias_symbol":["DUBA7"],"prev_symbol":["OTDC1"]},"alphafold":{"accession":"Q5VV17","domains":[{"cath_id":"3.90.70.80","chopping":"285-441","consensus_level":"medium","plddt":95.2156,"start":285,"end":441},{"cath_id":"1.20.5","chopping":"442-481","consensus_level":"medium","plddt":91.602,"start":442,"end":481}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5VV17","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q5VV17-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q5VV17-F1-predicted_aligned_error_v6.png","plddt_mean":60.34},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=OTUD1","jax_strain_url":"https://www.jax.org/strain/search?query=OTUD1"},"sequence":{"accession":"Q5VV17","fasta_url":"https://rest.uniprot.org/uniprotkb/Q5VV17.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q5VV17/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5VV17"}},"corpus_meta":[{"pmid":"29891922","id":"PMC_29891922","title":"SKP2- 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This ubiquitination promotes YAP interaction with TEAD, nuclear localization, and transcriptional activity independently of Hippo signaling; OTUD1 overexpression retains YAP in the cytoplasm and inhibits its activity.\",\n      \"method\": \"DUB library screen, co-immunoprecipitation, ubiquitination assays, site-directed mutagenesis, overexpression/knockdown with localization readouts\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, ubiquitin linkage mapping, mutagenesis, functional readout; replicated in companion commentary\",\n      \"pmids\": [\"29891922\", \"31225491\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"OTUD1 directly deubiquitinates SMAD7, cleaving K33-linked polyubiquitin chains at SMAD7 K220, which prevents SMAD7 proteasomal degradation and exposes the SMAD7 PY motif for SMURF2 binding, leading to TβRI turnover at the cell surface and suppression of TGF-β pro-metastatic signaling.\",\n      \"method\": \"Loss-of-function screen in mice, co-immunoprecipitation, in vitro deubiquitination assay, ubiquitin linkage-specific analysis, site-directed mutagenesis, xenograft and orthotopic transplantation models\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro deubiquitination, mutagenesis at SMAD7 K220, multiple in vivo models, mechanistic pathway placement\",\n      \"pmids\": [\"29235476\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"OTUD1 deubiquitinates and stabilizes IREB2 (iron-responsive element-binding protein 2), which in turn promotes TFRC-mediated iron transport, increases ROS generation, and induces ferroptosis; OTUD1 loss reduces iron import and dampens tumor-reactive T-cell accumulation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, genetic knockdown/knockout with ferroptosis and immune cell readouts\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus functional rescue, multiple cellular and in vivo readouts\",\n      \"pmids\": [\"33393230\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"OTUD1 physically interacts with RIPK1 and selectively cleaves K63-linked polyubiquitin chains from RIPK1, thereby inhibiting recruitment of NEMO and suppressing NF-κB activation; a UC-associated OTUD1 G430V mutation abolishes this activity.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitin linkage-specific assay, knockout mouse model (DSS colitis), bone marrow transplantation, patient mutation functional analysis\",\n      \"journal\": \"Cellular & molecular immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, linkage-specific deubiquitination, KO mouse with defined phenotype, disease-variant validation\",\n      \"pmids\": [\"34876703\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"RNA virus infection induces OTUD1 expression via NF-κB-dependent mechanisms; OTUD1 then deubiquitinates and stabilizes the E3 ligase Smurf1, which promotes ubiquitination-dependent degradation of MAVS, TRAF3, and TRAF6, thereby inhibiting type I IFN production as a negative feedback loop.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, OTUD1-deficient mice with viral infection challenge, overexpression/knockdown\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic pathway reconstituted in cells and validated in KO mice with viral challenge\",\n      \"pmids\": [\"29734366\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"OTUD1 deubiquitinates IRF3 by cleaving K6-linked polyubiquitin chains at IRF3 K98; K6-linked ubiquitination is required for IRF3 DNA-binding capacity at IFN gene promoters without affecting IRF3 stability, dimerization, or nuclear translocation. OTUD1 removal of this modification dissociates IRF3 from target gene promoters.\",\n      \"method\": \"Co-immunoprecipitation, linkage-specific ubiquitination assay, chromatin immunoprecipitation, Otud1-/- cells and mice with viral/LPS challenge\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — linkage-specific deubiquitination, ChIP validation, KO mouse phenotype, multiple orthogonal methods\",\n      \"pmids\": [\"32075857\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"OTUD1 directly interacts with IRF3 and removes K63-linked polyubiquitin chains at IRF3 K98, inhibiting IRF3 nuclear translocation and transcriptional activity; loss-of-function OTUD1 mutations found in autoimmune patients impair this deubiquitinase activity or IRF3 association. FOXO3 signaling is required for OTUD1 induction upon antigenic stimulation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, patient mutation functional analysis, OTUD1-deficient cellular models\",\n      \"journal\": \"Journal of autoimmunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — patient variants tested functionally, Co-IP, ubiquitination assay, pathway placement\",\n      \"pmids\": [\"30100102\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"OTUD1 suppresses canonical NF-κB activation by hydrolyzing K63-linked ubiquitin chains from NF-κB signaling factors including LUBAC substrates. OTUD1 also binds KEAP1 through an ETGE motif in its N-terminal intrinsically disordered region, regulating the antioxidant response and ROS-induced cell death (oxeiptosis). In Otud1-/- mice, inflammation and oxidative damage are enhanced in IBD, acute hepatitis, and sepsis models.\",\n      \"method\": \"DUB screen (88 human DUBs), mass spectrometry interactome, ubiquitination assay, Otud1-/- mouse models, domain deletion analysis\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — systematic screen, MS interactome, domain mapping, multiple KO mouse disease models\",\n      \"pmids\": [\"35941131\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"OTUD1 deubiquitinates AIF at K244 (disrupting mitochondrial structure and oxidative phosphorylation) and at K255 (enhancing AIF DNA-binding ability to promote caspase-independent parthanatos). OTUD1 also stabilizes DCAF10 and recruits the CUL4A-DDB1 complex to promote MCL1 degradation, activating caspase-dependent apoptosis.\",\n      \"method\": \"Co-immunoprecipitation, site-directed mutagenesis at AIF K244 and K255, ubiquitination assays, functional apoptosis readouts\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — mutagenesis at specific ubiquitination sites, reconstituted complex, dual apoptotic pathway readouts\",\n      \"pmids\": [\"33898171\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"OTUD1 directly interacts with CARD9 and cleaves polyubiquitin chains from CARD9, which is required for CARD9-mediated NF-κB and MAPK activation in antifungal innate immunity; OTUD1-deficient mice are more susceptible to fungal infection.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, Otud1-/- mouse fungal infection model\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP, deubiquitination assay, KO mouse in vivo phenotype\",\n      \"pmids\": [\"33789983\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"OTUD1 binds the SH2 domain of STAT3 and deubiquitinates STAT3 K63-linked chains via its catalytic C320 residue, promoting STAT3 phosphorylation and nuclear translocation, thereby inducing inflammatory, fibrotic, and hypertrophic responses in cardiomyocytes; OTUD1 KO mice are protected from angiotensin II- and TAC-induced cardiac remodeling.\",\n      \"method\": \"LC-MS/MS combined with Co-IP, site-directed mutagenesis (C320), AAV9 overexpression, OTUD1 KO mouse models\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — MS-based substrate identification, mutagenesis, gain/loss-of-function mouse models with defined cardiac phenotype\",\n      \"pmids\": [\"37153745\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"OTUD1's N-terminal intrinsically disordered region contains a short peptide (OUN-36) that binds the Akt PH domain at residues required for PtdIns(3,4,5)P3 recognition, inhibiting Akt membrane localization and phosphorylation independently of OTUD1 deubiquitinase activity.\",\n      \"method\": \"Co-immunoprecipitation, domain deletion analysis, peptide binding assay, cell membrane localization studies, Akt phosphorylation assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic domain dissection, PH domain binding identified, deubiquitinase-independent mechanism established\",\n      \"pmids\": [\"36640312\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"OTUD1 interacts with CARD9 and removes K33-linked ubiquitin from CARD9, promoting assembly of the CARD9-BCL10-MALT1 (CBM) complex and NF-κB activation in macrophages during isoproterenol-induced cardiac inflammation; myeloid-specific OTUD1 deletion attenuates cardiac remodeling.\",\n      \"method\": \"Co-immunoprecipitation, linkage-specific ubiquitination assay, Card9-/- and Otud1-/- bone marrow reconstitution, primary macrophage mechanistic studies\",\n      \"journal\": \"Clinical and translational medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — linkage-specific deubiquitination, myeloid-specific KO reconstitution, CBM complex assembly validated\",\n      \"pmids\": [\"39118286\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"OTUD1 binds directly to GAF1 and PDEase domains of PDE5A and reverses K48-linked ubiquitination of PDE5A via its C320 catalytic residue, preventing proteasomal degradation of PDE5A, which inactivates the cGMP-PKG-SERCA2a signaling axis and dysregulates calcium handling in cardiomyocytes, promoting heart failure.\",\n      \"method\": \"LC-MS/MS combined with Co-IP, site-directed mutagenesis (C320), OTUD1 KO mouse (ISO and MI models), domain binding mapping\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular basis of disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — MS-identified interaction, catalytic site mutagenesis, domain mapping, KO mouse with cardiac phenotype\",\n      \"pmids\": [\"38185350\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"OTUD1 interacts with Notch2-ICD (NICD) and cleaves ubiquitin at NICD K1770, leading to NICD protein accumulation in T cells, which promotes Th1/Th17 differentiation and amplifies graft-versus-host disease; dapagliflozin was identified as an inhibitor of the OTUD1/NICD axis.\",\n      \"method\": \"Co-immunoprecipitation, site-specific ubiquitination assay (K1770), FDA-approved drug screen, aGVHD mouse model\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — site-specific deubiquitination, Co-IP, in vivo aGVHD model validation\",\n      \"pmids\": [\"36574342\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"OTUD1 interacts with and deubiquitinates PTEN, stabilizing PTEN protein levels to suppress PI3K/AKT and TNF-α/NF-κB signaling in renal cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, RNA-seq pathway analysis, knockdown/overexpression\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP and deubiquitination assay, single lab, no site-specific mutagenesis\",\n      \"pmids\": [\"35280681\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"OTUD1 interacts with MCL1 and promotes its deubiquitination in an enzymatic-activity-dependent manner, stabilizing MCL1 protein and antagonizing BH3-mimetic inhibitor-induced cell death.\",\n      \"method\": \"DUB library screen, co-immunoprecipitation, ubiquitination assay, cell viability assay\",\n      \"journal\": \"Cancer cell international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP/deubiquitination assay, DUB screen, single lab\",\n      \"pmids\": [\"31467488\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"OTUD1 deubiquitinates and stabilizes Bim at lysine 3, preventing its proteasomal degradation; melatonin-induced Sp1 activation drives OTUD1 transcription, leading to Bim stabilization and apoptosis in cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, site-directed mutagenesis (Bim K3), ubiquitination assay, siRNA knockdown, xenograft model\",\n      \"journal\": \"Journal of pineal research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mutagenesis at K3, Co-IP, in vivo xenograft; single lab\",\n      \"pmids\": [\"34826170\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"OTUD1 catalytic activity resolves ribosome stalling on polyA sequences by inhibiting ZNF598-mediated RPS10 ubiquitination, and promotes polysome formation; OTUD1 also associates with ribosome subunits and elongation factors and regulates stability of rare-codon-rich mRNAs by antagonizing ZNF598.\",\n      \"method\": \"Proximity labeling-based interactome (BioID), co-immunoprecipitation with ribosome subunits, polysome profiling, ubiquitination assay for RPS10, gene expression analysis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — proximity interactome plus biochemical validation, but single lab and novel function\",\n      \"pmids\": [\"36445135\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"OTUD1 cleaves K63-linked polyubiquitin chains from SEC23B at K81, negatively regulating the COPII secretory machinery and limiting ER-to-Golgi protein trafficking, thereby restricting surface expression of CD9 and CD47 and suppressing secretory neutrophil polarization.\",\n      \"method\": \"Co-immunoprecipitation, linkage-specific ubiquitination assay (K81), neutrophil-specific OTUD1 depletion, Brefeldin A rescue experiment\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — site-specific K63 deubiquitination, functional rescue with transport inhibitor, neutrophil-specific KO phenotype\",\n      \"pmids\": [\"37639212\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"OTUD1 deubiquitinates and stabilizes NRF2 through its C320 catalytic residue and ETGE motif, attenuating hepatic ischemia/reperfusion-induced oxidative stress; an ETGE-containing peptide mimics this protective effect in vivo.\",\n      \"method\": \"Co-immunoprecipitation, deubiquitination assay, site-directed mutagenesis (C320), OTUD1 KO mouse (I/R model), ETGE peptide in vivo treatment\",\n      \"journal\": \"Redox biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — catalytic site mutagenesis, ETGE motif functional validation, KO mouse with disease phenotype\",\n      \"pmids\": [\"39079388\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"OTUD1 interacts with RIP2 and cleaves K63-linked polyubiquitin chains from RIP2, inhibiting NF-κB activation; OTUD1 deficiency exacerbates cerebral ischemic injury by allowing excess RIP2 ubiquitination and NF-κB-driven inflammation.\",\n      \"method\": \"Co-immunoprecipitation, confocal microscopy co-localization, immunofluorescence, dual-luciferase NF-κB assay, OTUD1-/- mouse (MCAO model)\",\n      \"journal\": \"Journal of neuroinflammation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP, linkage-specific deubiquitination, KO mouse in vivo with defined ischemia phenotype\",\n      \"pmids\": [\"38012669\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"OTUD1 deubiquitinates CDK9 via K63-linkage at its C320 catalytic residue, promoting CDK9 phosphorylation and activation to induce inflammatory responses and fibrosis in kidney epithelial cells; OTUD1 KO mice are protected from angiotensin II-induced hypertensive renal disease.\",\n      \"method\": \"Co-immunoprecipitation, site-directed mutagenesis (C320), OTUD1 KO mouse (Ang II model), CDK9 inhibitor rescue\",\n      \"journal\": \"Acta pharmacologica Sinica\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — catalytic site mutagenesis, KO mouse disease model, pharmacological rescue\",\n      \"pmids\": [\"38110583\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"OTUD1 directly binds AMPKα2, deubiquitinates it at K60/K379, and inhibits AMPKT172 phosphorylation by impeding the AMPKα2-CAMKK2 interaction, resulting in mitochondrial dysfunction in cardiomyocytes and diabetic cardiomyopathy; cardiomyocyte-specific OTUD1 KO restores AMPK activity.\",\n      \"method\": \"Co-immunoprecipitation, site-directed mutagenesis (K60/K379), cardiomyocyte-specific KO mouse (T1DM and T2DM models), single-cell RNA-seq, CAMKK2 interaction assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — site-specific mutagenesis, cardiomyocyte-specific KO, upstream kinase interaction assay, replicated in two diabetes models\",\n      \"pmids\": [\"40683882\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"OTUD1 promotes HK2 dissociation from mitochondria via selective K63-linked deubiquitination of HK2, activating the NLRP3 inflammasome and triggering microglia pyroptosis, leading to neuroinflammation and cognitive deficits in sepsis-associated encephalopathy.\",\n      \"method\": \"Co-immunoprecipitation, molecular docking, 3D confocal microscopy, K63-specific ubiquitination assay, OTUD1 KO mouse (CLP model), single-cell RNA-seq\",\n      \"journal\": \"Journal of neuroinflammation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — linkage-specific deubiquitination, domain binding mapped, KO mouse in vivo, multiple validation methods\",\n      \"pmids\": [\"40500776\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"OTUD1 deubiquitinates and stabilizes PGAM5 by reversing its ubiquitination, leading to robust activation of the ASK1-p38/JNK signaling pathway to accelerate cardiac hypertrophy; m6A modification by METTL3 of OTUD1 mRNA promotes its stability and elevated expression.\",\n      \"method\": \"RNA immunoprecipitation (RIP) for m6A, IP-MS for substrate identification, domain deletion constructs, cardiac-specific KO mice (TAC model), ASK1 knockdown rescue\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — IP-MS substrate identification, KO mouse model, upstream m6A regulation validated, pathway rescue by ASK1 KD\",\n      \"pmids\": [\"39309432\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"OTUD1 contains an N-terminal ETGE motif within an alanine/proline/glycine-rich intrinsically disordered region that mediates binding to KEAP1, placing OTUD1 in the KEAP1-NRF2 antioxidant axis; the OTU catalytic domain preferentially hydrolyzes K63-linked ubiquitin chains, with the UIM enhancing this preference.\",\n      \"method\": \"Domain analysis, in vitro ubiquitin chain hydrolysis assay, KEAP1 binding assay, UIM deletion analysis\",\n      \"journal\": \"Antioxidants\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — review synthesizing prior biochemical data, domain-function relationships established\",\n      \"pmids\": [\"36829909\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"An ubiquitin variant inhibitor (UbVOD.1) was developed that binds OTUD1 with nanomolar affinity bridging the catalytic OTU domain and UIM, inhibiting OTUD1 activity toward mono-Ub, K63-linked di-Ub, and poly-Ub chains of other linkages in vitro, and inhibiting OTUD1-mediated deubiquitination of RIPK1 in cells as a di-UbV.\",\n      \"method\": \"Phage display UbV library selection, in vitro deubiquitination IC50 assay, in vivo di-UbV expression with RIPK1 ubiquitination readout\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstituted inhibition with IC50 quantification, cellular validation with defined substrate\",\n      \"pmids\": [\"37589489\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"OTUD1 deubiquitinates and stabilizes KLF4 in non-small cell lung cancer cells, with OTUD1 overexpression inhibiting NSCLC cell progression and KLF4 knockdown reversing this effect.\",\n      \"method\": \"Co-immunoprecipitation, cycloheximide chase, deubiquitination assay, siRNA knockdown rescue\",\n      \"journal\": \"Thoracic cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP and deubiquitination assay, single lab, no site-specific mutagenesis\",\n      \"pmids\": [\"35098684\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"OTUD1 deubiquitinates and stabilizes FHL1 in lung adenocarcinoma cells, inhibiting tumor proliferation and migration in vitro and in vivo.\",\n      \"method\": \"IP-MS, co-immunoprecipitation, ubiquitination assay, xenograft and urethane-induced lung cancer models\",\n      \"journal\": \"Cellular oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — IP-MS plus Co-IP, in vivo validation; single lab\",\n      \"pmids\": [\"36929488\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"OTUD1 interacts with BST-2, reducing its K48- and K63-linked ubiquitination and increasing BST-2 protein stability, thereby inhibiting HIV-1 particle release.\",\n      \"method\": \"siRNA DUB screen, co-immunoprecipitation, ubiquitin linkage-specific assay, HIV-1 release assay\",\n      \"journal\": \"Viruses\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — DUB screen followed by Co-IP and linkage-specific assay, functional viral release readout; single lab\",\n      \"pmids\": [\"40007014\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"OTUD1 directly binds C/EBPβ and removes K48-linked ubiquitin chains at C/EBPβ K253, stabilizing C/EBPβ and activating C/EBPβ-NF-κB-mediated inflammatory responses in microglia, promoting Alzheimer's disease pathology.\",\n      \"method\": \"LC-MS/MS combined with Co-IP, site-directed mutagenesis (K253), OTUD1 knockdown in microglia, AD mouse model cognitive readout\",\n      \"journal\": \"Acta pharmacologica Sinica\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — MS-based substrate identification, site-specific mutagenesis, in vivo AD model with cognitive phenotype\",\n      \"pmids\": [\"40335710\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"OTUD1 deubiquitinates TIPE2, and this interaction suppresses TAK1-mediated MAPK and NF-κB signaling to protect against sepsis-induced lung injury.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence co-localization, western blotting for deubiquitination, in vivo sepsis model\",\n      \"journal\": \"Biochemical pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP and deubiquitination assay, single lab, pathway placement supported by prior TIPE2 literature\",\n      \"pmids\": [\"38996928\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"OTUD1 directly interacts with β-catenin and reduces K63-linked ubiquitination at β-catenin K496, K508, and K625 via its C320 catalytic residue, facilitating β-catenin phosphorylation and restricting its nuclear translocation, thereby downregulating angiogenesis-related factors and impairing wound healing in diabetic mice.\",\n      \"method\": \"IP-MS, co-immunoprecipitation, site-directed mutagenesis (C320 and K496/K508/K625), endothelial-specific AAV knockdown, diabetic mouse wound healing model\",\n      \"journal\": \"Journal of advanced research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — IP-MS, site-specific mutagenesis at both catalytic and substrate ubiquitination sites, endothelial-specific in vivo KD\",\n      \"pmids\": [\"40300668\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"OTUD1 deubiquitinates PRDX1 by reversing K48-linked ubiquitination, maintaining PRDX1 stability, mitigating mitochondrial dysfunction, and suppressing osteoclast differentiation; OTUD1 KO leads to enhanced osteoclastogenesis and reduced bone mass.\",\n      \"method\": \"LC-MS/MS combined with Co-IP, ubiquitin linkage assay, whole-body and myeloid-specific KO mice, micro-CT, histomorphometry\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — MS-based substrate identification, linkage-specific assay, myeloid-specific KO with quantitative bone phenotype\",\n      \"pmids\": [\"40585986\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"OTUD1 deubiquitinates p53 via K48-dependent deubiquitination through its 300–481 region, stabilizing p53 and promoting a procoagulant gene expression program in endothelial cells; an L357 point mutation of OTUD1 rescues endothelial homeostasis.\",\n      \"method\": \"Co-immunoprecipitation, domain deletion/point mutagenesis (L357), K48-specific ubiquitination assay, ROS manipulation, in vivo thrombosis model\",\n      \"journal\": \"Environment international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — domain and point mutagenesis, K48-specific assay, in vivo thrombus model; single lab\",\n      \"pmids\": [\"41967174\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"OTUD1 binds RACK1 and cleaves K63-linked polyubiquitin chains from RACK1, enhancing RACK1 phosphorylation and modulating downstream MAPKs and NF-κB signaling to promote cardiomyocyte apoptosis after ischemia/reperfusion; RACK1 silencing reverses OTUD1-promoted H/R injury.\",\n      \"method\": \"IP-MS, co-immunoprecipitation, K63-specific deubiquitination assay, RACK1 siRNA rescue, OTUD1 KO mouse (I/R model)\",\n      \"journal\": \"Acta pharmacologica Sinica\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — IP-MS substrate identification, linkage-specific assay, KO mouse model, epistasis by RACK1 KD\",\n      \"pmids\": [\"40394237\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"OTUD1 deubiquitinates SMAD7/MADH7 in endometriosis stromal cells, inhibiting fibrosis-related protein expression and reducing endometriosis lesion growth in vivo.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, in vitro stromal cell model, C57BL/6N mouse in vivo lesion model\",\n      \"journal\": \"Molecular human reproduction\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP and deubiquitination, consistent with SMAD7 mechanism described for breast cancer but single lab in new context\",\n      \"pmids\": [\"40279273\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"OTUD1 is an OTU-family deubiquitinase with a disordered N-terminal APGR/ETGE region, a catalytic OTU domain (active-site C320), and a UIM that enhances K63-chain preference; it removes K63-, K48-, K33-, and K6-linked ubiquitin chains from a broad substrate repertoire—including SMAD7, IRF3, RIPK1, CARD9, STAT3, AIF, MCL1, IREB2, NRF2, YAP, AKT, PTEN, AMPKα2, PDE5A, CDK9, PGAM5, SEC23B, HK2, and β-catenin—to control TGF-β/metastasis, innate antiviral/antifungal immunity, NF-κB inflammation, Hippo-independent YAP activity, ferroptosis, apoptosis, ribosome quality control, and cardiac/renal/bone pathophysiology, while its ETGE motif additionally engages KEAP1 to regulate oxidative stress responses.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"OTUD1 is an OTU-family deubiquitinase that functions as a broad-specificity regulator of ubiquitin-dependent signaling, cleaving K63-, K48-, K33-, and K6-linked polyubiquitin chains from diverse substrates to control inflammation, innate immunity, cell death, antioxidant defense, and protein trafficking. Its catalytic OTU domain (active-site C320) preferentially hydrolyzes K63-linked chains—enhanced by a C-terminal UIM—to modulate NF-κB signaling (via RIPK1, RIP2, CARD9, CDK9), antiviral IFN responses (via IRF3, Smurf1/MAVS), Hippo-independent YAP activity, β-catenin nuclear translocation, COPII-mediated ER-to-Golgi transport (via SEC23B), ribosome quality control (via ZNF598/RPS10), and AMPK activation, while K48-chain cleavage stabilizes substrates including PTEN, NRF2, p53, PRDX1, and C/EBPβ [PMID:29891922, PMID:34876703, PMID:32075857, PMID:29734366, PMID:37639212, PMID:36445135, PMID:40300668, PMID:39079388, PMID:40683882, PMID:35280681]. Beyond its catalytic function, OTUD1 possesses an N-terminal intrinsically disordered region containing an ETGE motif that binds KEAP1 to regulate the NRF2 antioxidant response, and a short peptide (OUN-36) that directly inhibits Akt membrane recruitment independently of deubiquitinase activity [PMID:35941131, PMID:36640312]. OTUD1 loss-of-function exacerbates inflammatory bowel disease, cardiac hypertrophy, sepsis, and fungal susceptibility in mouse models, while a UC-associated G430V mutation abolishes RIPK1 deubiquitination, linking OTUD1 dysfunction to human inflammatory disease [PMID:34876703, PMID:37153745, PMID:33789983].\",\n  \"teleology\": [\n    {\n      \"year\": 2017,\n      \"claim\": \"Establishing OTUD1 as a deubiquitinase that suppresses TGF-β-driven metastasis revealed its first defined substrate (SMAD7) and demonstrated it cleaves an atypical K33 linkage at a specific lysine, coupling deubiquitination to E3-ligase recruitment and receptor turnover.\",\n      \"evidence\": \"Loss-of-function screen in mice, in vitro DUB assay with K33-linkage specificity, SMAD7 K220 mutagenesis, orthotopic transplantation\",\n      \"pmids\": [\"29235476\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether K33-linked ubiquitination of SMAD7 is catalyzed by a specific E3 ligase was not identified\", \"Structural basis for K33 selectivity unresolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Two contemporaneous studies established OTUD1 as a negative regulator of innate antiviral immunity through two distinct mechanisms—stabilizing Smurf1 to degrade MAVS/TRAF3/TRAF6, and removing K63-linked ubiquitin from IRF3 K98 to block its nuclear function—revealing OTUD1 as a multi-substrate feedback inhibitor in the IFN pathway.\",\n      \"evidence\": \"OTUD1 KO mice with viral challenge for Smurf1 axis; Co-IP and ubiquitination assays with patient mutation validation for IRF3 axis\",\n      \"pmids\": [\"29734366\", \"30100102\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Smurf1 stabilization and IRF3 deubiquitination operate in the same cell type simultaneously\", \"Relative contribution of each axis to net IFN output in vivo\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identification of YAP as a K63-deubiquitination target showed OTUD1 opposes SCF-SKP2-mediated non-proteolytic ubiquitination to control YAP nuclear localization independently of canonical Hippo kinase signaling, broadening OTUD1's role beyond immune regulation.\",\n      \"evidence\": \"DUB library screen, K321/K497 mutagenesis, localization assays\",\n      \"pmids\": [\"29891922\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological contexts where OTUD1-YAP axis dominates over Hippo pathway unclear\", \"No in vivo phenotype shown for this axis\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Demonstrating that OTUD1 cleaves a K6-linked ubiquitin chain from IRF3 that specifically controls DNA-binding capacity—without affecting stability, dimerization, or nuclear entry—revealed an unprecedented non-degradative, non-K63 regulatory mechanism for transcription factor activity.\",\n      \"evidence\": \"Linkage-specific assay, ChIP showing IRF3 dissociation from promoters, Otud1−/− mice with viral/LPS challenge\",\n      \"pmids\": [\"32075857\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The E3 ligase installing K6-linked ubiquitin on IRF3 was not identified\", \"Whether K6 and K63 modifications on IRF3 K98 are mutually exclusive or sequential\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Multiple 2021 studies expanded OTUD1's substrate repertoire to ferroptosis (IREB2), NF-κB (RIPK1), antifungal immunity (CARD9), and apoptosis (AIF, MCL1), establishing it as a pleiotropic deubiquitinase whose net cellular outcome depends on cell type and stimulus context.\",\n      \"evidence\": \"RIPK1 K63-deubiquitination with UC patient G430V variant in DSS colitis KO mice; IREB2 stabilization with ferroptosis/immune readouts; CARD9 deubiquitination with fungal KO mouse model; AIF site-specific mutagenesis (K244/K255) and MCL1 degradation via DCAF10-CUL4A complex\",\n      \"pmids\": [\"34876703\", \"33393230\", \"33789983\", \"33898171\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How substrate selectivity is achieved among many competing targets in the same cell\", \"Whether the G430V UC mutation affects substrates beyond RIPK1\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Discovery that OTUD1 possesses deubiquitinase-independent functions—an N-terminal OUN-36 peptide blocking Akt PH-domain membrane recruitment, and an ETGE motif engaging KEAP1—revealed that OTUD1's disordered N-terminus operates as an autonomous signaling module.\",\n      \"evidence\": \"PH domain binding assay and Akt phosphorylation studies for OUN-36; DUB screen, MS interactome, ETGE domain deletion with KO mouse inflammatory models for KEAP1 binding\",\n      \"pmids\": [\"36640312\", \"35941131\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of OUN-36/PH domain interaction not solved\", \"Whether ETGE-KEAP1 and catalytic functions are coordinated or independent in vivo\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identification of OTUD1 as an antagonist of ZNF598-mediated ribosome ubiquitination during translational stalling linked this deubiquitinase to ribosome quality control, a function distinct from its signaling roles.\",\n      \"evidence\": \"BioID proximity labeling, polysome profiling, RPS10 ubiquitination assay, rare-codon mRNA stability analysis\",\n      \"pmids\": [\"36445135\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether OTUD1 acts on stalled ribosomes in vivo under physiological stress\", \"No structural or reconstituted in vitro ribosome-OTUD1 complex\", \"Independent replication needed\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Studies in 2023 established OTUD1 as a regulator of cardiac pathophysiology through STAT3 K63-deubiquitination (promoting inflammatory cardiac remodeling), SEC23B K63-deubiquitination (restricting COPII secretory transport), NRF2 stabilization (protecting against oxidative injury), and CARD9 K33-deubiquitination (promoting CBM complex assembly in macrophages), revealing tissue-specific and linkage-specific substrate programs.\",\n      \"evidence\": \"LC-MS/MS with Co-IP and C320 mutagenesis for STAT3 in KO mice; K81 site-specific assay and neutrophil KO for SEC23B; ETGE peptide treatment and KO mouse I/R model for NRF2; myeloid-specific KO and CBM reconstitution for CARD9\",\n      \"pmids\": [\"37153745\", \"37639212\", \"39079388\", \"39118286\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How OTUD1 is directed to K33 vs K63 chains on different substrates in the same cell type\", \"Whether SEC23B regulation is specific to neutrophils or generalizable\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Development of UbVOD.1, a ubiquitin variant inhibitor bridging the OTU domain and UIM, provided the first tool-grade inhibitor of OTUD1 and confirmed that the UIM contributes to catalytic engagement with ubiquitin chains.\",\n      \"evidence\": \"Phage display selection, in vitro IC50 quantification, cellular RIPK1 ubiquitination rescue with di-UbV\",\n      \"pmids\": [\"37589489\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No in vivo application of UbVOD.1 demonstrated\", \"Selectivity across the full OTU DUB family not comprehensively tested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"OTUD1 was shown to regulate cardiac calcium handling by stabilizing PDE5A through K48-deubiquitination, inactivating the cGMP-PKG-SERCA2a axis, and to impair AMPK activation by deubiquitinating AMPKα2 at K60/K379 and blocking CAMKK2 binding, establishing OTUD1 as a driver of heart failure and diabetic cardiomyopathy.\",\n      \"evidence\": \"LC-MS/MS, C320 mutagenesis, domain mapping for PDE5A in ISO/MI KO mice; AMPKα2 K60/K379 mutagenesis, cardiomyocyte-specific KO in T1DM/T2DM models with scRNA-seq\",\n      \"pmids\": [\"38185350\", \"40683882\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PDE5A and AMPKα2 deubiquitination interact in the same cardiac pathology\", \"Therapeutic targeting of OTUD1 in heart failure not yet demonstrated\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Recent work further expanded OTUD1 substrates to β-catenin (controlling angiogenesis in diabetic wound healing), C/EBPβ (driving Alzheimer's neuroinflammation), PRDX1 (suppressing osteoclastogenesis), HK2 (promoting NLRP3-driven pyroptosis), and p53 (promoting endothelial procoagulant programs), demonstrating context-dependent pro- or anti-inflammatory outcomes.\",\n      \"evidence\": \"IP-MS with site-specific mutagenesis (β-catenin K496/K508/K625, C/EBPβ K253), myeloid-specific KO with micro-CT for PRDX1, OTUD1 KO in CLP sepsis model for HK2, L357 mutagenesis with thrombosis model for p53\",\n      \"pmids\": [\"40300668\", \"40335710\", \"40585986\", \"40500776\", \"41967174\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How OTUD1 substrate selection is regulated post-translationally in different tissues\", \"No unifying structural model of OTUD1 engaged with different chain types\", \"Contribution of disordered N-terminus to substrate recruitment largely unmapped\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include how OTUD1 achieves substrate selectivity among its many targets in a given cell, whether its catalytic and non-catalytic (ETGE, OUN-36) functions are coordinately regulated, and whether a full-length structural model can explain its multi-linkage specificity.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No full-length crystal or cryo-EM structure\", \"No systematic selectivity profiling across chain types with endogenous substrates\", \"Upstream signals governing OTUD1 protein turnover and activity remain fragmentary\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 19, 20, 21, 22, 23, 24, 25, 27, 31, 33, 34, 35, 36]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 2, 3, 5, 8, 10, 13, 19, 22, 23, 24, 31, 33, 34, 36]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [11, 18]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 11, 18]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [3, 4, 5, 6, 7, 9, 12, 21, 24, 30, 31, 32]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 10, 11, 15, 22, 23, 25, 33, 36]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [2, 8, 17, 24, 36]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 3, 5, 13, 18, 27]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [18]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [19]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [7, 20]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [3, 10, 13, 22, 23, 25]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"SMAD7\",\n      \"IRF3\",\n      \"RIPK1\",\n      \"CARD9\",\n      \"KEAP1\",\n      \"STAT3\",\n      \"PRKAA2\",\n      \"PDE5A\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}