{"gene":"OTULIN","run_date":"2026-06-10T05:19:53","timeline":{"discoveries":[{"year":2013,"finding":"OTULIN (FAM105B) is a deubiquitinase with exquisite specificity for Met1-linked (linear) polyubiquitin chains; crystal structures of the OTULIN catalytic domain in complex with diubiquitin revealed Met1-specific ubiquitin-binding sites and a mechanism of substrate-assisted catalysis in which the proximal ubiquitin activates the catalytic triad of the protease. Mutation of Ub Glu16 reduces OTULIN kcat 240-fold.","method":"Crystal structure of OTU catalytic domain–diubiquitin complex; in vitro DUB activity assays; site-directed mutagenesis of Ub Glu16","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus in vitro reconstitution with mutagenesis, two independent labs reporting same specificity simultaneously","pmids":["23746843"],"is_preprint":false},{"year":2013,"finding":"OTULIN binds LUBAC and overexpression of OTULIN prevents TNFα-induced NEMO association with ubiquitinated RIPK1, demonstrating that OTULIN antagonizes LUBAC-mediated linear ubiquitin signaling at the receptor complex level.","method":"Co-immunoprecipitation; overexpression and knockdown in cells; TNFα stimulation assays monitoring NEMO–RIPK1 ubiquitin association","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP plus functional knockdown/overexpression readouts, replicated across two simultaneous reports (PMID 23746843 and 23806334)","pmids":["23746843","23806334"],"is_preprint":false},{"year":2013,"finding":"OTULIN depletion augments NF-κB signaling downstream of NOD2, and affinity purification of Met1-ubiquitin followed by quantitative proteomics identified RIPK2 as the predominant NOD2-regulated Met1-ubiquitin substrate; OTULIN restricts Met1-Ub on RIPK2 and on LUBAC components spontaneously.","method":"siRNA knockdown; affinity purification of Met1-Ub chains coupled to quantitative mass spectrometry; immunoprecipitation; NF-κB reporter assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — quantitative proteomics substrate identification combined with functional knockdown and Co-IP, single lab but orthogonal methods","pmids":["23806334"],"is_preprint":false},{"year":2014,"finding":"OTULIN binds LUBAC via a conserved PUB-interacting motif (PIM) that docks onto the PUB domain of HOIP; crystal structures and NMR revealed the molecular basis of the high-affinity interaction. Phosphorylation of OTULIN Tyr56 within the PIM prevents HOIP binding, whereas unphosphorylated OTULIN is part of the endogenous LUBAC complex.","method":"Crystal structure of HOIP PUB–OTULIN PIM complex; NMR; Co-immunoprecipitation; phospho-mimetic/phospho-null mutagenesis; in vitro binding assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus NMR plus mutagenesis, independently reported by two labs simultaneously (PMID 24726323 and 24726327)","pmids":["24726323","24726327"],"is_preprint":false},{"year":2014,"finding":"OTULIN must be present on LUBAC (via HOIP PUB–PIM interaction) to restrict Met1-polyUb signaling; HOIP binding is required for recruitment of OTULIN to the TNF receptor complex to counteract HOIP-dependent NF-κB activation.","method":"NF-κB luciferase reporter assays; Co-immunoprecipitation; site-directed mutagenesis of OTULIN Tyr56 and HOIP PUB; TNF receptor complex purification","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — functional epistasis with mutagenesis plus receptor complex purification, replicated in two concurrent papers","pmids":["24726323","24726327"],"is_preprint":false},{"year":2014,"finding":"Both CYLD and OTULIN interact with LUBAC via the PUB domain of HOIP even in unstimulated cells, and their interaction with HOIP synergistically suppresses LUBAC-mediated linear polyubiquitination and NF-κB activation; OTULIN interaction with HOIP also suppresses canonical Wnt signaling activation by LUBAC.","method":"Co-immunoprecipitation; cell-free translation/binding assays; NF-κB reporter assays; HOIP-null cell reconstitution with binding-deficient HOIP mutants","journal":"Genes to cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus functional rescue in HOIP-null cells, single lab","pmids":["24461064"],"is_preprint":false},{"year":2016,"finding":"OTULIN is essential in vivo for preventing spontaneous M1-linked polyubiquitin accumulation and NF-κB activation in myeloid cells; OTULIN deficiency in B and T cells instead causes LUBAC degradation and downregulation of M1-polyUb signaling. A hypomorphic human OTULIN mutation causes OTULIN-related autoinflammatory syndrome (ORAS) treatable by anti-TNF.","method":"Four independent conditional OTULIN knockout mouse models; immunoprecipitation; Western blot for LUBAC levels; cytokine measurements; anti-TNF rescue experiments","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — four independent genetic mouse models with mechanistic pathway readouts, replicated across cell types","pmids":["27523608"],"is_preprint":false},{"year":2018,"finding":"OTULIN promotes LUBAC activity by preventing LUBAC auto-ubiquitination with linear polyubiquitin; catalytically inactive OTULIN knock-in mice resemble LUBAC-deficient mice and die midgestation from TNFR1- and RIPK1-kinase-dependent cell death. Embryonic lethality is rescued by combined loss of caspase-8 and RIPK3, showing OTULIN and LUBAC function in a linear pathway.","method":"Catalytic-dead OTULIN knock-in mice (constitutive and endothelial-specific); genetic epistasis with TNFR1-KO, RIPK1-kinase-dead KI, caspase-8-KO, RIPK3-KO; Western blot for LUBAC auto-ubiquitination","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal genetic epistasis models in vivo with defined cell-death pathway placement","pmids":["29950720"],"is_preprint":false},{"year":2019,"finding":"OTULIN interacts with SNX27 (sorting nexin 27) via its C-terminal PDZ-binding motif (PDZbm) engaging the cargo-binding PDZ domain of SNX27; a second interface between the OTULIN OTU domain and the SNX27 PDZ domain was revealed by crystal structure. Via this association, OTULIN antagonizes SNX27-dependent cargo loading and VPS26A-retromer binding, inhibiting endosome-to-plasma membrane trafficking in a catalysis-independent manner.","method":"Mass spectrometry identification of OTULIN interactor; crystal structure of OTU domain–PDZ domain complex; Co-immunoprecipitation; endosomal trafficking assays; mutagenesis of PDZbm","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure plus Co-IP plus functional trafficking assay, single lab but multiple orthogonal methods","pmids":["31541095"],"is_preprint":false},{"year":2019,"finding":"OTULIN is cleaved by caspase-3 at Asp-31 during apoptosis, generating a C-terminal fragment that restricts caspase activation and cell death. During necroptosis, OTULIN is hyper-phosphorylated at Tyr-56, which modulates RIPK1 ubiquitin dynamics and promotes cell death; this phosphorylation is counteracted by the phosphatase DUSP14, identified as an OTULIN phosphatase.","method":"In vitro caspase-3 cleavage assay; site-directed mutagenesis (D31A); Co-immunoprecipitation of DUSP14; phosphorylation assays; keratinocyte apoptosis/necroptosis induction assays","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro cleavage assay with mutagenesis plus Co-IP of phosphatase, single lab","pmids":["31825842"],"is_preprint":false},{"year":2020,"finding":"TRIM32 interacts with OTULIN and conjugates non-proteolytic (K48/K63-linked) polyubiquitin chains onto OTULIN, which blocks the OTULIN–HOIP interaction, thereby preventing OTULIN from suppressing LUBAC and promoting NF-κB activation. TRIM32 E3 ligase activity is required for this effect.","method":"Proteomics of OTULIN protein complex; Co-immunoprecipitation; TRIM32 E3 ligase domain mutagenesis; ubiquitination assay; NF-κB reporter assay; genetic complementation","journal":"Journal of molecular cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus in-cell ubiquitination assay plus E3-dead mutagenesis, single lab","pmids":["31504727"],"is_preprint":false},{"year":2020,"finding":"OTULIN deficiency in liver parenchymal cells triggers steatohepatitis via aberrant mTOR activation (independent of TNFR1 signaling); rapamycin administration significantly reduces liver pathology in hepatocyte-specific OTULIN-KO mice.","method":"Liver-specific OTULIN knockout mice; TNFR1-KO epistasis; mTOR pathway Western blot; rapamycin pharmacological rescue; histopathology","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO plus pharmacological rescue with pathway readout, single lab","pmids":["32231246"],"is_preprint":false},{"year":2020,"finding":"OTULIN deficiency in hepatocytes causes apoptosis through FADD-dependent and RIPK1-kinase-dependent pathways; genetic ablation of FADD completely rescues, and RIPK1 kinase-dead knockin significantly protects, mice from OTULIN-deficient liver disease. Type I interferons also contribute to disease in this model.","method":"Hepatocyte-specific OTULIN-KO mice; genetic epistasis with FADD-KO and RIPK1 kinase-dead KI; IFNAR epistasis; histopathology; TUNEL assays","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal genetic epistasis experiments definitively placing FADD and RIPK1 kinase activity downstream of OTULIN loss","pmids":["32075762"],"is_preprint":false},{"year":2020,"finding":"OTULIN inhibits linear ubiquitination of β-catenin, preventing its Lys48-linked ubiquitination and proteasomal degradation upon DNA damage. The association between OTULIN and β-catenin is enhanced by ABL1-dependent phosphorylation of OTULIN Tyr56, which is triggered by genotoxic stress.","method":"Co-immunoprecipitation; in-cell ubiquitination assay; proteasome inhibition; ABL1 kinase assay; phospho-mimetic/null OTULIN Tyr56 mutants; xenograft tumor models","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus kinase assay with mutagenesis, single lab","pmids":["32770022"],"is_preprint":false},{"year":2020,"finding":"LUBAC and OTULIN localize to the phagophore area; LUBAC component RNF31 translocates to LC3 puncta upon autophagy induction. OTULIN knockdown promotes autophagy initiation but blocks autophagosome maturation by causing excessive linear-ubiquitinated ATG13 accumulation at the phagophore.","method":"siRNA knockdown; confocal immunofluorescence co-localization with LC3; autophagy flux assays (GFP-RFP-LC3); Western blot for ATG13 ubiquitination","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct co-localization plus knockdown phenotype with defined substrate (ATG13), single lab","pmids":["32543267"],"is_preprint":false},{"year":2021,"finding":"OTULIN deubiquitinates ALK1 (Activin receptor-like kinase 1) to remove linear ubiquitin chains conjugated by LUBAC; linear ubiquitination of ALK1 inhibits its kinase activity and Smad1/5 activation. EC-specific or constitutive Otulin deletion causes arteriovenous malformations and embryonic lethality rescued by BMP9 pretreatment or constitutively active ALK1 knockin.","method":"EC-specific and constitutive Otulin-KO mice; in vitro ALK1 kinase assay with/without linear ubiquitination; Smad1/5 phosphorylation Western blot; Co-immunoprecipitation; ALK1Q200D knockin rescue; HOIP inhibitor treatment of HHT2 patient-derived ECs","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with multiple in vivo rescue experiments plus in vitro kinase activity assay and Co-IP demonstrating substrate-level mechanism","pmids":["34157307"],"is_preprint":false},{"year":2021,"finding":"SNX27 recruits OTULIN to the membrane-associated TNF receptor complex via their PDZ interaction; OTULIN deubiquitinates linear polyubiquitin at the TNF receptor complex, and chemical inhibition of SNX27-retromer translocation blocks OTULIN membrane localization and enhances TNFα-induced NF-κB signaling.","method":"Co-immunoprecipitation; TNF receptor complex purification; cholera toxin pharmacological inhibition; confocal imaging; NF-κB reporter assay","journal":"Cell & bioscience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus receptor complex purification plus pharmacological perturbation, single lab","pmids":["34315543"],"is_preprint":false},{"year":2021,"finding":"OTULIN deficiency in epidermis-specific KO mice causes TNFR1-dependent, RIPK1-kinase-activity-dependent keratinocyte death that is primarily necroptosis (requiring RIPK3/MLKL); combined loss of RIPK3 and FADD fully prevents skin lesions, indicating redundant roles of apoptosis and necroptosis. MyD88 deficiency suppresses skin inflammation, implicating TLR/IL-1 signaling.","method":"Epidermis-specific OTULIN KO mice; genetic epistasis with RIPK3-KO, MLKL-KO, FADD-KO, RIPK1 kinase-dead KI, TNFR1-KO, MyD88-KO; histopathology","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal genetic epistasis experiments in vivo with clear pathway placement","pmids":["34625557"],"is_preprint":false},{"year":2021,"finding":"Keratinocyte-specific OTULIN ablation causes inflammatory skin lesions driven by keratinocyte cell death; genetic deletion of Tnfr1, RIPK1 kinase-dead KI, or combined keratinocyte-specific deletion of FADD and MLKL completely rescues dermatitis. OTULIN-deficient keratinocytes display a type-I interferon and IL-1β response signature.","method":"Keratinocyte-specific OTULIN KO mice; genetic epistasis with Tnfr1-KO, Ripk1-KD KI, FADD/MLKL double KO; single-cell RNA-sequencing; cytokine inhibition","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal genetic epistasis experiments, single-cell transcriptomics, replicated pathways across two concurrent papers","pmids":["34625556"],"is_preprint":false},{"year":2021,"finding":"OTULIN deubiquitinates IRF3 to remove linear polyubiquitin chains, inhibiting RIPA (RIG-I-induced pathway of apoptosis). During virus infection, RIPA overcomes OTULIN inhibition by caspase-3 cleavage of OTULIN at D31 followed by proteasomal degradation of the cleaved fragment, preceded by K48-linked ubiquitination at K64 and K197 by HOIP.","method":"OTULIN overexpression/knockdown; caspase-3 in vitro cleavage assay; D31A/K64R/K197R mutagenesis; mass spectrometry identification of ubiquitination sites; Co-immunoprecipitation with LUBAC/HOIP; virus infection apoptosis assay","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro caspase cleavage assay with mutagenesis plus MS-identified ubiquitination sites plus Co-IP, single lab","pmids":["34545182"],"is_preprint":false},{"year":2021,"finding":"OTULIN deubiquitinates proteasome subunits; OTULIN deficiency causes proteasome dysregulation in cells, which is the mechanistic basis for elevated type I interferon signaling in OTULIN-deficient patients and cell lines.","method":"CRISPR-generated OTULIN-KO cell lines; proteasome activity assays; Co-immunoprecipitation of proteasome subunits with OTULIN; type I IFN pathway assays in patient PBMCs/monocytes","journal":"Science advances","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP identifying proteasome subunits as substrates plus functional proteasome assay in KO cells, single lab","pmids":["34797715"],"is_preprint":false},{"year":2022,"finding":"OTULIN undergoes self-deubiquitination intermolecularly via dimerization; Lys64/66 of OTULIN are linearly ubiquitinated in a LUBAC-dependent manner to maintain OTULIN–LUBAC interaction under unstressed conditions. Genotoxic stress induces OTULIN dimerization (via cysteine-mediated disulfide bonds under oxidative stress) and self-deubiquitination, leading to OTULIN–LUBAC dissociation and NF-κB overactivation.","method":"Mutagenesis of OTULIN K64/K66; Co-immunoprecipitation; in vitro ubiquitination assay; cross-linking/dimerization assays; genotoxic stress and oxidative stress treatments","journal":"PNAS","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis plus in vitro ubiquitination assay plus Co-IP, single lab","pmids":["35939695"],"is_preprint":false},{"year":2022,"finding":"OTULIN haploinsufficiency causes accumulation of caveolin-1 in dermal fibroblasts (but not leukocytes), which facilitates cytotoxic damage by staphylococcal α-toxin; TNFR-mediated NF-κB signaling remains intact in haploinsufficient fibroblasts.","method":"Patient-derived fibroblast studies; linear ubiquitin accumulation assay; caveolin-1 protein level measurement; α-toxin cytotoxicity assay; leukocyte phenotyping","journal":"Science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — patient-derived cells with defined molecular mechanism (caveolin-1 accumulation) and functional readout (α-toxin cytotoxicity), single study","pmids":["35587511"],"is_preprint":false},{"year":2023,"finding":"OTULIN deficiency in macrophages licenses RIPK3-mediated cell death, which activates the Nlrp3 inflammasome independently of gasdermin D-mediated pyroptosis, leading to RIPK3-dependent IL-1β secretion; elevated serum IL-1β in myeloid-specific OTULIN-KO mice is abolished by Ripk3 or Nlrp3 deletion.","method":"Myeloid-specific OTULIN KO mice; genetic epistasis with RIPK3-KO and Nlrp3-KO; gasdermin D knockdown; IL-1β ELISA; macrophage cell death assays","journal":"Science immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple genetic epistasis experiments in vivo and in vitro placing RIPK3 and Nlrp3 downstream of OTULIN loss","pmids":["38000038"],"is_preprint":false},{"year":2023,"finding":"OTULIN interacts with SCRIB via its C-terminal PDZ-binding motif; Met1-Ub chains associate with VANGL2 and PRICKLE1 but not SCRIB, directing VANGL2 surface presentation. OTULIN is recruited to VANGL2-enriched cell-cell contacts and its loss causes deficits in Wnt5a-induced filopodia extension and VANGL2 trafficking, implicating linear (de)ubiquitination in planar cell polarity signaling.","method":"HEK293 cell-based interactomic analysis; Co-immunoprecipitation; confocal imaging of OTULIN at cell-cell contacts; OTULIN-KO cells; Wnt5a-stimulated filopodia assay; VANGL2-GFP trafficking assay","journal":"Disease models & mechanisms","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus functional KO phenotype with direct localization, single lab","pmids":["37589075"],"is_preprint":false},{"year":2023,"finding":"STAT3 is a direct substrate of linear ubiquitination in glioblastoma stem-like cells; linear ubiquitination of STAT3 negatively regulates its activity by recruiting the phosphatase TC-PTP to STAT3. Preferential OTULIN expression in GSCs deubiquitinates STAT3 to drive persistent STAT3 signaling and maintain stemness.","method":"Bio-orthogonal linear ubiquitin probe (NAEK-Ub) for substrate identification; Co-immunoprecipitation; STAT3 activity assays; OTULIN knockdown in GSCs; TC-PTP recruitment assay","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — novel substrate identified with bio-orthogonal probe plus Co-IP and functional validation, single lab","pmids":["36660824"],"is_preprint":false},{"year":2023,"finding":"SPATA2 competes with OTULIN for binding to HOIP via its PUB-interacting motif (PIM) and zinc finger domain, thereby promoting LUBAC auto-ubiquitination. Cyld-/-Spata2-/- double mutant mice show perinatal lethality and elevated M1-linked ubiquitination dependent on OTULIN, indicating SPATA2 counteracts OTULIN-mediated LUBAC deubiquitination independently of CYLD.","method":"Cyld-/-, Spata2-/-, and Cyld-/-Spata2-/- double KO mice; competitive binding assays (Co-IP with PIM mutants); M1-Ub Western blot; genetic rescue by additional OTULIN deletion","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with double KO mice plus competitive binding assay with mutagenesis, single lab","pmids":["36640323"],"is_preprint":false},{"year":2024,"finding":"The dominant-negative OTULIN Cys129Ser mutation ablates deubiquitinase activity without affecting protein stability or LUBAC/linear-ubiquitin binding; loss of catalytic activity causes LUBAC auto-ubiquitination that impairs LUBAC recruitment to the TNF receptor signaling complex, promoting TNF-induced cell death.","method":"Patient-derived cell studies; in vitro DUB activity assay; Co-immunoprecipitation of LUBAC with TNF receptor complex; linear ubiquitin accumulation assay; TNF-induced cell death assay","journal":"Journal of experimental medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro activity assay plus receptor complex Co-IP plus functional cell death readout, single study","pmids":["38630025"],"is_preprint":false},{"year":2024,"finding":"OTULIN prevents GPX4 proteasomal degradation by reducing its ubiquitin level (linear deubiquitination of GPX4), thereby conferring resistance to cisplatin-induced mitochondrial apoptosis (not ferroptosis) in osteosarcoma cells.","method":"OTULIN overexpression/knockdown; GPX4 ubiquitination assay; proteasome inhibitor (MG132) rescue; cell death assays (apoptosis vs ferroptosis markers); xenograft model","journal":"Journal of experimental & clinical cancer research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP/ubiquitination assay with pharmacological rescue, single lab, no direct linear-Ub specificity confirmed for GPX4","pmids":["39721999"],"is_preprint":false},{"year":2025,"finding":"OTULIN regulates the ubiquitination of NCOA4, preventing its degradation and thereby enabling NCOA4-mediated FTH1 accumulation; this protects hepatocytes from APAP-induced ferroptosis by maintaining iron homeostasis.","method":"OTULIN stable cell lines; ubiquitination assay of NCOA4; Co-immunoprecipitation; ferroptosis markers (GSH, lipid peroxidation); mouse APAP injury model","journal":"International immunopharmacology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Co-IP plus ubiquitination assay, single lab, mechanistic chain inferred from multiple steps","pmids":["40158433"],"is_preprint":false},{"year":2026,"finding":"OTULIN removes linear ubiquitin chains from TRAF6 at K104, K142, and K371 to suppress RIG-I-dependent antiviral signaling; linear ubiquitination of TRAF6 by LUBAC promotes K63-linked ubiquitination of TRAF6 and strengthens its association with MAVS to amplify antiviral responses. OTULIN-KO mice exhibit enhanced antiviral immunity.","method":"OTULIN overexpression/knockout in HeLa and iBMDM cells; lentiviral reconstitution; TRAF6 ubiquitination mass spectrometry; K104R/K142R/K371R mutagenesis; MAVS Co-immunoprecipitation; IFN-β reporter assay; Otulin+/- mouse viral challenge","journal":"PNAS","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS-identified ubiquitination sites with mutagenesis plus Co-IP plus in vivo mouse data, single lab","pmids":["41802043"],"is_preprint":false},{"year":2025,"finding":"OTULIN interacts with AMPK, controls its M1-ubiquitination, and restricts AMPK activation in response to glucose starvation and allosteric activation; LUBAC promotes AMPK activation while OTULIN restricts it, placing the LUBAC-OTULIN axis as a regulator of metabolic signaling.","method":"LUBAC and OTULIN KO/KD cells; AMPK activity assays; Co-immunoprecipitation of OTULIN with AMPK; M1-ubiquitination assay of AMPK; metabolic flux measurements; Drosophila starvation survival (LUBAC-deficient)","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Co-IP plus activity assay, preprint not yet peer-reviewed, single lab","pmids":["bio_10.1101_2024.11.08.622598"],"is_preprint":true},{"year":2025,"finding":"In OTULIN-deficient keratinocytes, β-catenin accumulates linear ubiquitin chains promoting its K48-linked ubiquitination and proteasomal degradation, reducing Wnt/β-catenin signaling and causing TCF3 degradation; restoring β-catenin stabilization prevents progressive skin inflammation and keratinocyte death.","method":"Keratinocyte-specific OTULIN KO mice; β-catenin linear ubiquitination assay; TCF3 protein level assay; β-catenin stabilizing genetic/pharmacological interventions; skin inflammation rescue assays","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — preprint, single lab, ubiquitination assay plus genetic rescue, not yet peer-reviewed","pmids":["bio_10.1101_2025.01.08.631848"],"is_preprint":true},{"year":2026,"finding":"RNF6 E3 ligase ubiquitinates OTULIN and promotes its degradation; RNF6 knockdown prevents OTULIN ubiquitination/degradation and thereby suppresses TGF-β1-induced epithelial-mesenchymal transition in bronchial epithelial cells. Silencing RNF6 phenocopies OTULIN overexpression in blocking partial EMT.","method":"Label-free proteomics combined with Co-IP/mass spectrometry; Co-IP validation; cycloheximide and MG132 assays for protein stability; OTULIN overexpression rescue of RNF6 knockdown; EMT marker Western blot","journal":"Biochemical pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus proteasome inhibitor rescue plus functional rescue epistasis, single lab","pmids":["42055142"],"is_preprint":false},{"year":2026,"finding":"OTULIN directly interacts with mitochondrial fusion protein OPA1 and regulates its ubiquitination status; the E3 ligase RNF31/HOIP ubiquitinates OPA1, and OTULIN counteracts RNF31-mediated OPA1 destabilization, thereby maintaining mitochondrial homeostasis in alveolar epithelial cells under hyperoxic stress.","method":"Co-immunoprecipitation of OTULIN with OPA1; RNF31 knockdown and OTULIN overexpression/knockdown in alveolar epithelial cells; OPA1 ubiquitination assay; mitochondrial ROS and membrane potential measurements; hyperoxia neonatal mouse model","journal":"Cellular & molecular biology letters","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP plus knockdown, no in vitro reconstitution, single lab","pmids":["42218396"],"is_preprint":false}],"current_model":"OTULIN is the only mammalian deubiquitinase with absolute specificity for Met1-linked (linear) polyubiquitin chains, which it cleaves via a substrate-assisted catalytic mechanism (proximal Ub activates the catalytic triad) after docking through dedicated Met1-specific ubiquitin-binding sites revealed by crystal structure; it constitutively associates with the LUBAC E3 ligase (via a Tyr56-phosphorylation-regulated PIM–HOIP PUB domain interaction) to prevent LUBAC auto-ubiquitination and thereby sustain LUBAC activity, while simultaneously limiting linear ubiquitin accumulation on receptor complex components (RIPK1, RIPK2, NEMO, ALK1, TRAF6, STAT3, ATG13, and proteasome subunits) downstream of TNF, NOD2, and RIG-I receptors; dynamic post-translational regulation of OTULIN itself—including ABL1-mediated Tyr56 phosphorylation, TRIM32-mediated non-proteolytic ubiquitination at Lys64/66, LUBAC-dependent linear ubiquitination, caspase-3 cleavage at Asp31, and RNF6-mediated proteasomal degradation—controls its interaction with LUBAC, its association with β-catenin (Wnt), SCRIB/VANGL2 (planar cell polarity), and the SNX27-retromer (endosomal trafficking), such that loss of OTULIN causes spontaneous linear ubiquitin accumulation leading to NF-κB hyperactivation in myeloid cells and TNFR1/RIPK1-kinase-dependent apoptosis and necroptosis in epithelial cells, underscoring its essential role in preventing autoinflammatory disease, embryonic lethality, and organ-specific pathology."},"narrative":{"mechanistic_narrative":"OTULIN (FAM105B) is the mammalian deubiquitinase with absolute specificity for Met1-linked (linear) polyubiquitin, which it hydrolyzes through a substrate-assisted catalytic mechanism in which the proximal ubiquitin activates the protease catalytic triad, as defined by crystal structures of the OTU domain bound to diubiquitin [PMID:23746843]. It constitutively associates with the LUBAC E3 ligase by docking a PUB-interacting motif (PIM) onto the HOIP PUB domain, an interaction abolished by phosphorylation of OTULIN Tyr56 [PMID:24726323, PMID:24726327]. Through this association OTULIN restrains linear ubiquitin signaling at two levels: it prevents LUBAC auto-ubiquitination to sustain ligase activity [PMID:29950720], and it removes Met1-ubiquitin from receptor-complex substrates including RIPK1/NEMO downstream of TNF [PMID:23746843, PMID:23806334], RIPK2 downstream of NOD2 [PMID:23806334], and ALK1 in BMP9/Smad1/5 signaling [PMID:34157307]. OTULIN is essential in vivo: catalytically inactive knock-in mice phenocopy LUBAC deficiency and die mid-gestation from TNFR1- and RIPK1-kinase-dependent cell death rescued by combined caspase-8/RIPK3 loss [PMID:29950720], and tissue-specific loss drives autoinflammation through myeloid NF-κB hyperactivation [PMID:27523608] and epithelial RIPK1-kinase-dependent apoptosis and necroptosis [PMID:32075762, PMID:34625557]. A hypomorphic human mutation causes OTULIN-related autoinflammatory syndrome (ORAS), treatable with anti-TNF [PMID:27523608]. OTULIN activity is itself tuned by post-translational regulation—ABL1-dependent Tyr56 phosphorylation, TRIM32-mediated non-proteolytic ubiquitination, and LUBAC-dependent linear ubiquitination at Lys64/66—that governs its LUBAC engagement and substrate selection [PMID:31504727, PMID:32770022, PMID:35939695]. Beyond the NF-κB axis, OTULIN engages the SNX27-retromer through a C-terminal PDZ-binding motif to control endosomal trafficking independently of catalysis [PMID:31541095, PMID:34315543] and acts on additional substrates including IRF3 and TRAF6 in antiviral signaling [PMID:34545182, PMID:41802043].","teleology":[{"year":2013,"claim":"Establishing how a DUB could be Met1-specific answered why linear ubiquitin signaling has a dedicated eraser, defining OTULIN's catalytic chemistry and chain selectivity.","evidence":"Crystal structure of OTU domain–diubiquitin complex with in vitro DUB assays and Ub Glu16 mutagenesis","pmids":["23746843"],"confidence":"High","gaps":["Did not establish in vivo substrate repertoire","Catalytic mechanism shown in vitro, not the cellular regulation of activity"]},{"year":2013,"claim":"Linking OTULIN to LUBAC and showing it blocks TNF-induced NEMO–RIPK1 ubiquitin association placed OTULIN as a negative regulator of linear ubiquitin signaling at the receptor complex.","evidence":"Reciprocal Co-IP, overexpression/knockdown, and TNFα stimulation in cells","pmids":["23746843","23806334"],"confidence":"High","gaps":["Mode of recruitment to the receptor complex not defined","Did not resolve whether antagonism is catalytic or stoichiometric"]},{"year":2013,"claim":"Identifying RIPK2 as the dominant NOD2-regulated Met1-Ub substrate extended OTULIN's role beyond TNF to a second innate immune receptor pathway.","evidence":"siRNA knockdown, Met1-Ub affinity purification with quantitative MS, NF-κB reporters","pmids":["23806334"],"confidence":"High","gaps":["Did not establish in vivo NOD2 phenotype","Spontaneous restriction of LUBAC components not mechanistically separated from receptor signaling"]},{"year":2014,"claim":"Defining the HOIP PUB–OTULIN PIM interface and its phospho-regulation by Tyr56 explained how OTULIN is recruited to LUBAC and how that recruitment is switched off.","evidence":"Crystal structure, NMR, Co-IP, phospho-mimetic/null mutagenesis, receptor complex purification","pmids":["24726323","24726327","24461064"],"confidence":"High","gaps":["Identity of the Tyr56 kinase not established here","Did not address CYLD/SPATA2 competition for the same PUB domain"]},{"year":2016,"claim":"Genetic loss-of-function in mice and humans demonstrated OTULIN is non-redundantly required to prevent autoinflammation, with cell-type-specific outcomes on M1-Ub and LUBAC levels.","evidence":"Four conditional knockout mouse models, IP/Western for LUBAC, cytokines, anti-TNF rescue; ORAS patient mutation","pmids":["27523608"],"confidence":"High","gaps":["Did not distinguish whether disease is driven by NF-κB or cell death","Mechanism of LUBAC degradation in lymphocytes unresolved"]},{"year":2018,"claim":"Catalytic-dead knock-in mice showed OTULIN's DUB activity sustains LUBAC by preventing auto-ubiquitination, placing OTULIN and LUBAC in a single TNFR1/RIPK1-driven cell-death pathway.","evidence":"Catalytic-dead knock-in mice with epistasis (TNFR1-KO, RIPK1-KD, caspase-8-KO, RIPK3-KO), Western for LUBAC auto-ubiquitination","pmids":["29950720"],"confidence":"High","gaps":["Did not identify tissue-specific cell-death effectors beyond embryo","Counterintuitive role as LUBAC-promoting rather than purely antagonistic not fully reconciled with receptor-level data"]},{"year":2019,"claim":"Discovery of the SNX27-retromer interaction via OTULIN's PDZ-binding motif revealed a catalysis-independent role in endosomal trafficking, broadening OTULIN function beyond NF-κB.","evidence":"MS interactor identification, crystal structure of OTU–PDZ complex, Co-IP, trafficking assays, PDZbm mutagenesis","pmids":["31541095"],"confidence":"High","gaps":["Physiological significance of trafficking role in vivo not established","Relationship between trafficking and immune functions unclear"]},{"year":2019,"claim":"Characterizing caspase-3 cleavage at Asp31 and Tyr56 hyperphosphorylation with DUSP14 as counteracting phosphatase showed OTULIN is dynamically inactivated during cell death.","evidence":"In vitro caspase-3 cleavage, D31A mutagenesis, DUSP14 Co-IP, keratinocyte death assays","pmids":["31825842"],"confidence":"Medium","gaps":["Single lab; cleavage fragment function not independently confirmed","Kinase responsible for necroptotic Tyr56 phosphorylation not identified"]},{"year":2020,"claim":"Identifying TRIM32-mediated non-proteolytic ubiquitination that blocks OTULIN–HOIP binding added an upstream regulatory layer controlling OTULIN's access to LUBAC.","evidence":"OTULIN complex proteomics, Co-IP, TRIM32 E3-dead mutagenesis, ubiquitination and NF-κB reporter assays","pmids":["31504727"],"confidence":"Medium","gaps":["Single lab without in vivo validation","Ubiquitin chain linkage and exact lysines not fully resolved"]},{"year":2020,"claim":"Tissue-specific knockouts dissected the death effectors downstream of OTULIN loss, showing hepatocyte disease is FADD- and RIPK1-kinase-dependent with an mTOR-driven steatohepatitis component.","evidence":"Liver/hepatocyte-specific KO mice with FADD-KO, RIPK1-KD, IFNAR, TNFR1 epistasis; rapamycin rescue; histopathology","pmids":["32075762","32231246"],"confidence":"High","gaps":["How OTULIN loss drives mTOR activation independent of TNFR1 not defined","Relative contribution of apoptosis vs interferon to liver pathology not fully separated"]},{"year":2020,"claim":"Connecting OTULIN to β-catenin stability and to autophagy via ATG13 extended its substrate range to Wnt signaling and autophagosome maturation.","evidence":"Co-IP, in-cell ubiquitination assays, ABL1 kinase assay, Tyr56 mutants, xenografts; LC3 co-localization and autophagy flux assays","pmids":["32770022","32543267"],"confidence":"Medium","gaps":["Single-lab findings without in vivo genetic confirmation","Direct vs indirect action on β-catenin and ATG13 ubiquitin not fully resolved"]},{"year":2021,"claim":"Demonstrating OTULIN deubiquitinates ALK1, IRF3, TRAF6, and proteasome subunits, and that SNX27 recruits it to the TNF receptor complex, mapped substrate-level mechanisms across vascular, antiviral, and proteostasis pathways.","evidence":"EC-specific KO mice with BMP9/ALK1 rescue, in vitro kinase assays, bio-orthogonal Ub probes, MS site mapping, Co-IP, proteasome activity assays in KO cells","pmids":["34157307","34545182","34797715","34315543"],"confidence":"High","gaps":["Several substrate findings rest on single labs","How substrate selectivity is achieved among diverse targets unresolved"]},{"year":2021,"claim":"Epidermis- and keratinocyte-specific knockouts established that OTULIN loss in skin drives TNFR1/RIPK1-kinase-dependent necroptosis and apoptosis with type-I IFN and IL-1β signatures.","evidence":"Skin-specific KO mice with RIPK3-KO, MLKL-KO, FADD-KO, RIPK1-KD, TNFR1-KO, MyD88-KO epistasis; scRNA-seq","pmids":["34625557","34625556"],"confidence":"High","gaps":["Relative weighting of apoptosis vs necroptosis context-dependent","Source of MyD88/IL-1 input not pinpointed"]},{"year":2022,"claim":"Self-deubiquitination/dimerization control and the caveolin-1 haploinsufficiency phenotype refined how OTULIN abundance and LUBAC engagement are maintained and how partial loss manifests differently across cell types.","evidence":"K64/K66 mutagenesis, dimerization/cross-linking assays, in vitro ubiquitination; patient fibroblast caveolin-1 and α-toxin cytotoxicity assays","pmids":["35939695","35587511"],"confidence":"Medium","gaps":["Single-lab mechanistic models","Link between caveolin-1 accumulation and linear ubiquitin not fully mechanistic"]},{"year":2023,"claim":"Macrophage and substrate-level studies linked OTULIN loss to RIPK3-driven Nlrp3 inflammasome activation and to STAT3, SCRIB/VANGL2, and SPATA2-dependent LUBAC regulation, expanding its roles into inflammasome biology, stemness, and planar cell polarity.","evidence":"Myeloid-specific KO mice with RIPK3/Nlrp3 epistasis; bio-orthogonal Ub probe for STAT3; interactomics and KO phenotypes for SCRIB/VANGL2; double-KO mice for SPATA2","pmids":["38000038","36660824","37589075","36640323"],"confidence":"Medium","gaps":["STAT3, SCRIB/VANGL2 roles from single labs","Tissue relevance of PCP role not established in vivo"]},{"year":2024,"claim":"Patient-derived analysis of the dominant-negative Cys129Ser mutation showed catalytic loss impairs LUBAC recruitment to the TNF receptor complex via auto-ubiquitination, mechanistically uniting human disease with the catalytic-dead mouse model.","evidence":"Patient cells, in vitro DUB assay, LUBAC/receptor complex Co-IP, linear-Ub and TNF-induced death assays","pmids":["38630025"],"confidence":"Medium","gaps":["Single patient-derived study","Dominant-negative mechanism not tested in animal model"]},{"year":2026,"claim":"Mapping OTULIN action on TRAF6 (K104/K142/K371) and OPA1, plus RNF6-mediated OTULIN degradation, extended the LUBAC-OTULIN axis into antiviral amplification, mitochondrial homeostasis, and EMT control.","evidence":"MS site mapping with mutagenesis, MAVS Co-IP, IFN-β reporters, Otulin+/- viral challenge; OPA1/RNF31 Co-IP and knockdown; RNF6 proteomics with proteasome-inhibitor and rescue assays","pmids":["41802043","42218396","42055142"],"confidence":"Medium","gaps":["OPA1 finding low-confidence and single-lab","In vivo significance of EMT and mitochondrial roles limited"]},{"year":null,"claim":"How OTULIN achieves substrate and context selectivity—choosing among RIPK1/2, ALK1, IRF3, TRAF6, STAT3, β-catenin, and proteasome subunits in different cell types—and how its many regulatory modifications are integrated remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying model for substrate targeting beyond LUBAC association","Several substrate and metabolic claims rest on single labs or preprints awaiting confirmation"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[0,1,2,15,19,20,30]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,2,15]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[4,7,5]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[8,16]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[1,4]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[16,24]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[8,16]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[1,2,6,19,30]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[7,12,17,18,23]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,4,15]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,7,20]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[8,16]}],"complexes":["LUBAC","SNX27-retromer"],"partners":["RNF31","SNX27","TRIM32","DUSP14","SPATA2","SCRIB","ABL1","RNF6"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q96BN8","full_name":"Ubiquitin thioesterase otulin","aliases":["Deubiquitinating enzyme otulin","OTU domain-containing deubiquitinase with linear linkage specificity","Ubiquitin thioesterase Gumby"],"length_aa":352,"mass_kda":40.3,"function":"Deubiquitinase that specifically removes linear ('Met-1'-linked) polyubiquitin chains to substrates and acts as a regulator of angiogenesis and innate immune response (PubMed:23708998, PubMed:23746843, PubMed:23806334, PubMed:23827681, PubMed:24726323, PubMed:24726327, PubMed:26997266, PubMed:27523608, PubMed:27559085, PubMed:28919039, PubMed:30804083, PubMed:35170849, PubMed:35587511, PubMed:38630025, PubMed:38652464). Required during angiogenesis, craniofacial and neuronal development by regulating the canonical Wnt signaling together with the LUBAC complex (PubMed:23708998). Acts as a negative regulator of NF-kappa-B by regulating the activity of the LUBAC complex (PubMed:23746843, PubMed:23806334). OTULIN function is mainly restricted to homeostasis of the LUBAC complex: acts by removing 'Met-1'-linked autoubiquitination of the LUBAC complex, thereby preventing inactivation of the LUBAC complex (PubMed:26670046). Acts as a key negative regulator of inflammation by restricting spontaneous inflammation and maintaining immune homeostasis (PubMed:27523608). In myeloid cell, required to prevent unwarranted secretion of cytokines leading to inflammation and autoimmunity by restricting linear polyubiquitin formation (PubMed:27523608). Plays a role in innate immune response by restricting linear polyubiquitin formation on LUBAC complex in response to NOD2 stimulation, probably to limit NOD2-dependent pro-inflammatory signaling (PubMed:23806334)","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q96BN8/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/OTULIN","classification":"Not Classified","n_dependent_lines":7,"n_total_lines":1208,"dependency_fraction":0.005794701986754967},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"SNX27","stoichiometry":4.0}],"url":"https://opencell.sf.czbiohub.org/search/OTULIN","total_profiled":1310},"omim":[{"mim_id":"621472","title":"OTU DEUBIQUITINASE WITH LINEAR LINKAGE SPECIFICITY-LIKE PROTEIN; OTULINL","url":"https://www.omim.org/entry/621472"},{"mim_id":"621030","title":"AUTOINFLAMMATION, PANNICULITIS, AND DERMATOSIS SYNDROME, AUTOSOMAL DOMINANT; AIPDSA","url":"https://www.omim.org/entry/621030"},{"mim_id":"619986","title":"IMMUNODEFICIENCY 107, SUSCEPTIBILITY TO INVASIVE STAPHYLOCOCCUS AUREUS INFECTION; IMD107","url":"https://www.omim.org/entry/619986"},{"mim_id":"617099","title":"AUTOINFLAMMATION, PANNICULITIS, AND DERMATOSIS SYNDROME, AUTOSOMAL RECESSIVE; AIPDSB","url":"https://www.omim.org/entry/617099"},{"mim_id":"615712","title":"OTU DEUBIQUITINASE WITH LINEAR LINKAGE SPECIFICITY; OTULIN","url":"https://www.omim.org/entry/615712"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Mitochondria","reliability":"Approved"},{"location":"Plasma membrane","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"bone marrow","ntpm":24.1}],"url":"https://www.proteinatlas.org/search/OTULIN"},"hgnc":{"alias_symbol":["FLJ34884"],"prev_symbol":["FAM105B"]},"alphafold":{"accession":"Q96BN8","domains":[{"cath_id":"-","chopping":"81-347","consensus_level":"medium","plddt":95.9518,"start":81,"end":347}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96BN8","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96BN8-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96BN8-F1-predicted_aligned_error_v6.png","plddt_mean":83.81},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=OTULIN","jax_strain_url":"https://www.jax.org/strain/search?query=OTULIN"},"sequence":{"accession":"Q96BN8","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96BN8.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96BN8/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96BN8"}},"corpus_meta":[{"pmid":"23746843","id":"PMC_23746843","title":"OTULIN antagonizes LUBAC signaling by specifically hydrolyzing Met1-linked polyubiquitin.","date":"2013","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/23746843","citation_count":407,"is_preprint":false},{"pmid":"27523608","id":"PMC_27523608","title":"The Deubiquitinase OTULIN Is an Essential Negative Regulator of Inflammation and Autoimmunity.","date":"2016","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/27523608","citation_count":277,"is_preprint":false},{"pmid":"28362430","id":"PMC_28362430","title":"CYLD, A20 and OTULIN deubiquitinases in NF-κB signaling and cell death: so similar, yet so different.","date":"2017","source":"Cell death and differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/28362430","citation_count":221,"is_preprint":false},{"pmid":"23806334","id":"PMC_23806334","title":"OTULIN restricts Met1-linked ubiquitination to control innate immune signaling.","date":"2013","source":"Molecular 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NCOA4-FTH1 complex to alleviate APAP-induced hepatocyte Ferroptosis.","date":"2025","source":"International immunopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/40158433","citation_count":5,"is_preprint":false},{"pmid":"38774872","id":"PMC_38774872","title":"OTULIN deficiency: focus on innate immune system impairment.","date":"2024","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/38774872","citation_count":4,"is_preprint":false},{"pmid":"38827742","id":"PMC_38827742","title":"OTULIN haploinsufficiency predisposes to environmentally directed inflammation.","date":"2024","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/38827742","citation_count":4,"is_preprint":false},{"pmid":"38561559","id":"PMC_38561559","title":"OTULIN Can Improve Spinal Cord Injury by the NF-κB and Wnt/β-Catenin Signaling Pathways.","date":"2024","source":"Molecular 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carcinoma","url":"https://pubmed.ncbi.nlm.nih.gov/40726617","citation_count":0,"is_preprint":false},{"pmid":"41802043","id":"PMC_41802043","title":"Deubiquitinase OTULIN dampens RIG-I-dependent antiviral signaling by removing linear ubiquitination from TRAF6.","date":"2026","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/41802043","citation_count":0,"is_preprint":false},{"pmid":"32944611","id":"PMC_32944611","title":"The plot thickens: OTULIN regulation in cell death.","date":"2020","source":"Molecular & cellular oncology","url":"https://pubmed.ncbi.nlm.nih.gov/32944611","citation_count":0,"is_preprint":false},{"pmid":"42218396","id":"PMC_42218396","title":"OTULIN protects hyperoxia-induced neonatal lung injury and modulates mitochondrial protein OPA1 in association with the E3 ubiquitin ligase RNF31.","date":"2026","source":"Cellular & molecular biology letters","url":"https://pubmed.ncbi.nlm.nih.gov/42218396","citation_count":0,"is_preprint":false},{"pmid":"42245667","id":"PMC_42245667","title":"Association of peripheral blood LUBAC and OTULIN expression with severity and outcome in acute ischemic stroke: a prospective cohort study.","date":"2026","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/42245667","citation_count":0,"is_preprint":false},{"pmid":"42055142","id":"PMC_42055142","title":"Regulation of OTULIN ubiquitination by RNF6 alleviates asthma via mitigating epithelial-mesenchymal transition‑like phenotypic changes.","date":"2026","source":"Biochemical pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/42055142","citation_count":0,"is_preprint":false},{"pmid":"39974971","id":"PMC_39974971","title":"OTULIN Interactome Reveals Immune Response and Autophagy Associated with Tauopathy in a Mouse Model.","date":"2025","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/39974971","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.11.18.689012","title":"Linear ubiquitin chain assembly complex contributes to NLRP3-mediated pyroptotic cell death","date":"2025-11-18","source":"bioRxiv","url":"https://doi.org/10.1101/2025.11.18.689012","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.03.04.25323351","title":"Homozygous<i>OTULIN</i>Variant Linked to OTULIN-Related Autoinflammatory Syndrome with Abscess Formation","date":"2025-03-05","source":"bioRxiv","url":"https://doi.org/10.1101/2025.03.04.25323351","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.01.08.631848","title":"Canonical Wnt induction by OTULIN prevents keratinocyte death and skin inflammation","date":"2025-01-08","source":"bioRxiv","url":"https://doi.org/10.1101/2025.01.08.631848","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.11.08.622598","title":"M1-linked Ubiquitination by LUBAC Regulates AMPK Activity and the Response to Energetic Stress","date":"2024-11-10","source":"bioRxiv","url":"https://doi.org/10.1101/2024.11.08.622598","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":36909,"output_tokens":9629,"usd":0.127581,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":19929,"output_tokens":6117,"usd":0.126285,"stage2_stop_reason":"end_turn"},"total_usd":0.253866,"stage1_batch_id":"msgbatch_013a8chtv3AWyTqv4z4sEF4C","stage2_batch_id":"msgbatch_01AHsuW63vinsvuTgzmQ3n62","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2013,\n      \"finding\": \"OTULIN (FAM105B) is a deubiquitinase with exquisite specificity for Met1-linked (linear) polyubiquitin chains; crystal structures of the OTULIN catalytic domain in complex with diubiquitin revealed Met1-specific ubiquitin-binding sites and a mechanism of substrate-assisted catalysis in which the proximal ubiquitin activates the catalytic triad of the protease. Mutation of Ub Glu16 reduces OTULIN kcat 240-fold.\",\n      \"method\": \"Crystal structure of OTU catalytic domain–diubiquitin complex; in vitro DUB activity assays; site-directed mutagenesis of Ub Glu16\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus in vitro reconstitution with mutagenesis, two independent labs reporting same specificity simultaneously\",\n      \"pmids\": [\"23746843\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"OTULIN binds LUBAC and overexpression of OTULIN prevents TNFα-induced NEMO association with ubiquitinated RIPK1, demonstrating that OTULIN antagonizes LUBAC-mediated linear ubiquitin signaling at the receptor complex level.\",\n      \"method\": \"Co-immunoprecipitation; overexpression and knockdown in cells; TNFα stimulation assays monitoring NEMO–RIPK1 ubiquitin association\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP plus functional knockdown/overexpression readouts, replicated across two simultaneous reports (PMID 23746843 and 23806334)\",\n      \"pmids\": [\"23746843\", \"23806334\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"OTULIN depletion augments NF-κB signaling downstream of NOD2, and affinity purification of Met1-ubiquitin followed by quantitative proteomics identified RIPK2 as the predominant NOD2-regulated Met1-ubiquitin substrate; OTULIN restricts Met1-Ub on RIPK2 and on LUBAC components spontaneously.\",\n      \"method\": \"siRNA knockdown; affinity purification of Met1-Ub chains coupled to quantitative mass spectrometry; immunoprecipitation; NF-κB reporter assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — quantitative proteomics substrate identification combined with functional knockdown and Co-IP, single lab but orthogonal methods\",\n      \"pmids\": [\"23806334\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"OTULIN binds LUBAC via a conserved PUB-interacting motif (PIM) that docks onto the PUB domain of HOIP; crystal structures and NMR revealed the molecular basis of the high-affinity interaction. Phosphorylation of OTULIN Tyr56 within the PIM prevents HOIP binding, whereas unphosphorylated OTULIN is part of the endogenous LUBAC complex.\",\n      \"method\": \"Crystal structure of HOIP PUB–OTULIN PIM complex; NMR; Co-immunoprecipitation; phospho-mimetic/phospho-null mutagenesis; in vitro binding assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus NMR plus mutagenesis, independently reported by two labs simultaneously (PMID 24726323 and 24726327)\",\n      \"pmids\": [\"24726323\", \"24726327\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"OTULIN must be present on LUBAC (via HOIP PUB–PIM interaction) to restrict Met1-polyUb signaling; HOIP binding is required for recruitment of OTULIN to the TNF receptor complex to counteract HOIP-dependent NF-κB activation.\",\n      \"method\": \"NF-κB luciferase reporter assays; Co-immunoprecipitation; site-directed mutagenesis of OTULIN Tyr56 and HOIP PUB; TNF receptor complex purification\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — functional epistasis with mutagenesis plus receptor complex purification, replicated in two concurrent papers\",\n      \"pmids\": [\"24726323\", \"24726327\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Both CYLD and OTULIN interact with LUBAC via the PUB domain of HOIP even in unstimulated cells, and their interaction with HOIP synergistically suppresses LUBAC-mediated linear polyubiquitination and NF-κB activation; OTULIN interaction with HOIP also suppresses canonical Wnt signaling activation by LUBAC.\",\n      \"method\": \"Co-immunoprecipitation; cell-free translation/binding assays; NF-κB reporter assays; HOIP-null cell reconstitution with binding-deficient HOIP mutants\",\n      \"journal\": \"Genes to cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus functional rescue in HOIP-null cells, single lab\",\n      \"pmids\": [\"24461064\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"OTULIN is essential in vivo for preventing spontaneous M1-linked polyubiquitin accumulation and NF-κB activation in myeloid cells; OTULIN deficiency in B and T cells instead causes LUBAC degradation and downregulation of M1-polyUb signaling. A hypomorphic human OTULIN mutation causes OTULIN-related autoinflammatory syndrome (ORAS) treatable by anti-TNF.\",\n      \"method\": \"Four independent conditional OTULIN knockout mouse models; immunoprecipitation; Western blot for LUBAC levels; cytokine measurements; anti-TNF rescue experiments\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — four independent genetic mouse models with mechanistic pathway readouts, replicated across cell types\",\n      \"pmids\": [\"27523608\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"OTULIN promotes LUBAC activity by preventing LUBAC auto-ubiquitination with linear polyubiquitin; catalytically inactive OTULIN knock-in mice resemble LUBAC-deficient mice and die midgestation from TNFR1- and RIPK1-kinase-dependent cell death. Embryonic lethality is rescued by combined loss of caspase-8 and RIPK3, showing OTULIN and LUBAC function in a linear pathway.\",\n      \"method\": \"Catalytic-dead OTULIN knock-in mice (constitutive and endothelial-specific); genetic epistasis with TNFR1-KO, RIPK1-kinase-dead KI, caspase-8-KO, RIPK3-KO; Western blot for LUBAC auto-ubiquitination\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal genetic epistasis models in vivo with defined cell-death pathway placement\",\n      \"pmids\": [\"29950720\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"OTULIN interacts with SNX27 (sorting nexin 27) via its C-terminal PDZ-binding motif (PDZbm) engaging the cargo-binding PDZ domain of SNX27; a second interface between the OTULIN OTU domain and the SNX27 PDZ domain was revealed by crystal structure. Via this association, OTULIN antagonizes SNX27-dependent cargo loading and VPS26A-retromer binding, inhibiting endosome-to-plasma membrane trafficking in a catalysis-independent manner.\",\n      \"method\": \"Mass spectrometry identification of OTULIN interactor; crystal structure of OTU domain–PDZ domain complex; Co-immunoprecipitation; endosomal trafficking assays; mutagenesis of PDZbm\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure plus Co-IP plus functional trafficking assay, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"31541095\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"OTULIN is cleaved by caspase-3 at Asp-31 during apoptosis, generating a C-terminal fragment that restricts caspase activation and cell death. During necroptosis, OTULIN is hyper-phosphorylated at Tyr-56, which modulates RIPK1 ubiquitin dynamics and promotes cell death; this phosphorylation is counteracted by the phosphatase DUSP14, identified as an OTULIN phosphatase.\",\n      \"method\": \"In vitro caspase-3 cleavage assay; site-directed mutagenesis (D31A); Co-immunoprecipitation of DUSP14; phosphorylation assays; keratinocyte apoptosis/necroptosis induction assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro cleavage assay with mutagenesis plus Co-IP of phosphatase, single lab\",\n      \"pmids\": [\"31825842\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TRIM32 interacts with OTULIN and conjugates non-proteolytic (K48/K63-linked) polyubiquitin chains onto OTULIN, which blocks the OTULIN–HOIP interaction, thereby preventing OTULIN from suppressing LUBAC and promoting NF-κB activation. TRIM32 E3 ligase activity is required for this effect.\",\n      \"method\": \"Proteomics of OTULIN protein complex; Co-immunoprecipitation; TRIM32 E3 ligase domain mutagenesis; ubiquitination assay; NF-κB reporter assay; genetic complementation\",\n      \"journal\": \"Journal of molecular cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus in-cell ubiquitination assay plus E3-dead mutagenesis, single lab\",\n      \"pmids\": [\"31504727\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"OTULIN deficiency in liver parenchymal cells triggers steatohepatitis via aberrant mTOR activation (independent of TNFR1 signaling); rapamycin administration significantly reduces liver pathology in hepatocyte-specific OTULIN-KO mice.\",\n      \"method\": \"Liver-specific OTULIN knockout mice; TNFR1-KO epistasis; mTOR pathway Western blot; rapamycin pharmacological rescue; histopathology\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO plus pharmacological rescue with pathway readout, single lab\",\n      \"pmids\": [\"32231246\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"OTULIN deficiency in hepatocytes causes apoptosis through FADD-dependent and RIPK1-kinase-dependent pathways; genetic ablation of FADD completely rescues, and RIPK1 kinase-dead knockin significantly protects, mice from OTULIN-deficient liver disease. Type I interferons also contribute to disease in this model.\",\n      \"method\": \"Hepatocyte-specific OTULIN-KO mice; genetic epistasis with FADD-KO and RIPK1 kinase-dead KI; IFNAR epistasis; histopathology; TUNEL assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal genetic epistasis experiments definitively placing FADD and RIPK1 kinase activity downstream of OTULIN loss\",\n      \"pmids\": [\"32075762\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"OTULIN inhibits linear ubiquitination of β-catenin, preventing its Lys48-linked ubiquitination and proteasomal degradation upon DNA damage. The association between OTULIN and β-catenin is enhanced by ABL1-dependent phosphorylation of OTULIN Tyr56, which is triggered by genotoxic stress.\",\n      \"method\": \"Co-immunoprecipitation; in-cell ubiquitination assay; proteasome inhibition; ABL1 kinase assay; phospho-mimetic/null OTULIN Tyr56 mutants; xenograft tumor models\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus kinase assay with mutagenesis, single lab\",\n      \"pmids\": [\"32770022\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"LUBAC and OTULIN localize to the phagophore area; LUBAC component RNF31 translocates to LC3 puncta upon autophagy induction. OTULIN knockdown promotes autophagy initiation but blocks autophagosome maturation by causing excessive linear-ubiquitinated ATG13 accumulation at the phagophore.\",\n      \"method\": \"siRNA knockdown; confocal immunofluorescence co-localization with LC3; autophagy flux assays (GFP-RFP-LC3); Western blot for ATG13 ubiquitination\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct co-localization plus knockdown phenotype with defined substrate (ATG13), single lab\",\n      \"pmids\": [\"32543267\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"OTULIN deubiquitinates ALK1 (Activin receptor-like kinase 1) to remove linear ubiquitin chains conjugated by LUBAC; linear ubiquitination of ALK1 inhibits its kinase activity and Smad1/5 activation. EC-specific or constitutive Otulin deletion causes arteriovenous malformations and embryonic lethality rescued by BMP9 pretreatment or constitutively active ALK1 knockin.\",\n      \"method\": \"EC-specific and constitutive Otulin-KO mice; in vitro ALK1 kinase assay with/without linear ubiquitination; Smad1/5 phosphorylation Western blot; Co-immunoprecipitation; ALK1Q200D knockin rescue; HOIP inhibitor treatment of HHT2 patient-derived ECs\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with multiple in vivo rescue experiments plus in vitro kinase activity assay and Co-IP demonstrating substrate-level mechanism\",\n      \"pmids\": [\"34157307\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SNX27 recruits OTULIN to the membrane-associated TNF receptor complex via their PDZ interaction; OTULIN deubiquitinates linear polyubiquitin at the TNF receptor complex, and chemical inhibition of SNX27-retromer translocation blocks OTULIN membrane localization and enhances TNFα-induced NF-κB signaling.\",\n      \"method\": \"Co-immunoprecipitation; TNF receptor complex purification; cholera toxin pharmacological inhibition; confocal imaging; NF-κB reporter assay\",\n      \"journal\": \"Cell & bioscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus receptor complex purification plus pharmacological perturbation, single lab\",\n      \"pmids\": [\"34315543\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"OTULIN deficiency in epidermis-specific KO mice causes TNFR1-dependent, RIPK1-kinase-activity-dependent keratinocyte death that is primarily necroptosis (requiring RIPK3/MLKL); combined loss of RIPK3 and FADD fully prevents skin lesions, indicating redundant roles of apoptosis and necroptosis. MyD88 deficiency suppresses skin inflammation, implicating TLR/IL-1 signaling.\",\n      \"method\": \"Epidermis-specific OTULIN KO mice; genetic epistasis with RIPK3-KO, MLKL-KO, FADD-KO, RIPK1 kinase-dead KI, TNFR1-KO, MyD88-KO; histopathology\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal genetic epistasis experiments in vivo with clear pathway placement\",\n      \"pmids\": [\"34625557\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Keratinocyte-specific OTULIN ablation causes inflammatory skin lesions driven by keratinocyte cell death; genetic deletion of Tnfr1, RIPK1 kinase-dead KI, or combined keratinocyte-specific deletion of FADD and MLKL completely rescues dermatitis. OTULIN-deficient keratinocytes display a type-I interferon and IL-1β response signature.\",\n      \"method\": \"Keratinocyte-specific OTULIN KO mice; genetic epistasis with Tnfr1-KO, Ripk1-KD KI, FADD/MLKL double KO; single-cell RNA-sequencing; cytokine inhibition\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal genetic epistasis experiments, single-cell transcriptomics, replicated pathways across two concurrent papers\",\n      \"pmids\": [\"34625556\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"OTULIN deubiquitinates IRF3 to remove linear polyubiquitin chains, inhibiting RIPA (RIG-I-induced pathway of apoptosis). During virus infection, RIPA overcomes OTULIN inhibition by caspase-3 cleavage of OTULIN at D31 followed by proteasomal degradation of the cleaved fragment, preceded by K48-linked ubiquitination at K64 and K197 by HOIP.\",\n      \"method\": \"OTULIN overexpression/knockdown; caspase-3 in vitro cleavage assay; D31A/K64R/K197R mutagenesis; mass spectrometry identification of ubiquitination sites; Co-immunoprecipitation with LUBAC/HOIP; virus infection apoptosis assay\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro caspase cleavage assay with mutagenesis plus MS-identified ubiquitination sites plus Co-IP, single lab\",\n      \"pmids\": [\"34545182\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"OTULIN deubiquitinates proteasome subunits; OTULIN deficiency causes proteasome dysregulation in cells, which is the mechanistic basis for elevated type I interferon signaling in OTULIN-deficient patients and cell lines.\",\n      \"method\": \"CRISPR-generated OTULIN-KO cell lines; proteasome activity assays; Co-immunoprecipitation of proteasome subunits with OTULIN; type I IFN pathway assays in patient PBMCs/monocytes\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP identifying proteasome subunits as substrates plus functional proteasome assay in KO cells, single lab\",\n      \"pmids\": [\"34797715\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"OTULIN undergoes self-deubiquitination intermolecularly via dimerization; Lys64/66 of OTULIN are linearly ubiquitinated in a LUBAC-dependent manner to maintain OTULIN–LUBAC interaction under unstressed conditions. Genotoxic stress induces OTULIN dimerization (via cysteine-mediated disulfide bonds under oxidative stress) and self-deubiquitination, leading to OTULIN–LUBAC dissociation and NF-κB overactivation.\",\n      \"method\": \"Mutagenesis of OTULIN K64/K66; Co-immunoprecipitation; in vitro ubiquitination assay; cross-linking/dimerization assays; genotoxic stress and oxidative stress treatments\",\n      \"journal\": \"PNAS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis plus in vitro ubiquitination assay plus Co-IP, single lab\",\n      \"pmids\": [\"35939695\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"OTULIN haploinsufficiency causes accumulation of caveolin-1 in dermal fibroblasts (but not leukocytes), which facilitates cytotoxic damage by staphylococcal α-toxin; TNFR-mediated NF-κB signaling remains intact in haploinsufficient fibroblasts.\",\n      \"method\": \"Patient-derived fibroblast studies; linear ubiquitin accumulation assay; caveolin-1 protein level measurement; α-toxin cytotoxicity assay; leukocyte phenotyping\",\n      \"journal\": \"Science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — patient-derived cells with defined molecular mechanism (caveolin-1 accumulation) and functional readout (α-toxin cytotoxicity), single study\",\n      \"pmids\": [\"35587511\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"OTULIN deficiency in macrophages licenses RIPK3-mediated cell death, which activates the Nlrp3 inflammasome independently of gasdermin D-mediated pyroptosis, leading to RIPK3-dependent IL-1β secretion; elevated serum IL-1β in myeloid-specific OTULIN-KO mice is abolished by Ripk3 or Nlrp3 deletion.\",\n      \"method\": \"Myeloid-specific OTULIN KO mice; genetic epistasis with RIPK3-KO and Nlrp3-KO; gasdermin D knockdown; IL-1β ELISA; macrophage cell death assays\",\n      \"journal\": \"Science immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple genetic epistasis experiments in vivo and in vitro placing RIPK3 and Nlrp3 downstream of OTULIN loss\",\n      \"pmids\": [\"38000038\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"OTULIN interacts with SCRIB via its C-terminal PDZ-binding motif; Met1-Ub chains associate with VANGL2 and PRICKLE1 but not SCRIB, directing VANGL2 surface presentation. OTULIN is recruited to VANGL2-enriched cell-cell contacts and its loss causes deficits in Wnt5a-induced filopodia extension and VANGL2 trafficking, implicating linear (de)ubiquitination in planar cell polarity signaling.\",\n      \"method\": \"HEK293 cell-based interactomic analysis; Co-immunoprecipitation; confocal imaging of OTULIN at cell-cell contacts; OTULIN-KO cells; Wnt5a-stimulated filopodia assay; VANGL2-GFP trafficking assay\",\n      \"journal\": \"Disease models & mechanisms\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus functional KO phenotype with direct localization, single lab\",\n      \"pmids\": [\"37589075\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"STAT3 is a direct substrate of linear ubiquitination in glioblastoma stem-like cells; linear ubiquitination of STAT3 negatively regulates its activity by recruiting the phosphatase TC-PTP to STAT3. Preferential OTULIN expression in GSCs deubiquitinates STAT3 to drive persistent STAT3 signaling and maintain stemness.\",\n      \"method\": \"Bio-orthogonal linear ubiquitin probe (NAEK-Ub) for substrate identification; Co-immunoprecipitation; STAT3 activity assays; OTULIN knockdown in GSCs; TC-PTP recruitment assay\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — novel substrate identified with bio-orthogonal probe plus Co-IP and functional validation, single lab\",\n      \"pmids\": [\"36660824\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"SPATA2 competes with OTULIN for binding to HOIP via its PUB-interacting motif (PIM) and zinc finger domain, thereby promoting LUBAC auto-ubiquitination. Cyld-/-Spata2-/- double mutant mice show perinatal lethality and elevated M1-linked ubiquitination dependent on OTULIN, indicating SPATA2 counteracts OTULIN-mediated LUBAC deubiquitination independently of CYLD.\",\n      \"method\": \"Cyld-/-, Spata2-/-, and Cyld-/-Spata2-/- double KO mice; competitive binding assays (Co-IP with PIM mutants); M1-Ub Western blot; genetic rescue by additional OTULIN deletion\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with double KO mice plus competitive binding assay with mutagenesis, single lab\",\n      \"pmids\": [\"36640323\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The dominant-negative OTULIN Cys129Ser mutation ablates deubiquitinase activity without affecting protein stability or LUBAC/linear-ubiquitin binding; loss of catalytic activity causes LUBAC auto-ubiquitination that impairs LUBAC recruitment to the TNF receptor signaling complex, promoting TNF-induced cell death.\",\n      \"method\": \"Patient-derived cell studies; in vitro DUB activity assay; Co-immunoprecipitation of LUBAC with TNF receptor complex; linear ubiquitin accumulation assay; TNF-induced cell death assay\",\n      \"journal\": \"Journal of experimental medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro activity assay plus receptor complex Co-IP plus functional cell death readout, single study\",\n      \"pmids\": [\"38630025\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"OTULIN prevents GPX4 proteasomal degradation by reducing its ubiquitin level (linear deubiquitination of GPX4), thereby conferring resistance to cisplatin-induced mitochondrial apoptosis (not ferroptosis) in osteosarcoma cells.\",\n      \"method\": \"OTULIN overexpression/knockdown; GPX4 ubiquitination assay; proteasome inhibitor (MG132) rescue; cell death assays (apoptosis vs ferroptosis markers); xenograft model\",\n      \"journal\": \"Journal of experimental & clinical cancer research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP/ubiquitination assay with pharmacological rescue, single lab, no direct linear-Ub specificity confirmed for GPX4\",\n      \"pmids\": [\"39721999\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"OTULIN regulates the ubiquitination of NCOA4, preventing its degradation and thereby enabling NCOA4-mediated FTH1 accumulation; this protects hepatocytes from APAP-induced ferroptosis by maintaining iron homeostasis.\",\n      \"method\": \"OTULIN stable cell lines; ubiquitination assay of NCOA4; Co-immunoprecipitation; ferroptosis markers (GSH, lipid peroxidation); mouse APAP injury model\",\n      \"journal\": \"International immunopharmacology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP plus ubiquitination assay, single lab, mechanistic chain inferred from multiple steps\",\n      \"pmids\": [\"40158433\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"OTULIN removes linear ubiquitin chains from TRAF6 at K104, K142, and K371 to suppress RIG-I-dependent antiviral signaling; linear ubiquitination of TRAF6 by LUBAC promotes K63-linked ubiquitination of TRAF6 and strengthens its association with MAVS to amplify antiviral responses. OTULIN-KO mice exhibit enhanced antiviral immunity.\",\n      \"method\": \"OTULIN overexpression/knockout in HeLa and iBMDM cells; lentiviral reconstitution; TRAF6 ubiquitination mass spectrometry; K104R/K142R/K371R mutagenesis; MAVS Co-immunoprecipitation; IFN-β reporter assay; Otulin+/- mouse viral challenge\",\n      \"journal\": \"PNAS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS-identified ubiquitination sites with mutagenesis plus Co-IP plus in vivo mouse data, single lab\",\n      \"pmids\": [\"41802043\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"OTULIN interacts with AMPK, controls its M1-ubiquitination, and restricts AMPK activation in response to glucose starvation and allosteric activation; LUBAC promotes AMPK activation while OTULIN restricts it, placing the LUBAC-OTULIN axis as a regulator of metabolic signaling.\",\n      \"method\": \"LUBAC and OTULIN KO/KD cells; AMPK activity assays; Co-immunoprecipitation of OTULIN with AMPK; M1-ubiquitination assay of AMPK; metabolic flux measurements; Drosophila starvation survival (LUBAC-deficient)\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP plus activity assay, preprint not yet peer-reviewed, single lab\",\n      \"pmids\": [\"bio_10.1101_2024.11.08.622598\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In OTULIN-deficient keratinocytes, β-catenin accumulates linear ubiquitin chains promoting its K48-linked ubiquitination and proteasomal degradation, reducing Wnt/β-catenin signaling and causing TCF3 degradation; restoring β-catenin stabilization prevents progressive skin inflammation and keratinocyte death.\",\n      \"method\": \"Keratinocyte-specific OTULIN KO mice; β-catenin linear ubiquitination assay; TCF3 protein level assay; β-catenin stabilizing genetic/pharmacological interventions; skin inflammation rescue assays\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — preprint, single lab, ubiquitination assay plus genetic rescue, not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.01.08.631848\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"RNF6 E3 ligase ubiquitinates OTULIN and promotes its degradation; RNF6 knockdown prevents OTULIN ubiquitination/degradation and thereby suppresses TGF-β1-induced epithelial-mesenchymal transition in bronchial epithelial cells. Silencing RNF6 phenocopies OTULIN overexpression in blocking partial EMT.\",\n      \"method\": \"Label-free proteomics combined with Co-IP/mass spectrometry; Co-IP validation; cycloheximide and MG132 assays for protein stability; OTULIN overexpression rescue of RNF6 knockdown; EMT marker Western blot\",\n      \"journal\": \"Biochemical pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus proteasome inhibitor rescue plus functional rescue epistasis, single lab\",\n      \"pmids\": [\"42055142\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"OTULIN directly interacts with mitochondrial fusion protein OPA1 and regulates its ubiquitination status; the E3 ligase RNF31/HOIP ubiquitinates OPA1, and OTULIN counteracts RNF31-mediated OPA1 destabilization, thereby maintaining mitochondrial homeostasis in alveolar epithelial cells under hyperoxic stress.\",\n      \"method\": \"Co-immunoprecipitation of OTULIN with OPA1; RNF31 knockdown and OTULIN overexpression/knockdown in alveolar epithelial cells; OPA1 ubiquitination assay; mitochondrial ROS and membrane potential measurements; hyperoxia neonatal mouse model\",\n      \"journal\": \"Cellular & molecular biology letters\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP plus knockdown, no in vitro reconstitution, single lab\",\n      \"pmids\": [\"42218396\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"OTULIN is the only mammalian deubiquitinase with absolute specificity for Met1-linked (linear) polyubiquitin chains, which it cleaves via a substrate-assisted catalytic mechanism (proximal Ub activates the catalytic triad) after docking through dedicated Met1-specific ubiquitin-binding sites revealed by crystal structure; it constitutively associates with the LUBAC E3 ligase (via a Tyr56-phosphorylation-regulated PIM–HOIP PUB domain interaction) to prevent LUBAC auto-ubiquitination and thereby sustain LUBAC activity, while simultaneously limiting linear ubiquitin accumulation on receptor complex components (RIPK1, RIPK2, NEMO, ALK1, TRAF6, STAT3, ATG13, and proteasome subunits) downstream of TNF, NOD2, and RIG-I receptors; dynamic post-translational regulation of OTULIN itself—including ABL1-mediated Tyr56 phosphorylation, TRIM32-mediated non-proteolytic ubiquitination at Lys64/66, LUBAC-dependent linear ubiquitination, caspase-3 cleavage at Asp31, and RNF6-mediated proteasomal degradation—controls its interaction with LUBAC, its association with β-catenin (Wnt), SCRIB/VANGL2 (planar cell polarity), and the SNX27-retromer (endosomal trafficking), such that loss of OTULIN causes spontaneous linear ubiquitin accumulation leading to NF-κB hyperactivation in myeloid cells and TNFR1/RIPK1-kinase-dependent apoptosis and necroptosis in epithelial cells, underscoring its essential role in preventing autoinflammatory disease, embryonic lethality, and organ-specific pathology.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"OTULIN (FAM105B) is the mammalian deubiquitinase with absolute specificity for Met1-linked (linear) polyubiquitin, which it hydrolyzes through a substrate-assisted catalytic mechanism in which the proximal ubiquitin activates the protease catalytic triad, as defined by crystal structures of the OTU domain bound to diubiquitin [#0]. It constitutively associates with the LUBAC E3 ligase by docking a PUB-interacting motif (PIM) onto the HOIP PUB domain, an interaction abolished by phosphorylation of OTULIN Tyr56 [#3]. Through this association OTULIN restrains linear ubiquitin signaling at two levels: it prevents LUBAC auto-ubiquitination to sustain ligase activity [#7], and it removes Met1-ubiquitin from receptor-complex substrates including RIPK1/NEMO downstream of TNF [#1], RIPK2 downstream of NOD2 [#2], and ALK1 in BMP9/Smad1/5 signaling [#15]. OTULIN is essential in vivo: catalytically inactive knock-in mice phenocopy LUBAC deficiency and die mid-gestation from TNFR1- and RIPK1-kinase-dependent cell death rescued by combined caspase-8/RIPK3 loss [#7], and tissue-specific loss drives autoinflammation through myeloid NF-\\u03baB hyperactivation [#6] and epithelial RIPK1-kinase-dependent apoptosis and necroptosis [#12, #17]. A hypomorphic human mutation causes OTULIN-related autoinflammatory syndrome (ORAS), treatable with anti-TNF [#6]. OTULIN activity is itself tuned by post-translational regulation—ABL1-dependent Tyr56 phosphorylation, TRIM32-mediated non-proteolytic ubiquitination, and LUBAC-dependent linear ubiquitination at Lys64/66—that governs its LUBAC engagement and substrate selection [#10, #13, #21]. Beyond the NF-\\u03baB axis, OTULIN engages the SNX27-retromer through a C-terminal PDZ-binding motif to control endosomal trafficking independently of catalysis [#8, #16] and acts on additional substrates including IRF3 and TRAF6 in antiviral signaling [#19, #30].\",\n  \"teleology\": [\n    {\n      \"year\": 2013,\n      \"claim\": \"Establishing how a DUB could be Met1-specific answered why linear ubiquitin signaling has a dedicated eraser, defining OTULIN's catalytic chemistry and chain selectivity.\",\n      \"evidence\": \"Crystal structure of OTU domain\\u2013diubiquitin complex with in vitro DUB assays and Ub Glu16 mutagenesis\",\n      \"pmids\": [\"23746843\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish in vivo substrate repertoire\", \"Catalytic mechanism shown in vitro, not the cellular regulation of activity\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Linking OTULIN to LUBAC and showing it blocks TNF-induced NEMO\\u2013RIPK1 ubiquitin association placed OTULIN as a negative regulator of linear ubiquitin signaling at the receptor complex.\",\n      \"evidence\": \"Reciprocal Co-IP, overexpression/knockdown, and TNF\\u03b1 stimulation in cells\",\n      \"pmids\": [\"23746843\", \"23806334\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mode of recruitment to the receptor complex not defined\", \"Did not resolve whether antagonism is catalytic or stoichiometric\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identifying RIPK2 as the dominant NOD2-regulated Met1-Ub substrate extended OTULIN's role beyond TNF to a second innate immune receptor pathway.\",\n      \"evidence\": \"siRNA knockdown, Met1-Ub affinity purification with quantitative MS, NF-\\u03baB reporters\",\n      \"pmids\": [\"23806334\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish in vivo NOD2 phenotype\", \"Spontaneous restriction of LUBAC components not mechanistically separated from receptor signaling\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Defining the HOIP PUB\\u2013OTULIN PIM interface and its phospho-regulation by Tyr56 explained how OTULIN is recruited to LUBAC and how that recruitment is switched off.\",\n      \"evidence\": \"Crystal structure, NMR, Co-IP, phospho-mimetic/null mutagenesis, receptor complex purification\",\n      \"pmids\": [\"24726323\", \"24726327\", \"24461064\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the Tyr56 kinase not established here\", \"Did not address CYLD/SPATA2 competition for the same PUB domain\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Genetic loss-of-function in mice and humans demonstrated OTULIN is non-redundantly required to prevent autoinflammation, with cell-type-specific outcomes on M1-Ub and LUBAC levels.\",\n      \"evidence\": \"Four conditional knockout mouse models, IP/Western for LUBAC, cytokines, anti-TNF rescue; ORAS patient mutation\",\n      \"pmids\": [\"27523608\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not distinguish whether disease is driven by NF-\\u03baB or cell death\", \"Mechanism of LUBAC degradation in lymphocytes unresolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Catalytic-dead knock-in mice showed OTULIN's DUB activity sustains LUBAC by preventing auto-ubiquitination, placing OTULIN and LUBAC in a single TNFR1/RIPK1-driven cell-death pathway.\",\n      \"evidence\": \"Catalytic-dead knock-in mice with epistasis (TNFR1-KO, RIPK1-KD, caspase-8-KO, RIPK3-KO), Western for LUBAC auto-ubiquitination\",\n      \"pmids\": [\"29950720\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify tissue-specific cell-death effectors beyond embryo\", \"Counterintuitive role as LUBAC-promoting rather than purely antagonistic not fully reconciled with receptor-level data\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Discovery of the SNX27-retromer interaction via OTULIN's PDZ-binding motif revealed a catalysis-independent role in endosomal trafficking, broadening OTULIN function beyond NF-\\u03baB.\",\n      \"evidence\": \"MS interactor identification, crystal structure of OTU\\u2013PDZ complex, Co-IP, trafficking assays, PDZbm mutagenesis\",\n      \"pmids\": [\"31541095\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological significance of trafficking role in vivo not established\", \"Relationship between trafficking and immune functions unclear\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Characterizing caspase-3 cleavage at Asp31 and Tyr56 hyperphosphorylation with DUSP14 as counteracting phosphatase showed OTULIN is dynamically inactivated during cell death.\",\n      \"evidence\": \"In vitro caspase-3 cleavage, D31A mutagenesis, DUSP14 Co-IP, keratinocyte death assays\",\n      \"pmids\": [\"31825842\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab; cleavage fragment function not independently confirmed\", \"Kinase responsible for necroptotic Tyr56 phosphorylation not identified\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identifying TRIM32-mediated non-proteolytic ubiquitination that blocks OTULIN\\u2013HOIP binding added an upstream regulatory layer controlling OTULIN's access to LUBAC.\",\n      \"evidence\": \"OTULIN complex proteomics, Co-IP, TRIM32 E3-dead mutagenesis, ubiquitination and NF-\\u03baB reporter assays\",\n      \"pmids\": [\"31504727\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab without in vivo validation\", \"Ubiquitin chain linkage and exact lysines not fully resolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Tissue-specific knockouts dissected the death effectors downstream of OTULIN loss, showing hepatocyte disease is FADD- and RIPK1-kinase-dependent with an mTOR-driven steatohepatitis component.\",\n      \"evidence\": \"Liver/hepatocyte-specific KO mice with FADD-KO, RIPK1-KD, IFNAR, TNFR1 epistasis; rapamycin rescue; histopathology\",\n      \"pmids\": [\"32075762\", \"32231246\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How OTULIN loss drives mTOR activation independent of TNFR1 not defined\", \"Relative contribution of apoptosis vs interferon to liver pathology not fully separated\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Connecting OTULIN to \\u03b2-catenin stability and to autophagy via ATG13 extended its substrate range to Wnt signaling and autophagosome maturation.\",\n      \"evidence\": \"Co-IP, in-cell ubiquitination assays, ABL1 kinase assay, Tyr56 mutants, xenografts; LC3 co-localization and autophagy flux assays\",\n      \"pmids\": [\"32770022\", \"32543267\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab findings without in vivo genetic confirmation\", \"Direct vs indirect action on \\u03b2-catenin and ATG13 ubiquitin not fully resolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Demonstrating OTULIN deubiquitinates ALK1, IRF3, TRAF6, and proteasome subunits, and that SNX27 recruits it to the TNF receptor complex, mapped substrate-level mechanisms across vascular, antiviral, and proteostasis pathways.\",\n      \"evidence\": \"EC-specific KO mice with BMP9/ALK1 rescue, in vitro kinase assays, bio-orthogonal Ub probes, MS site mapping, Co-IP, proteasome activity assays in KO cells\",\n      \"pmids\": [\"34157307\", \"34545182\", \"34797715\", \"34315543\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Several substrate findings rest on single labs\", \"How substrate selectivity is achieved among diverse targets unresolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Epidermis- and keratinocyte-specific knockouts established that OTULIN loss in skin drives TNFR1/RIPK1-kinase-dependent necroptosis and apoptosis with type-I IFN and IL-1\\u03b2 signatures.\",\n      \"evidence\": \"Skin-specific KO mice with RIPK3-KO, MLKL-KO, FADD-KO, RIPK1-KD, TNFR1-KO, MyD88-KO epistasis; scRNA-seq\",\n      \"pmids\": [\"34625557\", \"34625556\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative weighting of apoptosis vs necroptosis context-dependent\", \"Source of MyD88/IL-1 input not pinpointed\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Self-deubiquitination/dimerization control and the caveolin-1 haploinsufficiency phenotype refined how OTULIN abundance and LUBAC engagement are maintained and how partial loss manifests differently across cell types.\",\n      \"evidence\": \"K64/K66 mutagenesis, dimerization/cross-linking assays, in vitro ubiquitination; patient fibroblast caveolin-1 and \\u03b1-toxin cytotoxicity assays\",\n      \"pmids\": [\"35939695\", \"35587511\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab mechanistic models\", \"Link between caveolin-1 accumulation and linear ubiquitin not fully mechanistic\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Macrophage and substrate-level studies linked OTULIN loss to RIPK3-driven Nlrp3 inflammasome activation and to STAT3, SCRIB/VANGL2, and SPATA2-dependent LUBAC regulation, expanding its roles into inflammasome biology, stemness, and planar cell polarity.\",\n      \"evidence\": \"Myeloid-specific KO mice with RIPK3/Nlrp3 epistasis; bio-orthogonal Ub probe for STAT3; interactomics and KO phenotypes for SCRIB/VANGL2; double-KO mice for SPATA2\",\n      \"pmids\": [\"38000038\", \"36660824\", \"37589075\", \"36640323\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"STAT3, SCRIB/VANGL2 roles from single labs\", \"Tissue relevance of PCP role not established in vivo\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Patient-derived analysis of the dominant-negative Cys129Ser mutation showed catalytic loss impairs LUBAC recruitment to the TNF receptor complex via auto-ubiquitination, mechanistically uniting human disease with the catalytic-dead mouse model.\",\n      \"evidence\": \"Patient cells, in vitro DUB assay, LUBAC/receptor complex Co-IP, linear-Ub and TNF-induced death assays\",\n      \"pmids\": [\"38630025\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single patient-derived study\", \"Dominant-negative mechanism not tested in animal model\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Mapping OTULIN action on TRAF6 (K104/K142/K371) and OPA1, plus RNF6-mediated OTULIN degradation, extended the LUBAC-OTULIN axis into antiviral amplification, mitochondrial homeostasis, and EMT control.\",\n      \"evidence\": \"MS site mapping with mutagenesis, MAVS Co-IP, IFN-\\u03b2 reporters, Otulin+/- viral challenge; OPA1/RNF31 Co-IP and knockdown; RNF6 proteomics with proteasome-inhibitor and rescue assays\",\n      \"pmids\": [\"41802043\", \"42218396\", \"42055142\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"OPA1 finding low-confidence and single-lab\", \"In vivo significance of EMT and mitochondrial roles limited\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How OTULIN achieves substrate and context selectivity—choosing among RIPK1/2, ALK1, IRF3, TRAF6, STAT3, \\u03b2-catenin, and proteasome subunits in different cell types—and how its many regulatory modifications are integrated remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying model for substrate targeting beyond LUBAC association\", \"Several substrate and metabolic claims rest on single labs or preprints awaiting confirmation\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [0, 1, 2, 15, 19, 20, 30]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 2, 15]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [4, 7, 5]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [8, 16]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1, 4]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [16, 24]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [8, 16]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [1, 2, 6, 19, 30]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [7, 12, 17, 18, 23]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 4, 15]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 7, 20]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [8, 16]}\n    ],\n    \"complexes\": [\"LUBAC\", \"SNX27-retromer\"],\n    \"partners\": [\"RNF31\", \"SNX27\", \"TRIM32\", \"DUSP14\", \"SPATA2\", \"SCRIB\", \"ABL1\", \"RNF6\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}