{"gene":"RNF31","run_date":"2026-04-28T19:45:45","timeline":{"discoveries":[{"year":2012,"finding":"HOIP (RNF31) is the catalytically active subunit of LUBAC and uses a two-step RING/HECT-hybrid mechanism: RING1-IBR catalyzes transfer of ubiquitin from E2 onto a covalent HECT-like thioester intermediate at RING2, then a unique C-terminal Linear ubiquitin chain Determining Domain (LDD) coordinates the acceptor ubiquitin N-terminus for Met1-linked (linear) chain formation.","method":"In vitro ubiquitination assays, active-site mutagenesis, biochemical dissection of RING1-IBR and RING2-LDD sub-domains, cellular NF-κB reporter assays","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with mutagenesis identifying catalytic mechanism; replicated by structural studies","pmids":["22863777"],"is_preprint":false},{"year":2013,"finding":"Crystal structures of the HOIP catalytic core in apo form and in complex with ubiquitin revealed a novel C-terminal fold and zinc-finger that form a ubiquitin-binding platform orienting the acceptor ubiquitin α-amino group for nucleophilic attack on the E3~ubiquitin thioester, explaining linear chain specificity.","method":"X-ray crystallography (apo and ubiquitin-bound structures), mutagenesis, cellular NF-κB assays","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 — crystal structures with functional mutagenesis validation in cells","pmids":["24141947"],"is_preprint":false},{"year":2016,"finding":"Crystal structure of fully active HOIP RBR in complex with an E2~ubiquitin conjugate showed that active HOIP adopts a conformation markedly different from auto-inhibited RBRs, binds E2~ubiquitin in an elongated fashion aligning catalytic centres for HECT-like transfer, and revealed three helix-IBR-fold ubiquitin-binding regions that engage the activated (donor) ubiquitin and an additional regulatory ubiquitin.","method":"X-ray crystallography of HOIP RBR/E2~ubiquitin transfer complex, structural comparison with Parkin and HHARI","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 — first structure of a fully active RBR E3 in transfer complex, high-resolution with mechanistic implications","pmids":["26789245"],"is_preprint":false},{"year":2014,"finding":"HOIP (RNF31) PUB domain binds the PUB-interacting motif (PIM) of the linear deubiquitinase OTULIN; structural studies defined critical contacts (OTULIN Tyr56); phosphorylation of OTULIN Tyr56 negatively regulates this interaction. HOIP binding to OTULIN recruits OTULIN to the TNF receptor complex to counteract HOIP-dependent NF-κB activation.","method":"Co-IP, structural studies (crystal/NMR), phosphorylation site mutagenesis, TNF receptor complex recruitment assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 — structural data plus reciprocal interaction mapping and functional recruitment assays","pmids":["24726327"],"is_preprint":false},{"year":2016,"finding":"SPATA2 is a constitutive direct binding partner of HOIP that bridges the interaction between the deubiquitinase CYLD and HOIP; SPATA2 is required for CYLD recruitment to TNFR1- and NOD2-signaling complexes, and its loss diminishes TNF-induced necroptosis similarly to CYLD loss.","method":"Co-IP, signaling complex (TNFR1/NOD2) pull-down, SPATA2 knockout/knockdown with necroptosis readout","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, complex recruitment assay, loss-of-function with defined phenotype","pmids":["27545878"],"is_preprint":false},{"year":2014,"finding":"HOIP (RNF31) deficiency causes aberrant TNFR1-mediated endothelial cell death via aberrant complex-II formation, leading to vascularization defects and embryonic lethality; TNFR1 ablation rescues this phenotype, and HOIP catalytic activity is required to prevent TNF-induced cell death.","method":"Conditional (Tie2-Cre) and constitutive Hoip knockout mice, genetic epistasis with TNFR1 knockout, cell death assays, complex-II immunoprecipitation","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis in vivo with defined molecular mechanism (complex-II formation)","pmids":["25284787"],"is_preprint":false},{"year":2014,"finding":"RNF31 (HOIP) associates with estrogen receptor α (ERα) primarily in the cytosol, increases ERα stability and mono-ubiquitination in a ubiquitin-ligase-activity-dependent manner, and is required for ERα-stimulated proliferation and downstream target gene expression in breast cancer cells.","method":"Co-immunoprecipitation, siRNA knockdown, cycloheximide chase, cell-cycle analysis, ubiquitination assays with catalytic mutant","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP with functional ligase-activity mutant and multiple cellular phenotypes; single lab","pmids":["24441041"],"is_preprint":false},{"year":2009,"finding":"RNF31 forms an in vivo corepressor complex with DAX-1 at the promoters of steroidogenic genes (StAR, CYP19), stabilizes DAX-1 via monoubiquitination, and is required for DAX-1-mediated repression of SF-1-dependent steroidogenic gene transcription.","method":"ChIP, Co-IP, siRNA knockdown, gene expression analysis, co-expression studies in steroidogenic tissues","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP demonstrating complex at endogenous promoters plus functional knockdown; single lab","pmids":["19237537"],"is_preprint":false},{"year":2010,"finding":"HOIP (RNF31/PAUL/ZIBRA) is recruited to the CD40 signaling complex in a TRAF2-dependent manner; its ubiquitin ligase activity is required for CD40-mediated NF-κB activation, and a catalytic-dead HOIP mutant acts as a dominant negative.","method":"Mass spectrometry of CD40 signaling complexes, Co-IP, dominant-negative ligase-dead mutant, NF-κB reporter assay","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2-3 — MS-based complex identification and catalytic mutant functional test; single lab","pmids":["20614026"],"is_preprint":false},{"year":2011,"finding":"HOIP is essential for CD40 signaling: HOIP-deficient B cells fail to activate NF-κB and JNK via CD40, exhibit impaired IKK recruitment to the CD40 signaling complex, and show defective CD80 upregulation and germline IgE transcription; HOIP re-expression restores all defects.","method":"Somatic gene targeting to generate HOIP-deficient B cell lines, signaling assays (NF-κB, JNK, IKK recruitment), rescue by re-expression","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 — clean KO with multiple orthogonal functional readouts and rescue experiment","pmids":["21829693"],"is_preprint":false},{"year":2016,"finding":"HOIP cleavage by caspases 3 and 6 at Asp348, Asp387, and Asp390 during apoptosis reduces linear ubiquitination of NEMO and FADD; the N-terminal cleavage fragment retains binding to OTULIN and CYLD-SPATA2 while the C-terminal fragment retains NF-κB activity; cleavage-site mutation inhibits TNF-α-induced apoptosis.","method":"Caspase cleavage assays, site-directed mutagenesis of cleavage sites, linear ubiquitination assays, Co-IP with deubiquitinases, apoptosis assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1-2 — biochemical identification of cleavage sites with mutagenesis and functional apoptosis readout","pmids":["27669734"],"is_preprint":false},{"year":2017,"finding":"FADD is identified as a substrate for LUBAC linear ubiquitination in addition to NEMO; HOIP is cleaved predominantly by caspase at Asp390 during apoptosis and is subsequently subject to proteasomal degradation, decreasing linear ubiquitination of both NEMO and FADD.","method":"Linear ubiquitination assays, caspase cleavage assays, proteasome inhibitor experiments, Co-IP","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 — direct biochemical demonstration of FADD as LUBAC substrate with caspase cleavage mapping; single lab, partial overlap with prior study","pmids":["28189684"],"is_preprint":false},{"year":2015,"finding":"Ubiquitination of HOIP at Lys1056 (C-terminal lysine) induces a conformational change that suppresses HOIP catalytic activity and terminates TLR4/LPS-induced NF-κB signaling, but does not affect CD40-induced NF-κB activation, identifying a stimulus-specific regulatory PTM.","method":"Site-directed mutagenesis (K1056R), in vitro ubiquitination assays, NF-κB reporter assays with TLR4 and CD40 stimulation","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 — mutagenesis with functional signaling readout in cells; single lab","pmids":["26578682"],"is_preprint":false},{"year":2018,"finding":"HOIP (and cIAP1) are recruited to the TNFR2 signaling complex; HOIP mediates M1-linked polyubiquitination at this complex; cIAP1 antagonism prevents HOIP recruitment; both HOIP and cIAP1 are required for TNFR2-induced canonical NF-κB activation.","method":"Signaling complex immunoprecipitation, M1/K63 linkage-specific ubiquitin antibodies, cIAP antagonist treatment, NF-κB activation assays","journal":"Biochemical pharmacology","confidence":"Medium","confidence_rationale":"Tier 2-3 — complex recruitment and ubiquitination shown biochemically; single lab","pmids":["29378181"],"is_preprint":false},{"year":2014,"finding":"HOIP-depleted cells are hypersensitive to cisplatin-induced apoptosis through enhanced caspase-8/caspase-3-dependent apoptosis that requires ATM (but not ATR) checkpoint activation; basal and cisplatin-induced JNK activity is enhanced in HOIP-depleted cells, and JNK inhibition reverses apoptotic hyperactivation.","method":"siRNA screen, siRNA knockdown, caspase activation assays, ATM/ATR inhibitors, JNK inhibitor rescue, apoptosis assays","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 — epistasis via pharmacological inhibition with defined mechanistic pathway placement; single lab","pmids":["24686174"],"is_preprint":false},{"year":2022,"finding":"LUBAC (HOIP) binds and stabilizes GPx4 via linear ubiquitination both basally and under oxidative stress; LUBAC deficiency sensitizes cells to ferroptosis by promoting GPx4 degradation and downstream lipid peroxidation, identifying GPx4 as a key LUBAC substrate.","method":"Co-IP, linear ubiquitination assays, ferroptosis assays (lipid peroxidation), LUBAC-deficient cells, GPx4 stability (cycloheximide chase)","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP and ubiquitination assays with cellular ferroptosis phenotype; single lab","pmids":["36279464"],"is_preprint":false},{"year":2022,"finding":"RNF31 (HOIP) protects tumor cells from TNF-mediated caspase-8 cleavage and apoptosis; CRISPR knockout of Rnf31 sensitizes pancreatic cancer cells to CD8+ T cell killing in a TNF-dependent manner, increases intratumoral CD8+ T cell infiltration and effector function in vivo, and reduces tumor growth.","method":"CRISPR-Cas9 in vitro and in vivo screens, genetic knockout, caspase-8 cleavage assays, orthotopic transplantation into immune-competent mice","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — in vitro and in vivo CRISPR KO with defined molecular mechanism and immune phenotype","pmids":["35379808"],"is_preprint":false},{"year":2022,"finding":"Genetic and pharmacological ablation of RNF31 sensitizes cancer cells to NK and CD8+ T cell killing in a TNF-dependent manner by causing loss of A20 and non-canonical IKK complexes from TNF receptor complex I; a small-molecule RNF31 inhibitor sensitizes colon carcinoma organoids to TNF.","method":"Genome-wide CRISPR-Cas9 screens under NK and CD8+ T cell pressure, genetic knockout, pharmacologic inhibition, TNF receptor complex immunoprecipitation, organoid killing assays","journal":"Cell reports. Medicine","confidence":"High","confidence_rationale":"Tier 2 — genome-wide screen validated by orthogonal genetic and pharmacological approaches with mechanistic complex analysis","pmids":["35688159"],"is_preprint":false},{"year":2021,"finding":"HOIP-deficient tumor cells show increased sensitivity to combined TNF and IFN-γ, engaging both intrinsic and extrinsic apoptotic machinery through a transcription-dependent cell death pathway; both genetic deletion and pharmacological inhibition of HOIP augment this sensitivity.","method":"CRISPR/Cas9 screening, genetic deletion, pharmacological inhibition, NK/CD8+ T cell killing assays, cytokine (TNF+IFN-γ) treatment, apoptosis pathway dissection","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 2 — CRISPR screen followed by genetic and pharmacologic validation; single lab","pmids":["34467615"],"is_preprint":false},{"year":2018,"finding":"Epidermis-specific knockout of RNF31 in mice causes postnatal lethality due to severe skin inflammation triggered by TNF-α-induced apoptosis in keratinocytes; RNF31 deficiency impairs TNF-α-induced NF-κB activation and increases apoptosis; deletion of TNFR1 rescues lethality and skin inflammation.","method":"Tissue-specific conditional knockout, genetic epistasis with TNFR1 knockout, NF-κB activation assays, apoptosis assays in primary keratinocytes","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 — tissue-specific KO with genetic epistasis and mechanistic signaling readouts","pmids":["29728512"],"is_preprint":false},{"year":2016,"finding":"HOIP ubiquitin ligase activity is required for mature T cell survival; T-HOIPΔlinear mice show reduced CD4+/CD8+ T cell numbers and defective NKT development; HOIP-deficient CD4+ T cells fail to phosphorylate IκBα and JNK upon TCR stimulation; reduced CD127 expression causes mature T cell apoptosis, and enforced CD127 expression rescues CD8+ T cell development.","method":"Conditional knockout (ubiquitin ligase-dead knock-in), flow cytometry, signaling assays, apoptosis assays, CD127 rescue experiment","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 — clean catalytic-domain KO with mechanistic signaling readout and rescue; single lab","pmids":["27786304"],"is_preprint":false},{"year":2024,"finding":"STING activation recruits LUBAC (HOIP) to LC3B-associated Golgi membranes where it synthesizes M1-linked ubiquitin chains; loss of HOIP prevents M1-Ub chain formation and reduces both NF-κB and IRF3 signaling downstream of STING without affecting STING activation itself.","method":"HOIP knockout in THP1 monocytes and mouse BMDMs, M1-ubiquitin chain immunoprecipitation, STING activation assays, fractionation/localization to Golgi membranes","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 — genetic KO with biochemical ubiquitin chain detection and signaling readouts; single study","pmids":["39578541"],"is_preprint":false},{"year":2021,"finding":"RNF31 interacts with A20 via its RBR structural domain, promotes K63-linked ubiquitination of A20 leading to A20 proteasomal degradation, thereby activating the TLR4/MyD88/NF-κB pathway and aggravating hepatocyte apoptosis and inflammation in acute liver injury.","method":"Co-IP, actinomycin tracing, proteasome inhibitor experiments, ubiquitination assays, siRNA knockdown, mouse model of acute liver injury","journal":"Chemico-biological interactions","confidence":"Medium","confidence_rationale":"Tier 2-3 — biochemical interaction, ubiquitination and degradation assays with in vivo validation; single lab","pmids":["34416243"],"is_preprint":false},{"year":2019,"finding":"Fragment-based covalent ligand screening targeting the active-site cysteine of HOIP identified electrophilic fragments that inhibit HOIP catalytic activity; crystal structures of HOIP-inhibitor complexes enabled structure-based development; cell-permeable compounds inhibit HOIP and NF-κB activation in mammalian cells as shown by chemoproteomics.","method":"Fragment-based covalent screening, protein LC-MS, in vitro ubiquitination assays, protein crystallography, cell-based NF-κB assays, chemoproteomics","journal":"Journal of the American Chemical Society","confidence":"High","confidence_rationale":"Tier 1 — crystal structures of covalent inhibitor complexes at active-site cysteine with biochemical and cellular validation","pmids":["30657686"],"is_preprint":false},{"year":2022,"finding":"RNF31 associates with YAP protein, facilitates YAP poly-ubiquitination and proteasomal degradation at K76, and thereby suppresses Hippo/YAP/PD-L1 signaling in triple-negative breast cancer cells.","method":"Co-IP, ubiquitination assays, site-specific mutagenesis (YAP K76), immunoblots, gene expression profiling, xenograft models","journal":"Journal of experimental & clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP with site-specific mutagenesis; single lab","pmids":["36581998"],"is_preprint":false},{"year":2021,"finding":"RNF31 interacts with p53 via its PUB domain, promotes p53 ubiquitination and proteasomal degradation; RNF31 depletion alleviates p53 degradation (inhibited by MG132), and this RNF31/p53 axis drives colorectal cancer cell proliferation.","method":"Co-IP with truncated RNF31 domain constructs, cycloheximide chase, proteasome inhibitor (MG132), ubiquitination assays, siRNA knockdown","journal":"Cell death discovery","confidence":"Medium","confidence_rationale":"Tier 2-3 — domain-mapping Co-IP with degradation assays; single lab","pmids":["33824292"],"is_preprint":false},{"year":2024,"finding":"RNF31 promotes p53 ubiquitination and proteasomal degradation in hepatocytes; reduced p53 levels increase BNIP3 expression, promoting mitophagy and reducing hepatic steatosis; RNF31-containing small extracellular vesicles from mesenchymal stem cells reduce steatosis in HFD-fed mice.","method":"Ubiquitination assays, co-IP, siRNA knockdown, mt-Keima mitophagy imaging, RNA-seq, in vivo HFD mouse model with sEV delivery","journal":"Free radical biology & medicine","confidence":"Medium","confidence_rationale":"Tier 2-3 — multiple methods (ubiquitination, mitophagy imaging, in vivo) but single lab","pmids":["38615890"],"is_preprint":false},{"year":2022,"finding":"RBCK1 (HOIL-1L) interacts with RNF31 (HOIP) and represses its ubiquitination and proteasomal degradation, thereby stabilizing RNF31 protein levels in hepatocellular carcinoma cells.","method":"Co-IP, ubiquitination assays, proteasome inhibitor experiments, siRNA/overexpression in HCC cells","journal":"Cell death discovery","confidence":"Medium","confidence_rationale":"Tier 3 — Co-IP plus degradation assays; single lab","pmids":["35869046"],"is_preprint":false},{"year":2023,"finding":"RNF31 interacts with NLRP3 through its RBR structural domain, promotes K63-linked ubiquitination of NLRP3 leading to NLRP3 stabilization, and thereby activates the NLRP3 inflammasome during ulcerative colitis.","method":"Co-IP with domain-mapping, K63-linkage specific ubiquitination assays, RNF31 knockdown in cells and mouse DSS-colitis model","journal":"International immunopharmacology","confidence":"Medium","confidence_rationale":"Tier 2-3 — domain Co-IP, linkage-specific ubiquitination, and in vivo KD; single lab","pmids":["37951199"],"is_preprint":false},{"year":2004,"finding":"RNF31 (PAUL/ZIBRA) was identified as an Ariadne-like RBR ubiquitin ligase that binds to the cytoplasmic domain of the muscle-specific receptor tyrosine kinase MuSK and is present at neuromuscular junctions.","method":"Yeast two-hybrid or pull-down (interaction with MuSK cytoplasmic domain), expression profiling in developing mouse tissues, immunolocalization at NMJs","journal":"Gene expression patterns","confidence":"Low","confidence_rationale":"Tier 3 — single interaction assay with localization; no functional mechanism established","pmids":["14678832"],"is_preprint":false},{"year":2019,"finding":"Chemically synthesized monoubiquitylated NEMO (NEMOCoZi-Ub) is accepted as a substrate for linear extension by the HOIP RBR domain alone, demonstrating that NEMO linear ubiquitylation occurs in two steps: an initial priming event and a separate extension step requiring different LUBAC components.","method":"Chemical ubiquitylation using ligation auxiliary, in vitro linear extension assay with recombinant HOIP RBR, biophysical affinity measurements","journal":"Communications chemistry","confidence":"Medium","confidence_rationale":"Tier 1 — in vitro reconstitution with chemically defined substrates; single study","pmids":["31942456"],"is_preprint":false},{"year":2021,"finding":"In dendritic cell-specific HOIP knockout mice, autoinflammation is not rescued by TNFR1 deletion but is rescued by antibiotic treatment or MyD88 deficiency, placing HOIP upstream of MyD88-dependent TLR signaling in preventing DC-mediated autoinflammation. LPS-stimulated HOIP-deficient DCs show enhanced cell death and elevated IL-1α/IL-1β.","method":"DC-specific conditional KO, genetic epistasis with TNFR1-KO and MyD88-KO, antibiotic treatment, LPS stimulation, cytokine assays","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 — genetic epistasis with multiple KO combinations and in vivo rescue; single lab","pmids":["34253576"],"is_preprint":false},{"year":2021,"finding":"HOIP inhibits cancer cell apoptosis by associating with PTEN and promoting PTEN proteasomal degradation; HOIP depletion causes cell cycle arrest and apoptosis that can be rescued by PTEN silencing, placing HOIP upstream of PTEN in the PI3K-AKT survival pathway.","method":"Co-IP, PTEN stability assay (cycloheximide chase + MG132), siRNA double knockdown (HOIP+PTEN), apoptosis/cell-cycle assays","journal":"Journal of Cancer","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP with epistasis rescue; single lab","pmids":["34659546"],"is_preprint":false}],"current_model":"RNF31 (HOIP) is the catalytic RBR E3 ubiquitin ligase subunit of LUBAC that employs a RING/HECT-hybrid two-step mechanism — receiving ubiquitin from an E2 via a HECT-like thioester intermediate at its RING2 active-site cysteine, then using its unique Linear ubiquitin chain Determining Domain (LDD) to orient the acceptor ubiquitin α-amino group for Met1-linked (linear) chain formation — thereby activating NF-κB signaling downstream of TNF, IL-1, TLR, CD40, and STING receptors, protecting cells from TNFR1/caspase-mediated and ferroptosis-related death, and additionally ubiquitinating substrates including NEMO, FADD, GPx4, ERα, p53, YAP, A20, and NLRP3 to regulate inflammation, immunity, and cell survival."},"narrative":{"teleology":[{"year":2004,"claim":"Initial identification of RNF31 as an RBR-family E3 ligase associated with MuSK at neuromuscular junctions provided the first evidence that the gene encodes a ubiquitin ligase, though no catalytic mechanism or signaling function was established.","evidence":"Yeast two-hybrid/pull-down with MuSK cytoplasmic domain and immunolocalization at NMJs","pmids":["14678832"],"confidence":"Low","gaps":["Single interaction assay without reciprocal validation","No substrate or chain-type specificity determined","Functional role at NMJ undefined"]},{"year":2009,"claim":"Discovery that RNF31 forms a corepressor complex with DAX-1 at steroidogenic gene promoters and stabilizes DAX-1 via monoubiquitination established its first characterized substrate relationship and linked it to transcriptional regulation.","evidence":"ChIP at StAR/CYP19 promoters, Co-IP, siRNA knockdown, gene expression analysis in steroidogenic cells","pmids":["19237537"],"confidence":"Medium","gaps":["Whether this represents LUBAC-dependent linear ubiquitination or an independent activity was unknown","Chain type of monoubiquitination not defined as M1-linked"]},{"year":2010,"claim":"Identification of HOIP at the CD40 signaling complex and demonstration that its ligase activity is required for CD40-mediated NF-κB activation established HOIP as an activating component of immune receptor signaling.","evidence":"Mass spectrometry of CD40 complexes, dominant-negative catalytic mutant, NF-κB reporter assay","pmids":["20614026"],"confidence":"Medium","gaps":["Chain type synthesized at CD40 complex not yet characterized","Whether HOIP acts as part of LUBAC in this context was unclear"]},{"year":2011,"claim":"HOIP-deficient B cells confirmed the essential, non-redundant role of HOIP in CD40 signaling by showing complete loss of NF-κB and JNK activation, IKK recruitment failure, and defective B cell functional responses, all rescued by re-expression.","evidence":"Somatic gene targeting of HOIP in B cell lines, rescue by re-expression, multiple signaling readouts","pmids":["21829693"],"confidence":"High","gaps":["Whether HOIP is required for other TNFR superfamily members beyond CD40 was untested","In vivo B cell immune response not examined"]},{"year":2012,"claim":"Biochemical dissection revealed HOIP's unique catalytic mechanism—a RING/HECT-hybrid two-step reaction using a thioester intermediate at RING2 and a C-terminal LDD domain that specifies linear (Met1) chain linkage—resolving how LUBAC achieves chain-type specificity.","evidence":"In vitro ubiquitination assays with active-site and domain mutants, cellular NF-κB reporters","pmids":["22863777"],"confidence":"High","gaps":["Structural basis for acceptor ubiquitin orientation not yet visualized","How LUBAC accessory subunits regulate HOIP catalysis unknown"]},{"year":2013,"claim":"Crystal structures of the HOIP catalytic core with and without ubiquitin revealed the structural basis for linear chain specificity: a zinc-finger and novel C-terminal fold create an acceptor ubiquitin-binding platform positioning the α-amino group for nucleophilic attack.","evidence":"X-ray crystallography (apo and ubiquitin-bound), functional mutagenesis in cells","pmids":["24141947"],"confidence":"High","gaps":["Structure of full-length HOIP in activated state with E2~Ub not yet solved","Donor ubiquitin engagement undefined structurally"]},{"year":2014,"claim":"Multiple advances defined HOIP's regulatory network and in vivo essentiality: structural characterization of the HOIP PUB–OTULIN interaction revealed phospho-regulated deubiquitinase recruitment; HOIP deficiency caused TNFR1-dependent embryonic-lethal vascular defects; and HOIP was linked to ERα stabilization and cisplatin sensitivity, broadening its substrate repertoire and physiological roles.","evidence":"Crystal/NMR structures of PUB–OTULIN; conditional Hoip KO mice with TNFR1 epistasis; Co-IP/ubiquitination assays for ERα; siRNA screen for cisplatin sensitivity","pmids":["24726327","25284787","24441041","24686174"],"confidence":"High","gaps":["How OTULIN and CYLD coordinate linear chain editing at receptor complexes unknown","ERα ubiquitination chain type not determined as linear","In vivo relevance of ERα stabilization unconfirmed"]},{"year":2016,"claim":"The crystal structure of fully active HOIP RBR bound to E2~ubiquitin revealed the complete catalytic transfer mechanism—an elongated conformation distinct from auto-inhibited RBRs with three ubiquitin-binding sites engaging donor, regulatory, and acceptor ubiquitins—while functional studies showed HOIP is required for T cell survival, is cleaved by caspases during apoptosis to limit linear ubiquitination, and is regulated by Lys1056 ubiquitination in a stimulus-specific manner.","evidence":"X-ray crystallography of HOIP/E2~Ub complex; T cell-specific catalytic-dead knock-in mice; caspase cleavage mapping with mutagenesis; K1056R mutagenesis with TLR4/CD40 signaling assays","pmids":["26789245","27786304","27669734","26578682","27545878"],"confidence":"High","gaps":["How auto-inhibition is relieved by HOIL-1L/SHARPIN in the full LUBAC complex not structurally resolved","Relative contributions of OTULIN vs CYLD–SPATA2 in different signaling contexts unclear","Whether caspase cleavage is a feedforward amplification loop in apoptosis not fully tested"]},{"year":2018,"claim":"Extension of HOIP's receptor signaling roles to TNFR2 (requiring cIAP1 for recruitment) and skin-specific knockout showing TNFR1-rescuable lethal dermatitis established HOIP as a pan-TNFR family signaling node whose loss triggers tissue-specific inflammatory cell death.","evidence":"TNFR2 complex IP with M1-Ub antibodies and cIAP antagonist; epidermis-specific Hoip KO with TNFR1 epistasis","pmids":["29378181","29728512"],"confidence":"High","gaps":["Whether HOIP engages all TNFR superfamily members or only specific subsets unknown","Mechanism of cIAP1-dependent HOIP recruitment to TNFR2 not defined"]},{"year":2019,"claim":"Fragment-based covalent screening targeting the HOIP active-site cysteine yielded the first cell-permeable HOIP inhibitors, validated by co-crystal structures and cellular NF-κB suppression, opening pharmacological targeting of linear ubiquitination.","evidence":"Fragment screen, protein LC-MS, co-crystal structures, chemoproteomics, cell-based NF-κB assays","pmids":["30657686"],"confidence":"High","gaps":["Inhibitor selectivity over other RBR ligases not comprehensively profiled","In vivo efficacy not demonstrated","No therapeutic window established"]},{"year":2019,"claim":"Chemically synthesized monoubiquitylated NEMO demonstrated that HOIP RBR alone can extend a pre-primed substrate, establishing a two-step model for NEMO linear ubiquitination where priming and extension have distinct LUBAC subunit requirements.","evidence":"Chemical ubiquitylation via ligation auxiliary, in vitro extension assay with recombinant HOIP RBR","pmids":["31942456"],"confidence":"Medium","gaps":["Identity of the priming E3 or priming mechanism in cells not established","Whether the two-step model applies to substrates beyond NEMO unknown"]},{"year":2021,"claim":"Studies in dendritic cell-specific HOIP knockouts, plus identification of new substrates (A20, p53, PTEN), revealed cell-type-specific HOIP functions: DC autoinflammation is driven by MyD88/TLR rather than TNFR1, while HOIP-mediated degradation of A20, p53, and PTEN connects linear ubiquitination to inflammation, cell cycle, and PI3K-AKT signaling.","evidence":"DC-specific KO with MyD88/TNFR1 epistasis; Co-IP and ubiquitination/degradation assays for A20, p53, PTEN; mouse colitis and liver injury models","pmids":["34253576","34416243","33824292","34659546"],"confidence":"Medium","gaps":["Whether A20 and p53 ubiquitination is linear or K48/K63-linked needs clarification","PTEN interaction awaits independent validation","How HOIP selects among diverse substrates is mechanistically unclear"]},{"year":2022,"claim":"Multiple genome-wide CRISPR screens converged on RNF31 as a critical tumor immune-evasion gene: HOIP loss sensitizes cancer cells to TNF-mediated caspase-8-dependent apoptosis and immune cell killing, while new substrates GPx4 (ferroptosis protection), YAP (Hippo pathway), and NLRP3 (inflammasome) expanded HOIP's substrate repertoire.","evidence":"In vivo and in vitro CRISPR screens in pancreatic and colon cancer; pharmacological HOIP inhibition in organoids; Co-IP/ubiquitination for GPx4, YAP, NLRP3; ferroptosis and inflammasome assays","pmids":["35379808","35688159","34467615","36279464","36581998","37951199"],"confidence":"High","gaps":["Whether GPx4, YAP, NLRP3 ubiquitination is linear or non-linear chain type needs definitive characterization","Therapeutic index of HOIP inhibition for cancer immunotherapy not established","Structural basis for substrate selection among diverse targets unknown"]},{"year":2024,"claim":"HOIP was placed in the STING innate immune pathway: upon STING activation, LUBAC is recruited to LC3B-associated Golgi membranes where HOIP synthesizes M1-linked chains required for downstream NF-κB and IRF3 signaling, establishing a new receptor-proximal context for linear ubiquitination.","evidence":"HOIP KO in THP1 and BMDMs, M1-Ub IP, STING agonist stimulation, Golgi fractionation","pmids":["39578541"],"confidence":"Medium","gaps":["How LUBAC is recruited to STING-containing Golgi membranes mechanistically undefined","Whether LC3B association is required for LUBAC activity at this site not tested","Whether STING-dependent LUBAC recruitment occurs in non-myeloid cells unknown"]},{"year":null,"claim":"Key unresolved questions include: how HOIP discriminates among its growing list of substrates for linear versus non-linear ubiquitination, the structural basis of full-length LUBAC auto-inhibition and activation by HOIL-1L and SHARPIN, and whether pharmacological HOIP inhibition can achieve a therapeutic window in vivo for cancer immunotherapy or inflammatory disease.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structure of full-length trimeric LUBAC in auto-inhibited or active state","Chain-type specificity for many reported substrates (ERα, p53, PTEN, YAP) not rigorously determined","In vivo pharmacology of HOIP inhibitors not reported"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,2,6,7,10,11,15,22,24,25,28,30]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,1,2]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[6]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[5,13,17]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[21]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,5,8,9,13,16,17,19,20,21]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[9,19,20,21,31]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[5,10,11,14,16,18,19]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1,2,30]}],"complexes":["LUBAC"],"partners":["RBCK1","SHARPIN","OTULIN","SPATA2","CYLD","NEMO","A20","NLRP3"],"other_free_text":[]},"mechanistic_narrative":"RNF31 (HOIP) is the catalytic E3 ubiquitin ligase subunit of the linear ubiquitin chain assembly complex (LUBAC) that synthesizes Met1-linked (linear) polyubiquitin chains to activate NF-κB signaling and suppress cell death across multiple immune and developmental contexts. HOIP employs a RING/HECT-hybrid two-step mechanism in which RING1-IBR transfers ubiquitin from an E2 onto a thioester intermediate at the RING2 active-site cysteine, and a unique C-terminal Linear ubiquitin chain Determining Domain (LDD) orients the acceptor ubiquitin α-amino group for linear chain formation, as defined by crystal structures of the catalytic core, the ubiquitin-bound state, and the E2~ubiquitin transfer complex [PMID:22863777, PMID:24141947, PMID:26789245]. HOIP-dependent linear ubiquitination of NEMO and FADD at TNFR1, TNFR2, CD40, TLR, and STING receptor complexes is essential for canonical NF-κB and JNK activation, and HOIP deficiency causes TNFR1-dependent endothelial lethality, skin inflammation, T cell loss, and sensitization of tumor cells to TNF/IFNγ-mediated and immune cell–mediated killing [PMID:25284787, PMID:21829693, PMID:29728512, PMID:27786304, PMID:35379808, PMID:35688159]. Beyond linear chain assembly, HOIP ubiquitinates additional substrates—including ERα, p53, GPx4, YAP, A20, and NLRP3—to regulate their stability, and its own activity is modulated by caspase cleavage during apoptosis, Lys1056 ubiquitination, and interactions with the deubiquitinases OTULIN and CYLD–SPATA2, which counterbalance linear ubiquitin signaling [PMID:24726327, PMID:27545878, PMID:27669734, PMID:26578682, PMID:36279464, PMID:24441041]."},"prefetch_data":{"uniprot":{"accession":"Q96EP0","full_name":"E3 ubiquitin-protein ligase RNF31","aliases":["HOIL-1-interacting protein","HOIP","RING finger protein 31","RING-type E3 ubiquitin transferase RNF31","Zinc in-between-RING-finger ubiquitin-associated domain protein"],"length_aa":1072,"mass_kda":119.7,"function":"E3 ubiquitin-protein ligase component of the LUBAC complex which conjugates linear ('Met-1'-linked) polyubiquitin chains to substrates and plays a key role in NF-kappa-B activation and regulation of inflammation (PubMed:17006537, PubMed:19136968, PubMed:20005846, PubMed:21455173, PubMed:21455180, PubMed:21455181, PubMed:22863777, PubMed:28189684, PubMed:28481331). LUBAC conjugates linear polyubiquitin to IKBKG and RIPK1 and is involved in activation of the canonical NF-kappa-B and the JNK signaling pathways (PubMed:17006537, PubMed:19136968, PubMed:20005846, PubMed:21455173, PubMed:21455180, PubMed:21455181, PubMed:22863777, PubMed:28189684). Linear ubiquitination mediated by the LUBAC complex interferes with TNF-induced cell death and thereby prevents inflammation (PubMed:21455173, PubMed:28189684). LUBAC is recruited to the TNF-R1 signaling complex (TNF-RSC) following polyubiquitination of TNF-RSC components by BIRC2 and/or BIRC3 and to conjugate linear polyubiquitin to IKBKG and possibly other components contributing to the stability of the complex (PubMed:20005846, PubMed:27458237). The LUBAC complex is also involved in innate immunity by conjugating linear polyubiquitin chains at the surface of bacteria invading the cytosol to form the ubiquitin coat surrounding bacteria (PubMed:28481331, PubMed:34012115). LUBAC is not able to initiate formation of the bacterial ubiquitin coat, and can only promote formation of linear polyubiquitins on pre-existing ubiquitin (PubMed:28481331). Recruited to the surface of bacteria by RNF213, which initiates the bacterial ubiquitin coat (PubMed:34012115). The bacterial ubiquitin coat acts as an 'eat-me' signal for xenophagy and promotes NF-kappa-B activation (PubMed:28481331, PubMed:34012115). Together with OTULIN, the LUBAC complex regulates the canonical Wnt signaling during angiogenesis (PubMed:23708998). RNF31 is required for linear ubiquitination of BCL10, thereby promoting TCR-induced NF-kappa-B activation (PubMed:27777308). Binds polyubiquitin of different linkage types (PubMed:23708998)","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q96EP0/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RNF31","classification":"Not Classified","n_dependent_lines":154,"n_total_lines":1208,"dependency_fraction":0.12748344370860928},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RNF31","total_profiled":1310},"omim":[{"mim_id":"620632","title":"IMMUNODEFICIENCY 115 WITH AUTOINFLAMMATION; IMD115","url":"https://www.omim.org/entry/620632"},{"mim_id":"615712","title":"OTU DEUBIQUITINASE WITH LINEAR LINKAGE SPECIFICITY; OTULIN","url":"https://www.omim.org/entry/615712"},{"mim_id":"612487","title":"RING FINGER PROTEIN 31; RNF31","url":"https://www.omim.org/entry/612487"},{"mim_id":"611885","title":"SHANK-ASSOCIATED RH DOMAIN INTERACTOR; SHARPIN","url":"https://www.omim.org/entry/611885"},{"mim_id":"603453","title":"RECEPTOR-INTERACTING SERINE/THREONINE KINASE 1; RIPK1","url":"https://www.omim.org/entry/603453"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Cytosol","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/RNF31"},"hgnc":{"alias_symbol":["ZIBRA","FLJ10111","FLJ23501","HOIP","Paul"],"prev_symbol":[]},"alphafold":{"accession":"Q96EP0","domains":[{"cath_id":"1.20.58.2190","chopping":"3-180","consensus_level":"high","plddt":87.1067,"start":3,"end":180},{"cath_id":"1.10.8.10","chopping":"497-620","consensus_level":"high","plddt":92.392,"start":497,"end":620},{"cath_id":"-","chopping":"641-688","consensus_level":"high","plddt":85.4654,"start":641,"end":688},{"cath_id":"-","chopping":"714-793","consensus_level":"medium","plddt":80.3265,"start":714,"end":793},{"cath_id":"-","chopping":"859-1020","consensus_level":"high","plddt":86.4485,"start":859,"end":1020}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96EP0","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96EP0-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96EP0-F1-predicted_aligned_error_v6.png","plddt_mean":77.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RNF31","jax_strain_url":"https://www.jax.org/strain/search?query=RNF31"},"sequence":{"accession":"Q96EP0","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96EP0.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96EP0/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96EP0"}},"corpus_meta":[{"pmid":"25284787","id":"PMC_25284787","title":"HOIP 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pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2012,\n      \"finding\": \"HOIP (RNF31) is the catalytically active subunit of LUBAC and uses a two-step RING/HECT-hybrid mechanism: RING1-IBR catalyzes transfer of ubiquitin from E2 onto a covalent HECT-like thioester intermediate at RING2, then a unique C-terminal Linear ubiquitin chain Determining Domain (LDD) coordinates the acceptor ubiquitin N-terminus for Met1-linked (linear) chain formation.\",\n      \"method\": \"In vitro ubiquitination assays, active-site mutagenesis, biochemical dissection of RING1-IBR and RING2-LDD sub-domains, cellular NF-κB reporter assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with mutagenesis identifying catalytic mechanism; replicated by structural studies\",\n      \"pmids\": [\"22863777\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Crystal structures of the HOIP catalytic core in apo form and in complex with ubiquitin revealed a novel C-terminal fold and zinc-finger that form a ubiquitin-binding platform orienting the acceptor ubiquitin α-amino group for nucleophilic attack on the E3~ubiquitin thioester, explaining linear chain specificity.\",\n      \"method\": \"X-ray crystallography (apo and ubiquitin-bound structures), mutagenesis, cellular NF-κB assays\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structures with functional mutagenesis validation in cells\",\n      \"pmids\": [\"24141947\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Crystal structure of fully active HOIP RBR in complex with an E2~ubiquitin conjugate showed that active HOIP adopts a conformation markedly different from auto-inhibited RBRs, binds E2~ubiquitin in an elongated fashion aligning catalytic centres for HECT-like transfer, and revealed three helix-IBR-fold ubiquitin-binding regions that engage the activated (donor) ubiquitin and an additional regulatory ubiquitin.\",\n      \"method\": \"X-ray crystallography of HOIP RBR/E2~ubiquitin transfer complex, structural comparison with Parkin and HHARI\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — first structure of a fully active RBR E3 in transfer complex, high-resolution with mechanistic implications\",\n      \"pmids\": [\"26789245\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"HOIP (RNF31) PUB domain binds the PUB-interacting motif (PIM) of the linear deubiquitinase OTULIN; structural studies defined critical contacts (OTULIN Tyr56); phosphorylation of OTULIN Tyr56 negatively regulates this interaction. HOIP binding to OTULIN recruits OTULIN to the TNF receptor complex to counteract HOIP-dependent NF-κB activation.\",\n      \"method\": \"Co-IP, structural studies (crystal/NMR), phosphorylation site mutagenesis, TNF receptor complex recruitment assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — structural data plus reciprocal interaction mapping and functional recruitment assays\",\n      \"pmids\": [\"24726327\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SPATA2 is a constitutive direct binding partner of HOIP that bridges the interaction between the deubiquitinase CYLD and HOIP; SPATA2 is required for CYLD recruitment to TNFR1- and NOD2-signaling complexes, and its loss diminishes TNF-induced necroptosis similarly to CYLD loss.\",\n      \"method\": \"Co-IP, signaling complex (TNFR1/NOD2) pull-down, SPATA2 knockout/knockdown with necroptosis readout\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, complex recruitment assay, loss-of-function with defined phenotype\",\n      \"pmids\": [\"27545878\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"HOIP (RNF31) deficiency causes aberrant TNFR1-mediated endothelial cell death via aberrant complex-II formation, leading to vascularization defects and embryonic lethality; TNFR1 ablation rescues this phenotype, and HOIP catalytic activity is required to prevent TNF-induced cell death.\",\n      \"method\": \"Conditional (Tie2-Cre) and constitutive Hoip knockout mice, genetic epistasis with TNFR1 knockout, cell death assays, complex-II immunoprecipitation\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis in vivo with defined molecular mechanism (complex-II formation)\",\n      \"pmids\": [\"25284787\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"RNF31 (HOIP) associates with estrogen receptor α (ERα) primarily in the cytosol, increases ERα stability and mono-ubiquitination in a ubiquitin-ligase-activity-dependent manner, and is required for ERα-stimulated proliferation and downstream target gene expression in breast cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, cycloheximide chase, cell-cycle analysis, ubiquitination assays with catalytic mutant\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP with functional ligase-activity mutant and multiple cellular phenotypes; single lab\",\n      \"pmids\": [\"24441041\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"RNF31 forms an in vivo corepressor complex with DAX-1 at the promoters of steroidogenic genes (StAR, CYP19), stabilizes DAX-1 via monoubiquitination, and is required for DAX-1-mediated repression of SF-1-dependent steroidogenic gene transcription.\",\n      \"method\": \"ChIP, Co-IP, siRNA knockdown, gene expression analysis, co-expression studies in steroidogenic tissues\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP demonstrating complex at endogenous promoters plus functional knockdown; single lab\",\n      \"pmids\": [\"19237537\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"HOIP (RNF31/PAUL/ZIBRA) is recruited to the CD40 signaling complex in a TRAF2-dependent manner; its ubiquitin ligase activity is required for CD40-mediated NF-κB activation, and a catalytic-dead HOIP mutant acts as a dominant negative.\",\n      \"method\": \"Mass spectrometry of CD40 signaling complexes, Co-IP, dominant-negative ligase-dead mutant, NF-κB reporter assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — MS-based complex identification and catalytic mutant functional test; single lab\",\n      \"pmids\": [\"20614026\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"HOIP is essential for CD40 signaling: HOIP-deficient B cells fail to activate NF-κB and JNK via CD40, exhibit impaired IKK recruitment to the CD40 signaling complex, and show defective CD80 upregulation and germline IgE transcription; HOIP re-expression restores all defects.\",\n      \"method\": \"Somatic gene targeting to generate HOIP-deficient B cell lines, signaling assays (NF-κB, JNK, IKK recruitment), rescue by re-expression\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with multiple orthogonal functional readouts and rescue experiment\",\n      \"pmids\": [\"21829693\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"HOIP cleavage by caspases 3 and 6 at Asp348, Asp387, and Asp390 during apoptosis reduces linear ubiquitination of NEMO and FADD; the N-terminal cleavage fragment retains binding to OTULIN and CYLD-SPATA2 while the C-terminal fragment retains NF-κB activity; cleavage-site mutation inhibits TNF-α-induced apoptosis.\",\n      \"method\": \"Caspase cleavage assays, site-directed mutagenesis of cleavage sites, linear ubiquitination assays, Co-IP with deubiquitinases, apoptosis assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — biochemical identification of cleavage sites with mutagenesis and functional apoptosis readout\",\n      \"pmids\": [\"27669734\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"FADD is identified as a substrate for LUBAC linear ubiquitination in addition to NEMO; HOIP is cleaved predominantly by caspase at Asp390 during apoptosis and is subsequently subject to proteasomal degradation, decreasing linear ubiquitination of both NEMO and FADD.\",\n      \"method\": \"Linear ubiquitination assays, caspase cleavage assays, proteasome inhibitor experiments, Co-IP\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct biochemical demonstration of FADD as LUBAC substrate with caspase cleavage mapping; single lab, partial overlap with prior study\",\n      \"pmids\": [\"28189684\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Ubiquitination of HOIP at Lys1056 (C-terminal lysine) induces a conformational change that suppresses HOIP catalytic activity and terminates TLR4/LPS-induced NF-κB signaling, but does not affect CD40-induced NF-κB activation, identifying a stimulus-specific regulatory PTM.\",\n      \"method\": \"Site-directed mutagenesis (K1056R), in vitro ubiquitination assays, NF-κB reporter assays with TLR4 and CD40 stimulation\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mutagenesis with functional signaling readout in cells; single lab\",\n      \"pmids\": [\"26578682\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"HOIP (and cIAP1) are recruited to the TNFR2 signaling complex; HOIP mediates M1-linked polyubiquitination at this complex; cIAP1 antagonism prevents HOIP recruitment; both HOIP and cIAP1 are required for TNFR2-induced canonical NF-κB activation.\",\n      \"method\": \"Signaling complex immunoprecipitation, M1/K63 linkage-specific ubiquitin antibodies, cIAP antagonist treatment, NF-κB activation assays\",\n      \"journal\": \"Biochemical pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — complex recruitment and ubiquitination shown biochemically; single lab\",\n      \"pmids\": [\"29378181\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"HOIP-depleted cells are hypersensitive to cisplatin-induced apoptosis through enhanced caspase-8/caspase-3-dependent apoptosis that requires ATM (but not ATR) checkpoint activation; basal and cisplatin-induced JNK activity is enhanced in HOIP-depleted cells, and JNK inhibition reverses apoptotic hyperactivation.\",\n      \"method\": \"siRNA screen, siRNA knockdown, caspase activation assays, ATM/ATR inhibitors, JNK inhibitor rescue, apoptosis assays\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — epistasis via pharmacological inhibition with defined mechanistic pathway placement; single lab\",\n      \"pmids\": [\"24686174\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"LUBAC (HOIP) binds and stabilizes GPx4 via linear ubiquitination both basally and under oxidative stress; LUBAC deficiency sensitizes cells to ferroptosis by promoting GPx4 degradation and downstream lipid peroxidation, identifying GPx4 as a key LUBAC substrate.\",\n      \"method\": \"Co-IP, linear ubiquitination assays, ferroptosis assays (lipid peroxidation), LUBAC-deficient cells, GPx4 stability (cycloheximide chase)\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP and ubiquitination assays with cellular ferroptosis phenotype; single lab\",\n      \"pmids\": [\"36279464\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RNF31 (HOIP) protects tumor cells from TNF-mediated caspase-8 cleavage and apoptosis; CRISPR knockout of Rnf31 sensitizes pancreatic cancer cells to CD8+ T cell killing in a TNF-dependent manner, increases intratumoral CD8+ T cell infiltration and effector function in vivo, and reduces tumor growth.\",\n      \"method\": \"CRISPR-Cas9 in vitro and in vivo screens, genetic knockout, caspase-8 cleavage assays, orthotopic transplantation into immune-competent mice\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vitro and in vivo CRISPR KO with defined molecular mechanism and immune phenotype\",\n      \"pmids\": [\"35379808\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Genetic and pharmacological ablation of RNF31 sensitizes cancer cells to NK and CD8+ T cell killing in a TNF-dependent manner by causing loss of A20 and non-canonical IKK complexes from TNF receptor complex I; a small-molecule RNF31 inhibitor sensitizes colon carcinoma organoids to TNF.\",\n      \"method\": \"Genome-wide CRISPR-Cas9 screens under NK and CD8+ T cell pressure, genetic knockout, pharmacologic inhibition, TNF receptor complex immunoprecipitation, organoid killing assays\",\n      \"journal\": \"Cell reports. Medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genome-wide screen validated by orthogonal genetic and pharmacological approaches with mechanistic complex analysis\",\n      \"pmids\": [\"35688159\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"HOIP-deficient tumor cells show increased sensitivity to combined TNF and IFN-γ, engaging both intrinsic and extrinsic apoptotic machinery through a transcription-dependent cell death pathway; both genetic deletion and pharmacological inhibition of HOIP augment this sensitivity.\",\n      \"method\": \"CRISPR/Cas9 screening, genetic deletion, pharmacological inhibition, NK/CD8+ T cell killing assays, cytokine (TNF+IFN-γ) treatment, apoptosis pathway dissection\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — CRISPR screen followed by genetic and pharmacologic validation; single lab\",\n      \"pmids\": [\"34467615\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Epidermis-specific knockout of RNF31 in mice causes postnatal lethality due to severe skin inflammation triggered by TNF-α-induced apoptosis in keratinocytes; RNF31 deficiency impairs TNF-α-induced NF-κB activation and increases apoptosis; deletion of TNFR1 rescues lethality and skin inflammation.\",\n      \"method\": \"Tissue-specific conditional knockout, genetic epistasis with TNFR1 knockout, NF-κB activation assays, apoptosis assays in primary keratinocytes\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — tissue-specific KO with genetic epistasis and mechanistic signaling readouts\",\n      \"pmids\": [\"29728512\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"HOIP ubiquitin ligase activity is required for mature T cell survival; T-HOIPΔlinear mice show reduced CD4+/CD8+ T cell numbers and defective NKT development; HOIP-deficient CD4+ T cells fail to phosphorylate IκBα and JNK upon TCR stimulation; reduced CD127 expression causes mature T cell apoptosis, and enforced CD127 expression rescues CD8+ T cell development.\",\n      \"method\": \"Conditional knockout (ubiquitin ligase-dead knock-in), flow cytometry, signaling assays, apoptosis assays, CD127 rescue experiment\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean catalytic-domain KO with mechanistic signaling readout and rescue; single lab\",\n      \"pmids\": [\"27786304\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"STING activation recruits LUBAC (HOIP) to LC3B-associated Golgi membranes where it synthesizes M1-linked ubiquitin chains; loss of HOIP prevents M1-Ub chain formation and reduces both NF-κB and IRF3 signaling downstream of STING without affecting STING activation itself.\",\n      \"method\": \"HOIP knockout in THP1 monocytes and mouse BMDMs, M1-ubiquitin chain immunoprecipitation, STING activation assays, fractionation/localization to Golgi membranes\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with biochemical ubiquitin chain detection and signaling readouts; single study\",\n      \"pmids\": [\"39578541\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"RNF31 interacts with A20 via its RBR structural domain, promotes K63-linked ubiquitination of A20 leading to A20 proteasomal degradation, thereby activating the TLR4/MyD88/NF-κB pathway and aggravating hepatocyte apoptosis and inflammation in acute liver injury.\",\n      \"method\": \"Co-IP, actinomycin tracing, proteasome inhibitor experiments, ubiquitination assays, siRNA knockdown, mouse model of acute liver injury\",\n      \"journal\": \"Chemico-biological interactions\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — biochemical interaction, ubiquitination and degradation assays with in vivo validation; single lab\",\n      \"pmids\": [\"34416243\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Fragment-based covalent ligand screening targeting the active-site cysteine of HOIP identified electrophilic fragments that inhibit HOIP catalytic activity; crystal structures of HOIP-inhibitor complexes enabled structure-based development; cell-permeable compounds inhibit HOIP and NF-κB activation in mammalian cells as shown by chemoproteomics.\",\n      \"method\": \"Fragment-based covalent screening, protein LC-MS, in vitro ubiquitination assays, protein crystallography, cell-based NF-κB assays, chemoproteomics\",\n      \"journal\": \"Journal of the American Chemical Society\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structures of covalent inhibitor complexes at active-site cysteine with biochemical and cellular validation\",\n      \"pmids\": [\"30657686\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RNF31 associates with YAP protein, facilitates YAP poly-ubiquitination and proteasomal degradation at K76, and thereby suppresses Hippo/YAP/PD-L1 signaling in triple-negative breast cancer cells.\",\n      \"method\": \"Co-IP, ubiquitination assays, site-specific mutagenesis (YAP K76), immunoblots, gene expression profiling, xenograft models\",\n      \"journal\": \"Journal of experimental & clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP with site-specific mutagenesis; single lab\",\n      \"pmids\": [\"36581998\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"RNF31 interacts with p53 via its PUB domain, promotes p53 ubiquitination and proteasomal degradation; RNF31 depletion alleviates p53 degradation (inhibited by MG132), and this RNF31/p53 axis drives colorectal cancer cell proliferation.\",\n      \"method\": \"Co-IP with truncated RNF31 domain constructs, cycloheximide chase, proteasome inhibitor (MG132), ubiquitination assays, siRNA knockdown\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — domain-mapping Co-IP with degradation assays; single lab\",\n      \"pmids\": [\"33824292\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RNF31 promotes p53 ubiquitination and proteasomal degradation in hepatocytes; reduced p53 levels increase BNIP3 expression, promoting mitophagy and reducing hepatic steatosis; RNF31-containing small extracellular vesicles from mesenchymal stem cells reduce steatosis in HFD-fed mice.\",\n      \"method\": \"Ubiquitination assays, co-IP, siRNA knockdown, mt-Keima mitophagy imaging, RNA-seq, in vivo HFD mouse model with sEV delivery\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — multiple methods (ubiquitination, mitophagy imaging, in vivo) but single lab\",\n      \"pmids\": [\"38615890\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RBCK1 (HOIL-1L) interacts with RNF31 (HOIP) and represses its ubiquitination and proteasomal degradation, thereby stabilizing RNF31 protein levels in hepatocellular carcinoma cells.\",\n      \"method\": \"Co-IP, ubiquitination assays, proteasome inhibitor experiments, siRNA/overexpression in HCC cells\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP plus degradation assays; single lab\",\n      \"pmids\": [\"35869046\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"RNF31 interacts with NLRP3 through its RBR structural domain, promotes K63-linked ubiquitination of NLRP3 leading to NLRP3 stabilization, and thereby activates the NLRP3 inflammasome during ulcerative colitis.\",\n      \"method\": \"Co-IP with domain-mapping, K63-linkage specific ubiquitination assays, RNF31 knockdown in cells and mouse DSS-colitis model\",\n      \"journal\": \"International immunopharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — domain Co-IP, linkage-specific ubiquitination, and in vivo KD; single lab\",\n      \"pmids\": [\"37951199\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"RNF31 (PAUL/ZIBRA) was identified as an Ariadne-like RBR ubiquitin ligase that binds to the cytoplasmic domain of the muscle-specific receptor tyrosine kinase MuSK and is present at neuromuscular junctions.\",\n      \"method\": \"Yeast two-hybrid or pull-down (interaction with MuSK cytoplasmic domain), expression profiling in developing mouse tissues, immunolocalization at NMJs\",\n      \"journal\": \"Gene expression patterns\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single interaction assay with localization; no functional mechanism established\",\n      \"pmids\": [\"14678832\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Chemically synthesized monoubiquitylated NEMO (NEMOCoZi-Ub) is accepted as a substrate for linear extension by the HOIP RBR domain alone, demonstrating that NEMO linear ubiquitylation occurs in two steps: an initial priming event and a separate extension step requiring different LUBAC components.\",\n      \"method\": \"Chemical ubiquitylation using ligation auxiliary, in vitro linear extension assay with recombinant HOIP RBR, biophysical affinity measurements\",\n      \"journal\": \"Communications chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with chemically defined substrates; single study\",\n      \"pmids\": [\"31942456\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In dendritic cell-specific HOIP knockout mice, autoinflammation is not rescued by TNFR1 deletion but is rescued by antibiotic treatment or MyD88 deficiency, placing HOIP upstream of MyD88-dependent TLR signaling in preventing DC-mediated autoinflammation. LPS-stimulated HOIP-deficient DCs show enhanced cell death and elevated IL-1α/IL-1β.\",\n      \"method\": \"DC-specific conditional KO, genetic epistasis with TNFR1-KO and MyD88-KO, antibiotic treatment, LPS stimulation, cytokine assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with multiple KO combinations and in vivo rescue; single lab\",\n      \"pmids\": [\"34253576\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"HOIP inhibits cancer cell apoptosis by associating with PTEN and promoting PTEN proteasomal degradation; HOIP depletion causes cell cycle arrest and apoptosis that can be rescued by PTEN silencing, placing HOIP upstream of PTEN in the PI3K-AKT survival pathway.\",\n      \"method\": \"Co-IP, PTEN stability assay (cycloheximide chase + MG132), siRNA double knockdown (HOIP+PTEN), apoptosis/cell-cycle assays\",\n      \"journal\": \"Journal of Cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP with epistasis rescue; single lab\",\n      \"pmids\": [\"34659546\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RNF31 (HOIP) is the catalytic RBR E3 ubiquitin ligase subunit of LUBAC that employs a RING/HECT-hybrid two-step mechanism — receiving ubiquitin from an E2 via a HECT-like thioester intermediate at its RING2 active-site cysteine, then using its unique Linear ubiquitin chain Determining Domain (LDD) to orient the acceptor ubiquitin α-amino group for Met1-linked (linear) chain formation — thereby activating NF-κB signaling downstream of TNF, IL-1, TLR, CD40, and STING receptors, protecting cells from TNFR1/caspase-mediated and ferroptosis-related death, and additionally ubiquitinating substrates including NEMO, FADD, GPx4, ERα, p53, YAP, A20, and NLRP3 to regulate inflammation, immunity, and cell survival.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"RNF31 (HOIP) is the catalytic E3 ubiquitin ligase subunit of the linear ubiquitin chain assembly complex (LUBAC) that synthesizes Met1-linked (linear) polyubiquitin chains to activate NF-κB signaling and suppress cell death across multiple immune and developmental contexts. HOIP employs a RING/HECT-hybrid two-step mechanism in which RING1-IBR transfers ubiquitin from an E2 onto a thioester intermediate at the RING2 active-site cysteine, and a unique C-terminal Linear ubiquitin chain Determining Domain (LDD) orients the acceptor ubiquitin α-amino group for linear chain formation, as defined by crystal structures of the catalytic core, the ubiquitin-bound state, and the E2~ubiquitin transfer complex [PMID:22863777, PMID:24141947, PMID:26789245]. HOIP-dependent linear ubiquitination of NEMO and FADD at TNFR1, TNFR2, CD40, TLR, and STING receptor complexes is essential for canonical NF-κB and JNK activation, and HOIP deficiency causes TNFR1-dependent endothelial lethality, skin inflammation, T cell loss, and sensitization of tumor cells to TNF/IFNγ-mediated and immune cell–mediated killing [PMID:25284787, PMID:21829693, PMID:29728512, PMID:27786304, PMID:35379808, PMID:35688159]. Beyond linear chain assembly, HOIP ubiquitinates additional substrates—including ERα, p53, GPx4, YAP, A20, and NLRP3—to regulate their stability, and its own activity is modulated by caspase cleavage during apoptosis, Lys1056 ubiquitination, and interactions with the deubiquitinases OTULIN and CYLD–SPATA2, which counterbalance linear ubiquitin signaling [PMID:24726327, PMID:27545878, PMID:27669734, PMID:26578682, PMID:36279464, PMID:24441041].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Initial identification of RNF31 as an RBR-family E3 ligase associated with MuSK at neuromuscular junctions provided the first evidence that the gene encodes a ubiquitin ligase, though no catalytic mechanism or signaling function was established.\",\n      \"evidence\": \"Yeast two-hybrid/pull-down with MuSK cytoplasmic domain and immunolocalization at NMJs\",\n      \"pmids\": [\"14678832\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single interaction assay without reciprocal validation\", \"No substrate or chain-type specificity determined\", \"Functional role at NMJ undefined\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Discovery that RNF31 forms a corepressor complex with DAX-1 at steroidogenic gene promoters and stabilizes DAX-1 via monoubiquitination established its first characterized substrate relationship and linked it to transcriptional regulation.\",\n      \"evidence\": \"ChIP at StAR/CYP19 promoters, Co-IP, siRNA knockdown, gene expression analysis in steroidogenic cells\",\n      \"pmids\": [\"19237537\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether this represents LUBAC-dependent linear ubiquitination or an independent activity was unknown\", \"Chain type of monoubiquitination not defined as M1-linked\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Identification of HOIP at the CD40 signaling complex and demonstration that its ligase activity is required for CD40-mediated NF-κB activation established HOIP as an activating component of immune receptor signaling.\",\n      \"evidence\": \"Mass spectrometry of CD40 complexes, dominant-negative catalytic mutant, NF-κB reporter assay\",\n      \"pmids\": [\"20614026\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Chain type synthesized at CD40 complex not yet characterized\", \"Whether HOIP acts as part of LUBAC in this context was unclear\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"HOIP-deficient B cells confirmed the essential, non-redundant role of HOIP in CD40 signaling by showing complete loss of NF-κB and JNK activation, IKK recruitment failure, and defective B cell functional responses, all rescued by re-expression.\",\n      \"evidence\": \"Somatic gene targeting of HOIP in B cell lines, rescue by re-expression, multiple signaling readouts\",\n      \"pmids\": [\"21829693\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether HOIP is required for other TNFR superfamily members beyond CD40 was untested\", \"In vivo B cell immune response not examined\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Biochemical dissection revealed HOIP's unique catalytic mechanism—a RING/HECT-hybrid two-step reaction using a thioester intermediate at RING2 and a C-terminal LDD domain that specifies linear (Met1) chain linkage—resolving how LUBAC achieves chain-type specificity.\",\n      \"evidence\": \"In vitro ubiquitination assays with active-site and domain mutants, cellular NF-κB reporters\",\n      \"pmids\": [\"22863777\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for acceptor ubiquitin orientation not yet visualized\", \"How LUBAC accessory subunits regulate HOIP catalysis unknown\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Crystal structures of the HOIP catalytic core with and without ubiquitin revealed the structural basis for linear chain specificity: a zinc-finger and novel C-terminal fold create an acceptor ubiquitin-binding platform positioning the α-amino group for nucleophilic attack.\",\n      \"evidence\": \"X-ray crystallography (apo and ubiquitin-bound), functional mutagenesis in cells\",\n      \"pmids\": [\"24141947\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of full-length HOIP in activated state with E2~Ub not yet solved\", \"Donor ubiquitin engagement undefined structurally\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Multiple advances defined HOIP's regulatory network and in vivo essentiality: structural characterization of the HOIP PUB–OTULIN interaction revealed phospho-regulated deubiquitinase recruitment; HOIP deficiency caused TNFR1-dependent embryonic-lethal vascular defects; and HOIP was linked to ERα stabilization and cisplatin sensitivity, broadening its substrate repertoire and physiological roles.\",\n      \"evidence\": \"Crystal/NMR structures of PUB–OTULIN; conditional Hoip KO mice with TNFR1 epistasis; Co-IP/ubiquitination assays for ERα; siRNA screen for cisplatin sensitivity\",\n      \"pmids\": [\"24726327\", \"25284787\", \"24441041\", \"24686174\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How OTULIN and CYLD coordinate linear chain editing at receptor complexes unknown\", \"ERα ubiquitination chain type not determined as linear\", \"In vivo relevance of ERα stabilization unconfirmed\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"The crystal structure of fully active HOIP RBR bound to E2~ubiquitin revealed the complete catalytic transfer mechanism—an elongated conformation distinct from auto-inhibited RBRs with three ubiquitin-binding sites engaging donor, regulatory, and acceptor ubiquitins—while functional studies showed HOIP is required for T cell survival, is cleaved by caspases during apoptosis to limit linear ubiquitination, and is regulated by Lys1056 ubiquitination in a stimulus-specific manner.\",\n      \"evidence\": \"X-ray crystallography of HOIP/E2~Ub complex; T cell-specific catalytic-dead knock-in mice; caspase cleavage mapping with mutagenesis; K1056R mutagenesis with TLR4/CD40 signaling assays\",\n      \"pmids\": [\"26789245\", \"27786304\", \"27669734\", \"26578682\", \"27545878\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How auto-inhibition is relieved by HOIL-1L/SHARPIN in the full LUBAC complex not structurally resolved\", \"Relative contributions of OTULIN vs CYLD–SPATA2 in different signaling contexts unclear\", \"Whether caspase cleavage is a feedforward amplification loop in apoptosis not fully tested\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Extension of HOIP's receptor signaling roles to TNFR2 (requiring cIAP1 for recruitment) and skin-specific knockout showing TNFR1-rescuable lethal dermatitis established HOIP as a pan-TNFR family signaling node whose loss triggers tissue-specific inflammatory cell death.\",\n      \"evidence\": \"TNFR2 complex IP with M1-Ub antibodies and cIAP antagonist; epidermis-specific Hoip KO with TNFR1 epistasis\",\n      \"pmids\": [\"29378181\", \"29728512\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether HOIP engages all TNFR superfamily members or only specific subsets unknown\", \"Mechanism of cIAP1-dependent HOIP recruitment to TNFR2 not defined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Fragment-based covalent screening targeting the HOIP active-site cysteine yielded the first cell-permeable HOIP inhibitors, validated by co-crystal structures and cellular NF-κB suppression, opening pharmacological targeting of linear ubiquitination.\",\n      \"evidence\": \"Fragment screen, protein LC-MS, co-crystal structures, chemoproteomics, cell-based NF-κB assays\",\n      \"pmids\": [\"30657686\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Inhibitor selectivity over other RBR ligases not comprehensively profiled\", \"In vivo efficacy not demonstrated\", \"No therapeutic window established\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Chemically synthesized monoubiquitylated NEMO demonstrated that HOIP RBR alone can extend a pre-primed substrate, establishing a two-step model for NEMO linear ubiquitination where priming and extension have distinct LUBAC subunit requirements.\",\n      \"evidence\": \"Chemical ubiquitylation via ligation auxiliary, in vitro extension assay with recombinant HOIP RBR\",\n      \"pmids\": [\"31942456\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Identity of the priming E3 or priming mechanism in cells not established\", \"Whether the two-step model applies to substrates beyond NEMO unknown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Studies in dendritic cell-specific HOIP knockouts, plus identification of new substrates (A20, p53, PTEN), revealed cell-type-specific HOIP functions: DC autoinflammation is driven by MyD88/TLR rather than TNFR1, while HOIP-mediated degradation of A20, p53, and PTEN connects linear ubiquitination to inflammation, cell cycle, and PI3K-AKT signaling.\",\n      \"evidence\": \"DC-specific KO with MyD88/TNFR1 epistasis; Co-IP and ubiquitination/degradation assays for A20, p53, PTEN; mouse colitis and liver injury models\",\n      \"pmids\": [\"34253576\", \"34416243\", \"33824292\", \"34659546\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether A20 and p53 ubiquitination is linear or K48/K63-linked needs clarification\", \"PTEN interaction awaits independent validation\", \"How HOIP selects among diverse substrates is mechanistically unclear\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Multiple genome-wide CRISPR screens converged on RNF31 as a critical tumor immune-evasion gene: HOIP loss sensitizes cancer cells to TNF-mediated caspase-8-dependent apoptosis and immune cell killing, while new substrates GPx4 (ferroptosis protection), YAP (Hippo pathway), and NLRP3 (inflammasome) expanded HOIP's substrate repertoire.\",\n      \"evidence\": \"In vivo and in vitro CRISPR screens in pancreatic and colon cancer; pharmacological HOIP inhibition in organoids; Co-IP/ubiquitination for GPx4, YAP, NLRP3; ferroptosis and inflammasome assays\",\n      \"pmids\": [\"35379808\", \"35688159\", \"34467615\", \"36279464\", \"36581998\", \"37951199\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether GPx4, YAP, NLRP3 ubiquitination is linear or non-linear chain type needs definitive characterization\", \"Therapeutic index of HOIP inhibition for cancer immunotherapy not established\", \"Structural basis for substrate selection among diverse targets unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"HOIP was placed in the STING innate immune pathway: upon STING activation, LUBAC is recruited to LC3B-associated Golgi membranes where HOIP synthesizes M1-linked chains required for downstream NF-κB and IRF3 signaling, establishing a new receptor-proximal context for linear ubiquitination.\",\n      \"evidence\": \"HOIP KO in THP1 and BMDMs, M1-Ub IP, STING agonist stimulation, Golgi fractionation\",\n      \"pmids\": [\"39578541\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How LUBAC is recruited to STING-containing Golgi membranes mechanistically undefined\", \"Whether LC3B association is required for LUBAC activity at this site not tested\", \"Whether STING-dependent LUBAC recruitment occurs in non-myeloid cells unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include: how HOIP discriminates among its growing list of substrates for linear versus non-linear ubiquitination, the structural basis of full-length LUBAC auto-inhibition and activation by HOIL-1L and SHARPIN, and whether pharmacological HOIP inhibition can achieve a therapeutic window in vivo for cancer immunotherapy or inflammatory disease.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structure of full-length trimeric LUBAC in auto-inhibited or active state\", \"Chain-type specificity for many reported substrates (ERα, p53, PTEN, YAP) not rigorously determined\", \"In vivo pharmacology of HOIP inhibitors not reported\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 2, 6, 7, 10, 11, 15, 22, 24, 25, 28, 30]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 1, 2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [5, 13, 17]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [21]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 5, 8, 9, 13, 16, 17, 19, 20, 21]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [9, 19, 20, 21, 31]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [5, 10, 11, 14, 16, 18, 19]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 2, 30]}\n    ],\n    \"complexes\": [\n      \"LUBAC\"\n    ],\n    \"partners\": [\n      \"RBCK1\",\n      \"SHARPIN\",\n      \"OTULIN\",\n      \"SPATA2\",\n      \"CYLD\",\n      \"NEMO\",\n      \"A20\",\n      \"NLRP3\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}