{"gene":"RNF31","run_date":"2026-06-10T06:43:37","timeline":{"discoveries":[{"year":2012,"finding":"HOIP (RNF31) is the catalytically active subunit of LUBAC and generates linear (Met1-linked) ubiquitin chains via a two-step RBR mechanism: RING1-IBR catalyzes transfer of ubiquitin from E2 onto RING2 to form a transient HECT-like thioester intermediate, after which ubiquitin is transferred to the N-terminus of an acceptor ubiquitin. A unique C-terminal region termed the Linear ubiquitin chain Determining Domain (LDD) coordinates the acceptor ubiquitin and confers specificity for linear chain formation.","method":"In vitro ubiquitination assays, active-site mutagenesis, cellular NF-κB reporter assays","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution of catalytic mechanism plus mutagenesis plus cellular validation in a single rigorous study","pmids":["22863777"],"is_preprint":false},{"year":2013,"finding":"Crystal structure of the catalytic core of HOIP in apo form and in complex with ubiquitin revealed a novel C-terminal fold that, together with a zinc-finger, forms a ubiquitin-binding platform orienting the acceptor ubiquitin for nucleophilic attack on the E3~ubiquitin thioester. The structure captured both donor and acceptor ubiquitin simultaneously, explaining determinants of linear chain specificity. Mutations in these interfaces impair NF-κB pathway activation in vivo.","method":"X-ray crystallography (apo and ubiquitin-bound), mutagenesis, NF-κB activation assays","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with functional validation by mutagenesis and cellular assays in one rigorous study","pmids":["24141947"],"is_preprint":false},{"year":2016,"finding":"Crystal structure of fully active HOIP RBR in complex with an E2~ubiquitin conjugate revealed that active HOIP adopts a conformation markedly different from auto-inhibited RBRs, binds the E2~ubiquitin conjugate in an elongated fashion with catalytic centres aligned for HECT-like ubiquitin transfer, and contains three helix-IBR-fold motifs that bind both the activated (donor) ubiquitin and an additional regulatory ubiquitin molecule.","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 / Strong — high-resolution crystal structure of the active transfer complex, first of its kind for any RBR E3 ligase","pmids":["26789245"],"is_preprint":false},{"year":2014,"finding":"The HOIP PUB domain directly binds the PUB-interacting motif (PIM) of the deubiquitinase OTULIN; structural studies defined the binding interface with OTULIN Tyr56 making critical contacts. Phosphorylation of OTULIN Tyr56 negatively regulates this interaction. HOIP–OTULIN interaction is required for recruitment of OTULIN to the TNF receptor complex and to counteract HOIP-dependent NF-κB activation.","method":"Structural studies (X-ray/NMR), co-immunoprecipitation, phospho-mimetic mutagenesis, TNF receptor complex pull-down assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Moderate — structural determination of PUB–PIM interface combined with mutagenesis and functional receptor-complex recruitment assays in a single study","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. Recruitment of SPATA2 to TNFR1 and NOD2 signaling complexes is HOIP-dependent; loss of SPATA2 abolishes CYLD recruitment and reduces TNF-induced necroptosis.","method":"Co-immunoprecipitation, signaling complex pull-down, SPATA2 knockout cell lines, necroptosis assays","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus receptor-complex pull-down plus genetic KO with defined functional readout","pmids":["27545878"],"is_preprint":false},{"year":2014,"finding":"HOIP (RNF31) associates with estrogen receptor α (ERα) predominantly in the cytosol, increases ERα stability and mono-ubiquitination in an E3-ligase-activity-dependent manner, and is required for ERα-stimulated breast cancer cell proliferation and downstream target gene expression (cyclin D1, c-myc). RNF31 depletion reduces S-phase entry and ERα protein levels.","method":"Co-immunoprecipitation, RNF31 siRNA knockdown, cycloheximide-chase stability assay, cell-cycle analysis (FACS), luciferase reporter assays","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus stability chase plus KD with multiple cellular phenotype readouts in a single lab study","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 and CYP19, stabilizes DAX-1 (linked to DAX-1 mono-ubiquitination), and is required for DAX-1-mediated repression of steroidogenic gene transcription.","method":"Co-immunoprecipitation, chromatin immunoprecipitation (ChIP), RNF31 siRNA knockdown, reporter assays, ubiquitination assays","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP plus Co-IP plus KD with transcriptional readouts in a single lab study","pmids":["19237537"],"is_preprint":false},{"year":2011,"finding":"HOIP (RNF31) is recruited to the CD40 signaling complex in a TRAF2-dependent manner and is essential for CD40-mediated NF-κB activation; a ubiquitin-ligase-dead HOIP mutant inhibits NF-κB, and HOIP-deficient B cells fail to activate NF-κB, c-Jun kinase, or recruit IKK proteins to the CD40 complex.","method":"Somatic gene targeting (HOIP KO B cell lines), dominant-negative mutant overexpression, Co-IP/signaling complex analysis, NF-κB reporter and kinase assays","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with rescue plus dominant-negative mutagenesis plus complex pull-down","pmids":["21829693"],"is_preprint":false},{"year":2010,"finding":"HOIP/RNF31 is recruited to the CD40 signaling complex in a TRAF2-dependent manner (identified by mass spectrometry of immunoprecipitated CD40 complexes); a catalytically inactive HOIP mutant inhibits CD40-mediated NF-κB activation, indicating that HOIP ubiquitin-ligase activity is required for proximal CD40 signaling.","method":"Stimulation/immunoprecipitation of CD40 complexes + mass spectrometry, dominant-negative HOIP mutant, NF-κB reporter assay","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS-based complex identification plus mutagenesis plus reporter assay","pmids":["20614026"],"is_preprint":false},{"year":2014,"finding":"HOIP is required to prevent TNF-induced cell death in endothelial cells during embryogenesis; its catalytic activity is necessary for this protective function. HOIP-deficient cells show aberrant TNFR1 complex-II formation and are hypersensitive to TNF and LTα-induced death. TNFR1 deletion rescues embryonic lethality of HOIP-null mice.","method":"Constitutive and Tie2-Cre conditional HOIP knockout mice, apoptosis assays, TNFR1 genetic rescue (double-KO), signaling complex analysis","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple conditional KO models with genetic epistasis rescue (TNFR1 KO) and catalytic-activity requirement demonstrated","pmids":["25284787"],"is_preprint":false},{"year":2015,"finding":"A hypomorphic missense mutation (L72P) in the HOIP PUB domain impairs HOIP protein expression and destabilizes the entire LUBAC complex, causing defective linear ubiquitination and NF-κB activation in response to IL-1β and TNF in patient fibroblasts, and impaired B-cell activation in response to CD40 engagement.","method":"Patient primary cell analysis, western blot for LUBAC complex integrity, NF-κB activation assays, B-cell functional assays","journal":"The Journal of experimental medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — human loss-of-function variant with multiple orthogonal functional assays in primary patient cells","pmids":["26008899"],"is_preprint":false},{"year":2017,"finding":"HOIP is cleaved by caspases predominantly at Asp390 during apoptosis and is subsequently degraded by the proteasome. Effector caspases 3 and 6 cleave at aspartates 348, 387, and 390. The N-terminal cleavage fragment retains binding to OTULIN and CYLD-SPATA2; the C-terminal fragment retains NF-κB activity, but overall linear ubiquitination of NEMO and FADD (both identified as LUBAC substrates) decreases upon apoptosis. FADD is identified as a novel substrate for linear ubiquitination by LUBAC.","method":"Caspase cleavage assays, site-directed mutagenesis of cleavage sites, western blot, NF-κB reporter assays, linear ubiquitination assays for NEMO and FADD","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis of caspase sites plus biochemical ubiquitination assays for novel substrate FADD, single lab","pmids":["28189684"],"is_preprint":false},{"year":2016,"finding":"RNF31/HOIP is cleaved by caspases 3 and 6 at aspartates 348, 387, and 390 under apoptotic conditions, and this cleavage suppresses its ability to activate NF-κB signaling. Mutation of caspase cleavage sites inhibits TNF-α-induced apoptosis, establishing a regulatory loop between cell death and survival signaling.","method":"In vitro caspase cleavage assays, site-directed mutagenesis, apoptosis assays, NF-κB reporter assays","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical caspase assays with mutagenesis and functional NF-κB/apoptosis readouts","pmids":["27669734"],"is_preprint":false},{"year":2015,"finding":"Ubiquitination of HOIP at C-terminal lysine 1056 negatively regulates its catalytic activity by inducing a conformational change that suppresses linear-chain-forming activity. HOIP K1056R mutation leads to persistent LUBAC activity and prolonged NF-κB activation induced by TLR4/LPS stimulation but not by CD40 stimulation, indicating pathway-specific regulation.","method":"Site-directed mutagenesis (K1056R), in vitro ubiquitination assays, NF-κB reporter assays, TLR4 signaling experiments","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — identification of regulatory ubiquitination site by mutagenesis with functional pathway specificity demonstrated","pmids":["26578682"],"is_preprint":false},{"year":2018,"finding":"HOIP (as LUBAC component) is recruited to the TNFR2 signaling complex in a cIAP1-dependent manner; loss of cIAP1 (via cIAP antagonist) prevents HOIP recruitment and HOIP-mediated M1-ubiquitination at the TNFR2 complex. Both HOIP and cIAP1 are required for TNFR2-induced canonical NF-κB activation.","method":"TNFR2 signaling complex immunoprecipitation, cIAP antagonist treatment, M1/K63 ubiquitin linkage-specific antibody blotting, NF-κB activation assays","journal":"Biochemical pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — receptor-complex pull-down with pharmacological inhibition and linkage-specific ubiquitin detection","pmids":["29378181"],"is_preprint":false},{"year":2018,"finding":"Epidermis-specific knockout of RNF31 in mice causes early postnatal lethality with severe skin inflammation driven by TNF-α-induced apoptosis in keratinocytes. RNF31 deficiency impairs TNF-α-induced NF-κB activation and increases apoptosis. Genetic deletion of TNFR1 rescues lethality and skin inflammation in RNF31 epidermis-KO mice.","method":"Epidermis-specific conditional KO mice, genetic epistasis (TNFR1 KO rescue), apoptosis assays, NF-κB activation assays","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — tissue-specific genetic KO with TNFR1 genetic rescue epistasis and multiple mechanistic readouts","pmids":["29728512"],"is_preprint":false},{"year":2016,"finding":"In T cell-specific HOIP-knockin mice expressing a catalytically inactive HOIP (HOIPΔlinear), CD4+ and CD8+ T cell numbers are markedly reduced, NKT cell development is severely impaired, and mature T cells undergo accelerated apoptosis. HOIPΔlinear CD4+ T cells fail to phosphorylate IκBα and JNK through TCR stimulation. Reduced CD127 expression contributes to apoptosis; enforced CD127 expression rescues mature CD8+ T cell development.","method":"Conditional knock-in mice with catalytically inactive HOIP, flow cytometry, TCR signaling (phospho-IκBα, phospho-JNK), apoptosis assays, CD127 rescue experiment","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — catalytic knock-in mouse model with multiple orthogonal phenotypic and signaling readouts plus mechanistic rescue","pmids":["27786304"],"is_preprint":false},{"year":2022,"finding":"LUBAC/HOIP binds GPx4 and stabilizes it by modulating its linear ubiquitination, protecting cells against ferroptosis. LUBAC deficiency sensitizes cells to ferroptosis by promoting GPx4 degradation and downstream lipid peroxidation. GPx4 is identified as a direct substrate of HOIP-mediated linear ubiquitination.","method":"Co-immunoprecipitation, linear ubiquitination assays, lipid peroxidation assays, ferroptosis sensitivity assays in LUBAC-deficient cells","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ubiquitination assays plus cellular ferroptosis phenotype, single lab study","pmids":["36279464"],"is_preprint":false},{"year":2022,"finding":"RNF31 genetic or pharmacologic ablation sensitizes cancer cells to NK cell and CD8+ T cell killing in a TNF-dependent manner, 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 and enhances bystander killing of MHC-deficient tumor cells.","method":"Genome-wide CRISPR-Cas9 knockout screens under NK/CD8+ T cell pressure, pharmacological RNF31 inhibitor, TNF receptor complex analysis, tumor organoid killing assays","journal":"Cell reports. Medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — CRISPR screen identification validated by genetic and pharmacologic ablation with mechanistic complex analysis and organoid model","pmids":["35688159"],"is_preprint":false},{"year":2022,"finding":"Loss of Rnf31 in pancreatic cancer cells removes protection from TNF-mediated caspase-8 cleavage and subsequent apoptosis, sensitizing tumor cells to CD8+ T cell killing. Rnf31-deficient orthotopic pancreatic tumors show increased CD8+ T cell infiltration and effector function in vivo.","method":"In vitro and in vivo CRISPR screening, caspase-8 cleavage assays, orthotopic transplantation into immune-competent mice, T cell functional assays, human PDA organoids","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — CRISPR screen plus mechanistic apoptosis assays plus in vivo orthotopic model with immune readouts","pmids":["35379808"],"is_preprint":false},{"year":2021,"finding":"HOIP-deficient melanoma cells are hypersensitive to combined TNF and IFN-γ killing by NK and CD8+ T cells. Both genetic deletion and pharmacological inhibition of HOIP increase tumor cell sensitivity to TNF+IFN-γ co-stimulation, which engages both intrinsic and extrinsic apoptotic machinery in a transcription-dependent manner.","method":"CRISPR/Cas9 HOIP KO, pharmacological HOIP inhibition, NK/CD8+ T cell co-culture killing assays, cytokine treatment experiments, apoptosis pathway analysis","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO plus pharmacological inhibition with mechanistic cytokine synergy and apoptosis pathway defined, single lab","pmids":["34467615"],"is_preprint":false},{"year":2021,"finding":"RNF31 interacts with A20 via its RBR structural domain (co-immunoprecipitation), promotes K48-linked ubiquitination of A20 and its proteasomal degradation, thereby activating the TLR4/MyD88/NF-κB signaling pathway and aggravating hepatocyte apoptosis and inflammatory cytokine production.","method":"Co-immunoprecipitation, ubiquitination assays, proteasome inhibitor experiments (actinomycin chase + MG132), siRNA knockdown, in vivo LPS/d-Gal acute liver injury model","journal":"Chemico-biological interactions","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ubiquitination/degradation assays plus in vivo model, single lab","pmids":["34416243"],"is_preprint":false},{"year":2021,"finding":"RNF31/HOIP associates with PTEN via co-immunoprecipitation and promotes PTEN ubiquitination and proteasomal degradation in cancer cells. HOIP depletion causes cell cycle arrest and apoptosis that can be rescued by PTEN silencing, implicating a HOIP–PTEN–PI3K/AKT axis in chemotherapy resistance.","method":"Co-immunoprecipitation, ubiquitination assays, PTEN knockdown rescue, cell cycle analysis, apoptosis assays","journal":"Journal of Cancer","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP plus ubiquitination assay plus KD rescue, replicated across multiple cancer cell lines but single lab","pmids":["34659546"],"is_preprint":false},{"year":2021,"finding":"RNF31 interacts with NLRP3 via its RBR domain and promotes K63-linked ubiquitination of NLRP3, stabilizing the NLRP3 inflammasome complex and enhancing IL-1β and IL-18 production. In vivo RNF31 knockdown attenuates DSS-induced colitis and reduces NLRP3 expression.","method":"Co-immunoprecipitation, ubiquitination assays (K63-linkage specific), RNF31 KD cell model, DSS-induced colitis mouse model","journal":"International immunopharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus linkage-specific ubiquitination assays plus in vivo model, single lab","pmids":["37951199"],"is_preprint":false},{"year":2022,"finding":"RBCK1 (HOIL-1L) interacts with RNF31 and represses RNF31 ubiquitination and proteasomal degradation, thereby stabilizing RNF31 protein in hepatocellular carcinoma cells.","method":"Co-immunoprecipitation, ubiquitination assays, proteasome inhibitor experiments, RNF31/RBCK1 KD cell lines","journal":"Cell death discovery","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP plus ubiquitination/stability assays, single lab","pmids":["35869046"],"is_preprint":false},{"year":2022,"finding":"RNF31 depletion in TNBC increases YAP protein levels; RNF31 associates with YAP, facilitates YAP poly-ubiquitination and proteasomal degradation specifically at YAP K76, and suppresses Hippo/YAP/PD-L1 axis, thereby repressing TNBC cell proliferation, migration, and immune checkpoint expression.","method":"Co-immunoprecipitation, ubiquitination assays, site-directed mutagenesis (YAP K76), xenograft models, genome-wide expression profiling, siRNA KD","journal":"Journal of experimental & clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus site-specific mutagenesis plus in vivo xenograft, single lab","pmids":["36581998"],"is_preprint":false},{"year":2021,"finding":"RNF31 interacts with p53 via its PUB domain (demonstrated by truncation Co-IP assays), promotes p53 ubiquitination and proteasomal degradation, and the PUB domain is the key structural determinant for p53 ubiquitination. RNF31 depletion stabilizes p53 and inhibits CRC cell growth.","method":"Co-immunoprecipitation with truncation constructs, cycloheximide-chase assay, MG132 proteasome inhibition, ubiquitination assays","journal":"Cell death discovery","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — domain-mapping Co-IP plus stability assays plus ubiquitination, single lab","pmids":["33824292"],"is_preprint":false},{"year":2024,"finding":"RNF31 enhances p53 ubiquitination and proteasomal degradation; reduced RNF31 leads to p53 accumulation, which represses BNIP3 expression, impairing mitophagy and promoting steatosis in hepatocytes. RNF31 delivery via mesenchymal stem cell-derived small extracellular vesicles reduces hepatic steatosis in HFD-fed mice.","method":"Co-immunoprecipitation, ubiquitination assays, mt-Keima fluorescence mitophagy assay, RNA-seq, HFD mouse model, sEV delivery experiments","journal":"Free radical biology & medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus mechanistic mitophagy imaging plus in vivo model, single lab","pmids":["38615890"],"is_preprint":false},{"year":2024,"finding":"STING activation induces recruitment of HOIP (LUBAC) to LC3B-associated Golgi membranes and synthesis of M1-linked (linear) ubiquitin chains. Loss of HOIP prevents M1-Ub chain formation and reduces STING-induced NF-κB and IRF3 signaling in human THP1 monocytes and mouse bone marrow-derived macrophages, without affecting upstream STING activation itself.","method":"HOIP KO in THP1 monocytes and BMDMs, M1-Ub chain detection, STING pathway signaling assays (IRF3/NF-κB), LC3B/Golgi co-localization imaging","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO in two cell types with signaling assays and localization imaging, single lab","pmids":["39578541"],"is_preprint":false},{"year":2019,"finding":"Fragment-based covalent ligand screening targeting the active-site cysteine of HOIP yielded the first structure-based covalent inhibitors for an RBR E3 ligase. Crystal structures of HOIP-inhibitor complexes were solved; cell-based and chemoproteomic assays confirmed cell-penetrant HOIP labeling and inhibition of NF-κB activation.","method":"Fragment covalent screening, protein LC-MS ubiquitination assays, protein crystallography, cell-based NF-κB reporter assays, chemoproteomics","journal":"Journal of the American Chemical Society","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structures plus in vitro biochemical assays plus cell-based functional validation in one integrated study","pmids":["30657686"],"is_preprint":false},{"year":2014,"finding":"HOIP (RNF31) is identified by siRNA screen as a key regulator of cisplatin-induced genotoxicity; HOIP-deficient cells exhibit hypersensitivity to cisplatin via increased caspase-8/caspase-3-dependent apoptosis that requires ATM (not ATR) checkpoint activation. JNK activity is enhanced in HOIP-depleted cells and JNK inhibition reverses apoptotic hyperactivation.","method":"siRNA library screen, caspase activity assays, ATM/ATR pathway inhibitors, JNK inhibition rescue, cisplatin-resistance assays","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA screen hit validated with multiple mechanistic pathway inhibitor epistasis experiments","pmids":["24686174"],"is_preprint":false},{"year":2021,"finding":"DC-specific deletion of HOIP causes spontaneous inflammation; HOIP deficiency in DCs does not affect TNF-α-induced NF-κB activation but enhances TNF-α-induced apoptosis and necroptosis. Crossing with TNFR1-KO does not rescue inflammation, whereas antibiotic treatment reduces it. MyD88 deficiency rescues the inflammatory phenotype in HOIP-deficient DC mice, establishing MyD88-dependent (TLR) signaling—not TNFR1 signaling—as the driver of autoinflammation.","method":"DC-specific conditional HOIP KO mice, genetic epistasis (TNFR1-KO, MyD88-KO crosses), antibiotic treatment, apoptosis/necroptosis assays, cytokine measurement","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple genetic epistasis crosses with clean mechanistic separation of TNFR1 vs. MyD88 pathways","pmids":["34253576"],"is_preprint":false},{"year":2019,"finding":"Chemical ubiquitylation of NEMO using a ligation auxiliary demonstrated that monoubiquitylated NEMO retains similar affinity for linear diubiquitin chains as unmodified NEMO. The proximal ubiquitin of chemically synthesized NEMO-Ub is accepted as substrate for linear extension by the HOIP RBR domain alone, indicating NEMO linear ubiquitylation involves a two-step mechanism: an initial priming event (requiring factors beyond HOIP alone) and a separate HOIP RBR-mediated extension step.","method":"Chemical ubiquitylation (ligation auxiliary), in vitro linear extension assays with HOIP RBR domain, ITC binding affinity measurements","journal":"Communications chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — reconstitution-based in vitro chemistry plus binding assays, single lab study","pmids":["31942456"],"is_preprint":false},{"year":2025,"finding":"RNF31 interacts with and ubiquitinates ALYREF (RNA export factor), facilitating ALYREF nuclear transport via importin 13 (IPO13) under paclitaxel treatment; nuclear ALYREF then mediates export of mRNAs encoding paclitaxel-resistance factors (TUBB3, STMN1, TAU), inducing resistance. RNF31 inhibition traps ALYREF in the cytoplasm and re-sensitizes resistant TNBC cells to paclitaxel.","method":"Co-immunoprecipitation, ubiquitination assays, immunofluorescence (ALYREF subcellular localization), mRNA export assays, siRNA KD, RNF31 inhibitor + paclitaxel in vivo and organoid models","journal":"Clinical and translational medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ubiquitination plus localization imaging plus in vivo/organoid functional validation, single lab","pmids":["39915011"],"is_preprint":false},{"year":2004,"finding":"RNF31/PAUL (identified as a putative Ariadne-like RBR E3 ubiquitin ligase) binds the cytoplasmic domain of the muscle-specific receptor tyrosine kinase MuSK and is expressed at neuromuscular junctions, suggesting a role in postsynaptic membrane formation.","method":"Yeast two-hybrid and binding assay identifying PAUL as MuSK-interacting protein, in situ hybridization and immunostaining for NMJ localization","journal":"Gene expression patterns","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single binding assay plus localization, no functional validation of the interaction","pmids":["14678832"],"is_preprint":false}],"current_model":"RNF31 (HOIP) is the catalytic RING-IBR-RING (RBR) E3 ubiquitin ligase subunit of the trimeric LUBAC complex (with HOIL-1L/RBCK1 and SHARPIN) that uniquely generates Met1-linked (linear) ubiquitin chains via a RING/HECT-hybrid mechanism: RING1-IBR accepts ubiquitin from E2 to form a HECT-like thioester on RING2's active-site cysteine, then the Linear ubiquitin chain Determining Domain (LDD) orients the acceptor ubiquitin's α-amino group for nucleophilic transfer; structural studies have captured apo, ubiquitin-bound, and E2~ubiquitin transfer-complex states. HOIP linearly ubiquitinates key NF-κB signaling substrates (NEMO, FADD) and also attaches non-canonical ubiquitin marks to other substrates (ERα mono-ubiquitination, GPx4 stabilization, YAP and PTEN degradation, A20 degradation, NLRP3 K63 stabilization, ALYREF nuclear transport), thereby controlling NF-κB-dependent survival signaling downstream of TNFR1, TNFR2, CD40, TLR4, NOD2, and STING; protecting endothelial cells, keratinocytes, T cells, and dendritic cells from TNF- or cytokine-induced apoptosis/necroptosis; and regulating steroidogenesis, ferroptosis, and immune evasion. HOIP activity is itself regulated by OTULIN (PUB-domain interaction, negatively controlled by OTULIN Tyr56 phosphorylation), CYLD (bridged via SPATA2), caspase-mediated cleavage at Asp348/387/390 during apoptosis, and auto-ubiquitination at Lys1056 that causes conformational inhibition specifically in TLR4 signaling."},"narrative":{"mechanistic_narrative":"RNF31 (HOIP) is the catalytically active RING-IBR-RING (RBR) E3 ubiquitin ligase subunit of the LUBAC complex and is the central node generating linear (Met1-linked) ubiquitin chains that drive NF-κB-dependent survival signaling [PMID:22863777]. It builds linear chains through a two-step RBR mechanism in which ubiquitin is transferred from an E2 onto the RING2 active-site cysteine to form a HECT-like thioester, after which a unique C-terminal Linear ubiquitin chain Determining Domain (LDD) orients the acceptor ubiquitin for nucleophilic attack on its N-terminus [PMID:22863777]; structural studies of the apo, ubiquitin-bound, and E2~ubiquitin transfer-complex states defined how donor and acceptor ubiquitin are simultaneously coordinated and how active HOIP departs from the auto-inhibited RBR conformation to align catalytic centers [PMID:24141947, PMID:26789245]. Through linear ubiquitination of substrates such as NEMO and FADD, HOIP controls NF-κB activation downstream of multiple receptors including CD40, TNFR2, and STING [PMID:28189684, PMID:21829693, PMID:29378181, PMID:39578541], and its activity is essential to protect cells from cytokine-induced death: catalytic-dead or tissue-specific HOIP loss sensitizes endothelial cells, keratinocytes, and T cells to TNF-driven apoptosis/necroptosis, phenotypes rescued by TNFR1 deletion [PMID:25284787, PMID:29728512, PMID:27786304]. HOIP activity is itself tuned by deubiquitinase recruitment via direct PUB-domain binding to OTULIN [PMID:24726327] and SPATA2-bridged engagement of CYLD [PMID:27545878], by caspase cleavage at Asp348/387/390 during apoptosis [PMID:28189684, PMID:27669734], and by auto-ubiquitination at Lys1056 that confers TLR4-specific conformational inhibition [PMID:26578682]. Beyond canonical LUBAC substrates, HOIP attaches non-degradative and degradative ubiquitin marks to a range of targets, stabilizing GPx4 to suppress ferroptosis [PMID:36279464] and NLRP3 via K63 chains [PMID:37951199] while driving K48-linked degradation of A20, PTEN, YAP, and p53 [PMID:34416243, PMID:34659546, PMID:36581998, PMID:33824292]. Loss or pharmacologic inhibition of RNF31 sensitizes tumor cells to TNF-dependent killing by NK and CD8+ T cells, establishing it as a target for immune-mediated cancer therapy [PMID:35688159, PMID:35379808, PMID:34467615]. A hypomorphic PUB-domain mutation (L72P) destabilizes LUBAC and causes defective linear ubiquitination and immunodeficiency in patients [PMID:26008899].","teleology":[{"year":2004,"claim":"An initial question was whether RNF31 had any defined molecular partner; identifying it as an Ariadne-like RBR protein binding the MuSK cytoplasmic domain gave the first functional context, though unrelated to its later-established LUBAC role.","evidence":"Yeast two-hybrid/binding assay and NMJ localization by in situ hybridization and immunostaining","pmids":["14678832"],"confidence":"Low","gaps":["Single binding assay with no functional validation of the MuSK interaction","No connection drawn to ubiquitin ligase activity or NF-κB signaling","Not reconciled with the canonical LUBAC function"]},{"year":2009,"claim":"Before its NF-κB role was defined, RNF31 was found to act as a transcriptional corepressor, establishing an early link between RNF31 and ubiquitin-dependent stabilization of a partner protein.","evidence":"Co-IP, ChIP, siRNA knockdown and reporter/ubiquitination assays on steroidogenic gene promoters with DAX-1","pmids":["19237537"],"confidence":"Medium","gaps":["Ubiquitin linkage type on DAX-1 not defined","Mechanism connecting stabilization to repression unresolved","Relationship to LUBAC complex unaddressed"]},{"year":2011,"claim":"To determine which receptors require HOIP, studies showed it is recruited TRAF2-dependently to the CD40 complex and is essential for CD40-driven NF-κB, establishing HOIP catalytic activity as required for proximal receptor signaling.","evidence":"Mass spectrometry of CD40 complexes, HOIP KO B-cell lines, dominant-negative catalytic mutant, NF-κB/kinase assays","pmids":["20614026","21829693"],"confidence":"Medium","gaps":["Direct CD40-complex substrate of HOIP not identified","Linear chain linkage at CD40 complex not directly demonstrated"]},{"year":2012,"claim":"The defining mechanistic question — how HOIP generates linear chains — was answered by showing a two-step RBR mechanism with a HECT-like thioester intermediate and an LDD that confers linear specificity.","evidence":"In vitro ubiquitination assays, active-site mutagenesis, cellular NF-κB reporter assays","pmids":["22863777"],"confidence":"High","gaps":["Atomic basis of acceptor orientation not yet visualized","Priming versus extension steps not distinguished"]},{"year":2016,"claim":"Structural studies progressively explained linear chain specificity and catalysis, capturing donor- and acceptor-ubiquitin coordination and the active transfer conformation distinct from auto-inhibited RBRs.","evidence":"X-ray crystallography of apo, ubiquitin-bound, and HOIP RBR/E2~ubiquitin transfer complexes with mutagenesis","pmids":["24141947","26789245"],"confidence":"High","gaps":["Full-length LUBAC architecture not resolved","Conformational transition kinetics not measured"]},{"year":2016,"claim":"How HOIP activity is restrained was addressed by defining deubiquitinase recruitment: direct PUB-domain binding to OTULIN (regulated by OTULIN Tyr56 phosphorylation) and SPATA2-bridged CYLD recruitment.","evidence":"Structural determination of the PUB-PIM interface, phospho-mimetic mutagenesis, reciprocal Co-IP, receptor-complex pull-down, SPATA2 KO necroptosis assays","pmids":["24726327","27545878"],"confidence":"High","gaps":["Quantitative balance between OTULIN and CYLD restraint unresolved","How phosphorylation is triggered in vivo not defined"]},{"year":2018,"claim":"Genetic models established the physiological imperative for HOIP catalytic activity in preventing cytokine-induced death across endothelium, epidermis, and T cells, with TNFR1 epistasis defining the death ligand.","evidence":"Constitutive, Tie2-Cre, epidermis-specific conditional KO and catalytic knock-in mice with TNFR1-KO genetic rescue and CD127 rescue","pmids":["25284787","29728512","27786304"],"confidence":"High","gaps":["The specific substrate whose linear ubiquitination prevents complex-II death not fully resolved per tissue","Necroptosis versus apoptosis contributions vary by context"]},{"year":2018,"claim":"Receptor scope was extended by showing cIAP1-dependent HOIP recruitment to the TNFR2 complex, broadening the set of NF-κB pathways routed through linear ubiquitination.","evidence":"TNFR2 complex IP, cIAP antagonist treatment, M1/K63 linkage-specific blotting, NF-κB assays","pmids":["29378181"],"confidence":"Medium","gaps":["TNFR2-complex substrate of M1 chains not identified","Single study"]},{"year":2017,"claim":"How HOIP is switched off during apoptosis and which pathway-specific regulation it undergoes were addressed by mapping caspase cleavage sites, the K1056 auto-ubiquitination switch, and identifying FADD as a linear ubiquitination substrate.","evidence":"Caspase cleavage and site mutagenesis, K1056R mutagenesis, in vitro ubiquitination and NF-κB/apoptosis assays, NEMO/FADD linear ubiquitination assays","pmids":["28189684","27669734","26578682"],"confidence":"Medium","gaps":["Physiological trigger of K1056 auto-ubiquitination in TLR4 signaling not defined","Fate of cleavage fragments in vivo unclear"]},{"year":2020,"claim":"Reconstitution chemistry refined the catalytic model by separating NEMO linear ubiquitylation into a priming event requiring factors beyond HOIP and a HOIP RBR-mediated extension step.","evidence":"Chemical ubiquitylation of NEMO, in vitro extension with HOIP RBR domain, ITC binding measurements","pmids":["31942456"],"confidence":"Medium","gaps":["Identity of the priming factor not established","Single-lab in vitro reconstitution"]},{"year":2024,"claim":"The repertoire of HOIP substrates and pathways was expanded substantially, revealing both linear (GPx4, STING-associated) and non-canonical chain functions (NLRP3 K63 stabilization; K48 degradation of A20, PTEN, YAP, p53; ERα/DAX-1 stabilization; ALYREF nuclear transport).","evidence":"Co-IP, linkage-specific and site-directed ubiquitination assays, knockdown/inhibitor rescue, in vivo disease and organoid models across ferroptosis, inflammasome, liver injury, mitophagy and chemoresistance","pmids":["36279464","37951199","34416243","34659546","36581998","33824292","38615890","24441041","39915011","39578541"],"confidence":"Medium","gaps":["Most non-canonical substrates rest on single-lab Co-IP plus ubiquitination assays","Chain linkage and direct-versus-indirect ubiquitination not uniformly confirmed","Reconciliation of degradative versus stabilizing roles per substrate incomplete"]},{"year":2022,"claim":"The translational question of whether HOIP can be exploited therapeutically was answered by structure-based covalent inhibitors and by CRISPR/pharmacologic studies showing RNF31 ablation sensitizes tumors to TNF-dependent immune killing.","evidence":"Fragment-based covalent inhibitor crystallography and chemoproteomics; genome-wide CRISPR screens under NK/CD8+ T cell pressure, inhibitor treatment, organoid and orthotopic tumor models","pmids":["30657686","35688159","35379808","34467615"],"confidence":"High","gaps":["In vivo selectivity and off-target effects of inhibitors not fully defined","Patient-level efficacy untested in the corpus"]},{"year":null,"claim":"It remains unresolved how HOIP selects among its many reported substrates and switches between generating linear, K48, and K63 linkages on different targets, and which priming factors and contextual cues dictate these outcomes in vivo.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying mechanism explains linkage-type switching across substrates","NEMO priming factor unidentified","Many non-canonical substrate claims await independent validation"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,1,2]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,17,21,25]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[33]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[5]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[28]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[6,33]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,7,14,28]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[18,19,20,31]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[9,15,16,17]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,21,22,25,26]}],"complexes":["LUBAC"],"partners":["RBCK1","OTULIN","SPATA2","CYLD","TRAF2","A20","NLRP3","YAP"],"other_free_text":[]}},"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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HOIP.","date":"2013","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/24141947","citation_count":179,"is_preprint":false},{"pmid":"24726327","id":"PMC_24726327","title":"Binding of OTULIN to the PUB domain of HOIP controls NF-κB signaling.","date":"2014","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/24726327","citation_count":153,"is_preprint":false},{"pmid":"26789245","id":"PMC_26789245","title":"Structure of a HOIP/E2~ubiquitin complex reveals RBR E3 ligase mechanism and regulation.","date":"2016","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/26789245","citation_count":152,"is_preprint":false},{"pmid":"16168833","id":"PMC_16168833","title":"Results of spot-scanning proton radiation therapy for chordoma and chondrosarcoma of the skull base: the Paul Scherrer Institut experience.","date":"2005","source":"International journal of radiation oncology, biology, 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Screening Enables Rapid Discovery of Inhibitors for the RBR E3 Ubiquitin Ligase HOIP.","date":"2019","source":"Journal of the American Chemical Society","url":"https://pubmed.ncbi.nlm.nih.gov/30657686","citation_count":98,"is_preprint":false},{"pmid":"24441041","id":"PMC_24441041","title":"The atypical ubiquitin ligase RNF31 stabilizes estrogen receptor α and modulates estrogen-stimulated breast cancer cell proliferation.","date":"2014","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/24441041","citation_count":82,"is_preprint":false},{"pmid":"30936877","id":"PMC_30936877","title":"Second Case of HOIP Deficiency Expands Clinical Features and Defines Inflammatory Transcriptome Regulated by LUBAC.","date":"2019","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/30936877","citation_count":65,"is_preprint":false},{"pmid":"24686174","id":"PMC_24686174","title":"E3 ubiquitin ligase HOIP attenuates apoptotic cell death induced by cisplatin.","date":"2014","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/24686174","citation_count":59,"is_preprint":false},{"pmid":"23582853","id":"PMC_23582853","title":"Spot-scanning proton radiation therapy for pediatric chordoma and chondrosarcoma: clinical outcome of 26 patients treated at paul scherrer institute.","date":"2013","source":"International journal of radiation oncology, biology, physics","url":"https://pubmed.ncbi.nlm.nih.gov/23582853","citation_count":59,"is_preprint":false},{"pmid":"29755466","id":"PMC_29755466","title":"Controlling Mast Cell Activation and Homeostasis: Work Influenced by Bill Paul That Continues Today.","date":"2018","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/29755466","citation_count":58,"is_preprint":false},{"pmid":"9130474","id":"PMC_9130474","title":"The Paul Kallos Memorial Lecture. The mast cell: a versatile effector cell for a challenging world.","date":"1997","source":"International archives of allergy and immunology","url":"https://pubmed.ncbi.nlm.nih.gov/9130474","citation_count":55,"is_preprint":false},{"pmid":"27756009","id":"PMC_27756009","title":"Magnetic nanoparticle-induced hyperthermia with appropriate payloads: Paul Ehrlich's \"magic (nano)bullet\" for cancer theranostics?","date":"2016","source":"Cancer treatment reviews","url":"https://pubmed.ncbi.nlm.nih.gov/27756009","citation_count":52,"is_preprint":false},{"pmid":"36279464","id":"PMC_36279464","title":"HOIP modulates the stability of GPx4 by linear ubiquitination.","date":"2022","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/36279464","citation_count":44,"is_preprint":false},{"pmid":"35688159","id":"PMC_35688159","title":"RNF31 inhibition sensitizes tumors to bystander killing by innate and adaptive immune cells.","date":"2022","source":"Cell reports. 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Fragments of a development, with special reference to the influence of Paul Ehrlich.","date":"1989","source":"Danish medical bulletin","url":"https://pubmed.ncbi.nlm.nih.gov/2651032","citation_count":38,"is_preprint":false},{"pmid":"27726791","id":"PMC_27726791","title":"Paul Ehrlich and the Early History of Granulocytes.","date":"2016","source":"Microbiology spectrum","url":"https://pubmed.ncbi.nlm.nih.gov/27726791","citation_count":36,"is_preprint":false},{"pmid":"29378181","id":"PMC_29378181","title":"The E3 ubiquitin ligases HOIP and cIAP1 are recruited to the TNFR2 signaling complex and mediate TNFR2-induced canonical NF-κB signaling.","date":"2018","source":"Biochemical pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/29378181","citation_count":35,"is_preprint":false},{"pmid":"18333255","id":"PMC_18333255","title":"Outcome following hepatic resection of metastatic renal tumors: the Paul Brousse Hospital experience.","date":"2006","source":"HPB : the official journal of the International Hepato Pancreato Biliary Association","url":"https://pubmed.ncbi.nlm.nih.gov/18333255","citation_count":34,"is_preprint":false},{"pmid":"28189684","id":"PMC_28189684","title":"Decreased linear ubiquitination of NEMO and FADD on apoptosis with caspase-mediated cleavage of HOIP.","date":"2017","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/28189684","citation_count":32,"is_preprint":false},{"pmid":"26231797","id":"PMC_26231797","title":"miR-503 suppresses tumor cell proliferation and metastasis by directly targeting RNF31 in prostate cancer.","date":"2015","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/26231797","citation_count":31,"is_preprint":false},{"pmid":"21829693","id":"PMC_21829693","title":"HOIL-1L interacting protein (HOIP) is essential for CD40 signaling.","date":"2011","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/21829693","citation_count":31,"is_preprint":false},{"pmid":"6333455","id":"PMC_6333455","title":"Aberrant expression of Forssman and Paul-Bunnell antigens on lymph node cells of MRL/Mp-lpr/lpr mice.","date":"1984","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/6333455","citation_count":30,"is_preprint":false},{"pmid":"23601931","id":"PMC_23601931","title":"Receptor-mediated endocytosis for drug delivery in African trypanosomes: fulfilling Paul Ehrlich's vision of chemotherapy.","date":"2013","source":"Trends in parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/23601931","citation_count":30,"is_preprint":false},{"pmid":"27460922","id":"PMC_27460922","title":"Atypical ubiquitin ligase RNF31: the nuclear factor modulator in breast cancer progression.","date":"2016","source":"BMC cancer","url":"https://pubmed.ncbi.nlm.nih.gov/27460922","citation_count":29,"is_preprint":false},{"pmid":"34467615","id":"PMC_34467615","title":"HOIP limits anti-tumor immunity by protecting against combined TNF and IFN-gamma-induced apoptosis.","date":"2021","source":"EMBO reports","url":"https://pubmed.ncbi.nlm.nih.gov/34467615","citation_count":29,"is_preprint":false},{"pmid":"20211283","id":"PMC_20211283","title":"The endogenous peptides of normal human serum extracted from the acetonitrile-insoluble precipitate using modified aqueous buffer with analysis by LC-ESI-Paul ion trap and Qq-TOF.","date":"2010","source":"Journal of proteomics","url":"https://pubmed.ncbi.nlm.nih.gov/20211283","citation_count":29,"is_preprint":false},{"pmid":"22427816","id":"PMC_22427816","title":"Knockdown of SF-1 and RNF31 affects components of steroidogenesis, TGFβ, and Wnt/β-catenin signaling in adrenocortical carcinoma cells.","date":"2012","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/22427816","citation_count":28,"is_preprint":false},{"pmid":"20614026","id":"PMC_20614026","title":"HOIL-1L interacting protein (HOIP) as an NF-kappaB regulating component of the CD40 signaling complex.","date":"2010","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/20614026","citation_count":26,"is_preprint":false},{"pmid":"9138526","id":"PMC_9138526","title":"Paul Ehrlich: pathfinder in cell biology. 1. 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ubiquitin chains via a two-step RBR mechanism: RING1-IBR catalyzes transfer of ubiquitin from E2 onto RING2 to form a transient HECT-like thioester intermediate, after which ubiquitin is transferred to the N-terminus of an acceptor ubiquitin. A unique C-terminal region termed the Linear ubiquitin chain Determining Domain (LDD) coordinates the acceptor ubiquitin and confers specificity for linear chain formation.\",\n      \"method\": \"In vitro ubiquitination assays, active-site mutagenesis, cellular NF-κB reporter assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution of catalytic mechanism plus mutagenesis plus cellular validation in a single rigorous study\",\n      \"pmids\": [\"22863777\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Crystal structure of the catalytic core of HOIP in apo form and in complex with ubiquitin revealed a novel C-terminal fold that, together with a zinc-finger, forms a ubiquitin-binding platform orienting the acceptor ubiquitin for nucleophilic attack on the E3~ubiquitin thioester. The structure captured both donor and acceptor ubiquitin simultaneously, explaining determinants of linear chain specificity. Mutations in these interfaces impair NF-κB pathway activation in vivo.\",\n      \"method\": \"X-ray crystallography (apo and ubiquitin-bound), mutagenesis, NF-κB activation assays\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with functional validation by mutagenesis and cellular assays in one rigorous study\",\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 revealed that active HOIP adopts a conformation markedly different from auto-inhibited RBRs, binds the E2~ubiquitin conjugate in an elongated fashion with catalytic centres aligned for HECT-like ubiquitin transfer, and contains three helix-IBR-fold motifs that bind both the activated (donor) ubiquitin and an additional regulatory ubiquitin molecule.\",\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 / Strong — high-resolution crystal structure of the active transfer complex, first of its kind for any RBR E3 ligase\",\n      \"pmids\": [\"26789245\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The HOIP PUB domain directly binds the PUB-interacting motif (PIM) of the deubiquitinase OTULIN; structural studies defined the binding interface with OTULIN Tyr56 making critical contacts. Phosphorylation of OTULIN Tyr56 negatively regulates this interaction. HOIP–OTULIN interaction is required for recruitment of OTULIN to the TNF receptor complex and to counteract HOIP-dependent NF-κB activation.\",\n      \"method\": \"Structural studies (X-ray/NMR), co-immunoprecipitation, phospho-mimetic mutagenesis, TNF receptor complex pull-down assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — structural determination of PUB–PIM interface combined with mutagenesis and functional receptor-complex recruitment assays in a single study\",\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. Recruitment of SPATA2 to TNFR1 and NOD2 signaling complexes is HOIP-dependent; loss of SPATA2 abolishes CYLD recruitment and reduces TNF-induced necroptosis.\",\n      \"method\": \"Co-immunoprecipitation, signaling complex pull-down, SPATA2 knockout cell lines, necroptosis assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus receptor-complex pull-down plus genetic KO with defined functional readout\",\n      \"pmids\": [\"27545878\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"HOIP (RNF31) associates with estrogen receptor α (ERα) predominantly in the cytosol, increases ERα stability and mono-ubiquitination in an E3-ligase-activity-dependent manner, and is required for ERα-stimulated breast cancer cell proliferation and downstream target gene expression (cyclin D1, c-myc). RNF31 depletion reduces S-phase entry and ERα protein levels.\",\n      \"method\": \"Co-immunoprecipitation, RNF31 siRNA knockdown, cycloheximide-chase stability assay, cell-cycle analysis (FACS), luciferase reporter assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus stability chase plus KD with multiple cellular phenotype readouts in a single lab study\",\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 and CYP19, stabilizes DAX-1 (linked to DAX-1 mono-ubiquitination), and is required for DAX-1-mediated repression of steroidogenic gene transcription.\",\n      \"method\": \"Co-immunoprecipitation, chromatin immunoprecipitation (ChIP), RNF31 siRNA knockdown, reporter assays, ubiquitination assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP plus Co-IP plus KD with transcriptional readouts in a single lab study\",\n      \"pmids\": [\"19237537\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"HOIP (RNF31) is recruited to the CD40 signaling complex in a TRAF2-dependent manner and is essential for CD40-mediated NF-κB activation; a ubiquitin-ligase-dead HOIP mutant inhibits NF-κB, and HOIP-deficient B cells fail to activate NF-κB, c-Jun kinase, or recruit IKK proteins to the CD40 complex.\",\n      \"method\": \"Somatic gene targeting (HOIP KO B cell lines), dominant-negative mutant overexpression, Co-IP/signaling complex analysis, NF-κB reporter and kinase assays\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with rescue plus dominant-negative mutagenesis plus complex pull-down\",\n      \"pmids\": [\"21829693\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"HOIP/RNF31 is recruited to the CD40 signaling complex in a TRAF2-dependent manner (identified by mass spectrometry of immunoprecipitated CD40 complexes); a catalytically inactive HOIP mutant inhibits CD40-mediated NF-κB activation, indicating that HOIP ubiquitin-ligase activity is required for proximal CD40 signaling.\",\n      \"method\": \"Stimulation/immunoprecipitation of CD40 complexes + mass spectrometry, dominant-negative HOIP mutant, NF-κB reporter assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS-based complex identification plus mutagenesis plus reporter assay\",\n      \"pmids\": [\"20614026\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"HOIP is required to prevent TNF-induced cell death in endothelial cells during embryogenesis; its catalytic activity is necessary for this protective function. HOIP-deficient cells show aberrant TNFR1 complex-II formation and are hypersensitive to TNF and LTα-induced death. TNFR1 deletion rescues embryonic lethality of HOIP-null mice.\",\n      \"method\": \"Constitutive and Tie2-Cre conditional HOIP knockout mice, apoptosis assays, TNFR1 genetic rescue (double-KO), signaling complex analysis\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple conditional KO models with genetic epistasis rescue (TNFR1 KO) and catalytic-activity requirement demonstrated\",\n      \"pmids\": [\"25284787\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"A hypomorphic missense mutation (L72P) in the HOIP PUB domain impairs HOIP protein expression and destabilizes the entire LUBAC complex, causing defective linear ubiquitination and NF-κB activation in response to IL-1β and TNF in patient fibroblasts, and impaired B-cell activation in response to CD40 engagement.\",\n      \"method\": \"Patient primary cell analysis, western blot for LUBAC complex integrity, NF-κB activation assays, B-cell functional assays\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — human loss-of-function variant with multiple orthogonal functional assays in primary patient cells\",\n      \"pmids\": [\"26008899\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"HOIP is cleaved by caspases predominantly at Asp390 during apoptosis and is subsequently degraded by the proteasome. Effector caspases 3 and 6 cleave at aspartates 348, 387, and 390. The N-terminal cleavage fragment retains binding to OTULIN and CYLD-SPATA2; the C-terminal fragment retains NF-κB activity, but overall linear ubiquitination of NEMO and FADD (both identified as LUBAC substrates) decreases upon apoptosis. FADD is identified as a novel substrate for linear ubiquitination by LUBAC.\",\n      \"method\": \"Caspase cleavage assays, site-directed mutagenesis of cleavage sites, western blot, NF-κB reporter assays, linear ubiquitination assays for NEMO and FADD\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis of caspase sites plus biochemical ubiquitination assays for novel substrate FADD, single lab\",\n      \"pmids\": [\"28189684\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"RNF31/HOIP is cleaved by caspases 3 and 6 at aspartates 348, 387, and 390 under apoptotic conditions, and this cleavage suppresses its ability to activate NF-κB signaling. Mutation of caspase cleavage sites inhibits TNF-α-induced apoptosis, establishing a regulatory loop between cell death and survival signaling.\",\n      \"method\": \"In vitro caspase cleavage assays, site-directed mutagenesis, apoptosis assays, NF-κB reporter assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical caspase assays with mutagenesis and functional NF-κB/apoptosis readouts\",\n      \"pmids\": [\"27669734\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Ubiquitination of HOIP at C-terminal lysine 1056 negatively regulates its catalytic activity by inducing a conformational change that suppresses linear-chain-forming activity. HOIP K1056R mutation leads to persistent LUBAC activity and prolonged NF-κB activation induced by TLR4/LPS stimulation but not by CD40 stimulation, indicating pathway-specific regulation.\",\n      \"method\": \"Site-directed mutagenesis (K1056R), in vitro ubiquitination assays, NF-κB reporter assays, TLR4 signaling experiments\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — identification of regulatory ubiquitination site by mutagenesis with functional pathway specificity demonstrated\",\n      \"pmids\": [\"26578682\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"HOIP (as LUBAC component) is recruited to the TNFR2 signaling complex in a cIAP1-dependent manner; loss of cIAP1 (via cIAP antagonist) prevents HOIP recruitment and HOIP-mediated M1-ubiquitination at the TNFR2 complex. Both HOIP and cIAP1 are required for TNFR2-induced canonical NF-κB activation.\",\n      \"method\": \"TNFR2 signaling complex immunoprecipitation, cIAP antagonist treatment, M1/K63 ubiquitin linkage-specific antibody blotting, NF-κB activation assays\",\n      \"journal\": \"Biochemical pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — receptor-complex pull-down with pharmacological inhibition and linkage-specific ubiquitin detection\",\n      \"pmids\": [\"29378181\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Epidermis-specific knockout of RNF31 in mice causes early postnatal lethality with severe skin inflammation driven by TNF-α-induced apoptosis in keratinocytes. RNF31 deficiency impairs TNF-α-induced NF-κB activation and increases apoptosis. Genetic deletion of TNFR1 rescues lethality and skin inflammation in RNF31 epidermis-KO mice.\",\n      \"method\": \"Epidermis-specific conditional KO mice, genetic epistasis (TNFR1 KO rescue), apoptosis assays, NF-κB activation assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — tissue-specific genetic KO with TNFR1 genetic rescue epistasis and multiple mechanistic readouts\",\n      \"pmids\": [\"29728512\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In T cell-specific HOIP-knockin mice expressing a catalytically inactive HOIP (HOIPΔlinear), CD4+ and CD8+ T cell numbers are markedly reduced, NKT cell development is severely impaired, and mature T cells undergo accelerated apoptosis. HOIPΔlinear CD4+ T cells fail to phosphorylate IκBα and JNK through TCR stimulation. Reduced CD127 expression contributes to apoptosis; enforced CD127 expression rescues mature CD8+ T cell development.\",\n      \"method\": \"Conditional knock-in mice with catalytically inactive HOIP, flow cytometry, TCR signaling (phospho-IκBα, phospho-JNK), apoptosis assays, CD127 rescue experiment\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — catalytic knock-in mouse model with multiple orthogonal phenotypic and signaling readouts plus mechanistic rescue\",\n      \"pmids\": [\"27786304\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"LUBAC/HOIP binds GPx4 and stabilizes it by modulating its linear ubiquitination, protecting cells against ferroptosis. LUBAC deficiency sensitizes cells to ferroptosis by promoting GPx4 degradation and downstream lipid peroxidation. GPx4 is identified as a direct substrate of HOIP-mediated linear ubiquitination.\",\n      \"method\": \"Co-immunoprecipitation, linear ubiquitination assays, lipid peroxidation assays, ferroptosis sensitivity assays in LUBAC-deficient cells\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ubiquitination assays plus cellular ferroptosis phenotype, single lab study\",\n      \"pmids\": [\"36279464\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RNF31 genetic or pharmacologic ablation sensitizes cancer cells to NK cell and CD8+ T cell killing in a TNF-dependent manner, 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 and enhances bystander killing of MHC-deficient tumor cells.\",\n      \"method\": \"Genome-wide CRISPR-Cas9 knockout screens under NK/CD8+ T cell pressure, pharmacological RNF31 inhibitor, TNF receptor complex analysis, tumor organoid killing assays\",\n      \"journal\": \"Cell reports. Medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — CRISPR screen identification validated by genetic and pharmacologic ablation with mechanistic complex analysis and organoid model\",\n      \"pmids\": [\"35688159\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Loss of Rnf31 in pancreatic cancer cells removes protection from TNF-mediated caspase-8 cleavage and subsequent apoptosis, sensitizing tumor cells to CD8+ T cell killing. Rnf31-deficient orthotopic pancreatic tumors show increased CD8+ T cell infiltration and effector function in vivo.\",\n      \"method\": \"In vitro and in vivo CRISPR screening, caspase-8 cleavage assays, orthotopic transplantation into immune-competent mice, T cell functional assays, human PDA organoids\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — CRISPR screen plus mechanistic apoptosis assays plus in vivo orthotopic model with immune readouts\",\n      \"pmids\": [\"35379808\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"HOIP-deficient melanoma cells are hypersensitive to combined TNF and IFN-γ killing by NK and CD8+ T cells. Both genetic deletion and pharmacological inhibition of HOIP increase tumor cell sensitivity to TNF+IFN-γ co-stimulation, which engages both intrinsic and extrinsic apoptotic machinery in a transcription-dependent manner.\",\n      \"method\": \"CRISPR/Cas9 HOIP KO, pharmacological HOIP inhibition, NK/CD8+ T cell co-culture killing assays, cytokine treatment experiments, apoptosis pathway analysis\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO plus pharmacological inhibition with mechanistic cytokine synergy and apoptosis pathway defined, single lab\",\n      \"pmids\": [\"34467615\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"RNF31 interacts with A20 via its RBR structural domain (co-immunoprecipitation), promotes K48-linked ubiquitination of A20 and its proteasomal degradation, thereby activating the TLR4/MyD88/NF-κB signaling pathway and aggravating hepatocyte apoptosis and inflammatory cytokine production.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, proteasome inhibitor experiments (actinomycin chase + MG132), siRNA knockdown, in vivo LPS/d-Gal acute liver injury model\",\n      \"journal\": \"Chemico-biological interactions\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ubiquitination/degradation assays plus in vivo model, single lab\",\n      \"pmids\": [\"34416243\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"RNF31/HOIP associates with PTEN via co-immunoprecipitation and promotes PTEN ubiquitination and proteasomal degradation in cancer cells. HOIP depletion causes cell cycle arrest and apoptosis that can be rescued by PTEN silencing, implicating a HOIP–PTEN–PI3K/AKT axis in chemotherapy resistance.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, PTEN knockdown rescue, cell cycle analysis, apoptosis assays\",\n      \"journal\": \"Journal of Cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP plus ubiquitination assay plus KD rescue, replicated across multiple cancer cell lines but single lab\",\n      \"pmids\": [\"34659546\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"RNF31 interacts with NLRP3 via its RBR domain and promotes K63-linked ubiquitination of NLRP3, stabilizing the NLRP3 inflammasome complex and enhancing IL-1β and IL-18 production. In vivo RNF31 knockdown attenuates DSS-induced colitis and reduces NLRP3 expression.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays (K63-linkage specific), RNF31 KD cell model, DSS-induced colitis mouse model\",\n      \"journal\": \"International immunopharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus linkage-specific ubiquitination assays plus in vivo model, single lab\",\n      \"pmids\": [\"37951199\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RBCK1 (HOIL-1L) interacts with RNF31 and represses RNF31 ubiquitination and proteasomal degradation, thereby stabilizing RNF31 protein in hepatocellular carcinoma cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, proteasome inhibitor experiments, RNF31/RBCK1 KD cell lines\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP plus ubiquitination/stability assays, single lab\",\n      \"pmids\": [\"35869046\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RNF31 depletion in TNBC increases YAP protein levels; RNF31 associates with YAP, facilitates YAP poly-ubiquitination and proteasomal degradation specifically at YAP K76, and suppresses Hippo/YAP/PD-L1 axis, thereby repressing TNBC cell proliferation, migration, and immune checkpoint expression.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, site-directed mutagenesis (YAP K76), xenograft models, genome-wide expression profiling, siRNA KD\",\n      \"journal\": \"Journal of experimental & clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus site-specific mutagenesis plus in vivo xenograft, single lab\",\n      \"pmids\": [\"36581998\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"RNF31 interacts with p53 via its PUB domain (demonstrated by truncation Co-IP assays), promotes p53 ubiquitination and proteasomal degradation, and the PUB domain is the key structural determinant for p53 ubiquitination. RNF31 depletion stabilizes p53 and inhibits CRC cell growth.\",\n      \"method\": \"Co-immunoprecipitation with truncation constructs, cycloheximide-chase assay, MG132 proteasome inhibition, ubiquitination assays\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — domain-mapping Co-IP plus stability assays plus ubiquitination, single lab\",\n      \"pmids\": [\"33824292\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RNF31 enhances p53 ubiquitination and proteasomal degradation; reduced RNF31 leads to p53 accumulation, which represses BNIP3 expression, impairing mitophagy and promoting steatosis in hepatocytes. RNF31 delivery via mesenchymal stem cell-derived small extracellular vesicles reduces hepatic steatosis in HFD-fed mice.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, mt-Keima fluorescence mitophagy assay, RNA-seq, HFD mouse model, sEV delivery experiments\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus mechanistic mitophagy imaging plus in vivo model, single lab\",\n      \"pmids\": [\"38615890\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"STING activation induces recruitment of HOIP (LUBAC) to LC3B-associated Golgi membranes and synthesis of M1-linked (linear) ubiquitin chains. Loss of HOIP prevents M1-Ub chain formation and reduces STING-induced NF-κB and IRF3 signaling in human THP1 monocytes and mouse bone marrow-derived macrophages, without affecting upstream STING activation itself.\",\n      \"method\": \"HOIP KO in THP1 monocytes and BMDMs, M1-Ub chain detection, STING pathway signaling assays (IRF3/NF-κB), LC3B/Golgi co-localization imaging\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO in two cell types with signaling assays and localization imaging, single lab\",\n      \"pmids\": [\"39578541\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Fragment-based covalent ligand screening targeting the active-site cysteine of HOIP yielded the first structure-based covalent inhibitors for an RBR E3 ligase. Crystal structures of HOIP-inhibitor complexes were solved; cell-based and chemoproteomic assays confirmed cell-penetrant HOIP labeling and inhibition of NF-κB activation.\",\n      \"method\": \"Fragment covalent screening, protein LC-MS ubiquitination assays, protein crystallography, cell-based NF-κB reporter assays, chemoproteomics\",\n      \"journal\": \"Journal of the American Chemical Society\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structures plus in vitro biochemical assays plus cell-based functional validation in one integrated study\",\n      \"pmids\": [\"30657686\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"HOIP (RNF31) is identified by siRNA screen as a key regulator of cisplatin-induced genotoxicity; HOIP-deficient cells exhibit hypersensitivity to cisplatin via increased caspase-8/caspase-3-dependent apoptosis that requires ATM (not ATR) checkpoint activation. JNK activity is enhanced in HOIP-depleted cells and JNK inhibition reverses apoptotic hyperactivation.\",\n      \"method\": \"siRNA library screen, caspase activity assays, ATM/ATR pathway inhibitors, JNK inhibition rescue, cisplatin-resistance assays\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA screen hit validated with multiple mechanistic pathway inhibitor epistasis experiments\",\n      \"pmids\": [\"24686174\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"DC-specific deletion of HOIP causes spontaneous inflammation; HOIP deficiency in DCs does not affect TNF-α-induced NF-κB activation but enhances TNF-α-induced apoptosis and necroptosis. Crossing with TNFR1-KO does not rescue inflammation, whereas antibiotic treatment reduces it. MyD88 deficiency rescues the inflammatory phenotype in HOIP-deficient DC mice, establishing MyD88-dependent (TLR) signaling—not TNFR1 signaling—as the driver of autoinflammation.\",\n      \"method\": \"DC-specific conditional HOIP KO mice, genetic epistasis (TNFR1-KO, MyD88-KO crosses), antibiotic treatment, apoptosis/necroptosis assays, cytokine measurement\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple genetic epistasis crosses with clean mechanistic separation of TNFR1 vs. MyD88 pathways\",\n      \"pmids\": [\"34253576\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Chemical ubiquitylation of NEMO using a ligation auxiliary demonstrated that monoubiquitylated NEMO retains similar affinity for linear diubiquitin chains as unmodified NEMO. The proximal ubiquitin of chemically synthesized NEMO-Ub is accepted as substrate for linear extension by the HOIP RBR domain alone, indicating NEMO linear ubiquitylation involves a two-step mechanism: an initial priming event (requiring factors beyond HOIP alone) and a separate HOIP RBR-mediated extension step.\",\n      \"method\": \"Chemical ubiquitylation (ligation auxiliary), in vitro linear extension assays with HOIP RBR domain, ITC binding affinity measurements\",\n      \"journal\": \"Communications chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstitution-based in vitro chemistry plus binding assays, single lab study\",\n      \"pmids\": [\"31942456\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RNF31 interacts with and ubiquitinates ALYREF (RNA export factor), facilitating ALYREF nuclear transport via importin 13 (IPO13) under paclitaxel treatment; nuclear ALYREF then mediates export of mRNAs encoding paclitaxel-resistance factors (TUBB3, STMN1, TAU), inducing resistance. RNF31 inhibition traps ALYREF in the cytoplasm and re-sensitizes resistant TNBC cells to paclitaxel.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, immunofluorescence (ALYREF subcellular localization), mRNA export assays, siRNA KD, RNF31 inhibitor + paclitaxel in vivo and organoid models\",\n      \"journal\": \"Clinical and translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ubiquitination plus localization imaging plus in vivo/organoid functional validation, single lab\",\n      \"pmids\": [\"39915011\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"RNF31/PAUL (identified as a putative Ariadne-like RBR E3 ubiquitin ligase) binds the cytoplasmic domain of the muscle-specific receptor tyrosine kinase MuSK and is expressed at neuromuscular junctions, suggesting a role in postsynaptic membrane formation.\",\n      \"method\": \"Yeast two-hybrid and binding assay identifying PAUL as MuSK-interacting protein, in situ hybridization and immunostaining for NMJ localization\",\n      \"journal\": \"Gene expression patterns\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single binding assay plus localization, no functional validation of the interaction\",\n      \"pmids\": [\"14678832\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RNF31 (HOIP) is the catalytic RING-IBR-RING (RBR) E3 ubiquitin ligase subunit of the trimeric LUBAC complex (with HOIL-1L/RBCK1 and SHARPIN) that uniquely generates Met1-linked (linear) ubiquitin chains via a RING/HECT-hybrid mechanism: RING1-IBR accepts ubiquitin from E2 to form a HECT-like thioester on RING2's active-site cysteine, then the Linear ubiquitin chain Determining Domain (LDD) orients the acceptor ubiquitin's α-amino group for nucleophilic transfer; structural studies have captured apo, ubiquitin-bound, and E2~ubiquitin transfer-complex states. HOIP linearly ubiquitinates key NF-κB signaling substrates (NEMO, FADD) and also attaches non-canonical ubiquitin marks to other substrates (ERα mono-ubiquitination, GPx4 stabilization, YAP and PTEN degradation, A20 degradation, NLRP3 K63 stabilization, ALYREF nuclear transport), thereby controlling NF-κB-dependent survival signaling downstream of TNFR1, TNFR2, CD40, TLR4, NOD2, and STING; protecting endothelial cells, keratinocytes, T cells, and dendritic cells from TNF- or cytokine-induced apoptosis/necroptosis; and regulating steroidogenesis, ferroptosis, and immune evasion. HOIP activity is itself regulated by OTULIN (PUB-domain interaction, negatively controlled by OTULIN Tyr56 phosphorylation), CYLD (bridged via SPATA2), caspase-mediated cleavage at Asp348/387/390 during apoptosis, and auto-ubiquitination at Lys1056 that causes conformational inhibition specifically in TLR4 signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RNF31 (HOIP) is the catalytically active RING-IBR-RING (RBR) E3 ubiquitin ligase subunit of the LUBAC complex and is the central node generating linear (Met1-linked) ubiquitin chains that drive NF-\\u03baB-dependent survival signaling [#0]. It builds linear chains through a two-step RBR mechanism in which ubiquitin is transferred from an E2 onto the RING2 active-site cysteine to form a HECT-like thioester, after which a unique C-terminal Linear ubiquitin chain Determining Domain (LDD) orients the acceptor ubiquitin for nucleophilic attack on its N-terminus [#0]; structural studies of the apo, ubiquitin-bound, and E2~ubiquitin transfer-complex states defined how donor and acceptor ubiquitin are simultaneously coordinated and how active HOIP departs from the auto-inhibited RBR conformation to align catalytic centers [#1, #2]. Through linear ubiquitination of substrates such as NEMO and FADD, HOIP controls NF-\\u03baB activation downstream of multiple receptors including CD40, TNFR2, and STING [#11, #7, #14, #28], and its activity is essential to protect cells from cytokine-induced death: catalytic-dead or tissue-specific HOIP loss sensitizes endothelial cells, keratinocytes, and T cells to TNF-driven apoptosis/necroptosis, phenotypes rescued by TNFR1 deletion [#9, #15, #16]. HOIP activity is itself tuned by deubiquitinase recruitment via direct PUB-domain binding to OTULIN [#3] and SPATA2-bridged engagement of CYLD [#4], by caspase cleavage at Asp348/387/390 during apoptosis [#11, #12], and by auto-ubiquitination at Lys1056 that confers TLR4-specific conformational inhibition [#13]. Beyond canonical LUBAC substrates, HOIP attaches non-degradative and degradative ubiquitin marks to a range of targets, stabilizing GPx4 to suppress ferroptosis [#17] and NLRP3 via K63 chains [#23] while driving K48-linked degradation of A20, PTEN, YAP, and p53 [#21, #22, #25, #26]. Loss or pharmacologic inhibition of RNF31 sensitizes tumor cells to TNF-dependent killing by NK and CD8+ T cells, establishing it as a target for immune-mediated cancer therapy [#18, #19, #20]. A hypomorphic PUB-domain mutation (L72P) destabilizes LUBAC and causes defective linear ubiquitination and immunodeficiency in patients [#10].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"An initial question was whether RNF31 had any defined molecular partner; identifying it as an Ariadne-like RBR protein binding the MuSK cytoplasmic domain gave the first functional context, though unrelated to its later-established LUBAC role.\",\n      \"evidence\": \"Yeast two-hybrid/binding assay and NMJ localization by in situ hybridization and immunostaining\",\n      \"pmids\": [\"14678832\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single binding assay with no functional validation of the MuSK interaction\", \"No connection drawn to ubiquitin ligase activity or NF-\\u03baB signaling\", \"Not reconciled with the canonical LUBAC function\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Before its NF-\\u03baB role was defined, RNF31 was found to act as a transcriptional corepressor, establishing an early link between RNF31 and ubiquitin-dependent stabilization of a partner protein.\",\n      \"evidence\": \"Co-IP, ChIP, siRNA knockdown and reporter/ubiquitination assays on steroidogenic gene promoters with DAX-1\",\n      \"pmids\": [\"19237537\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ubiquitin linkage type on DAX-1 not defined\", \"Mechanism connecting stabilization to repression unresolved\", \"Relationship to LUBAC complex unaddressed\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"To determine which receptors require HOIP, studies showed it is recruited TRAF2-dependently to the CD40 complex and is essential for CD40-driven NF-\\u03baB, establishing HOIP catalytic activity as required for proximal receptor signaling.\",\n      \"evidence\": \"Mass spectrometry of CD40 complexes, HOIP KO B-cell lines, dominant-negative catalytic mutant, NF-\\u03baB/kinase assays\",\n      \"pmids\": [\"20614026\", \"21829693\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct CD40-complex substrate of HOIP not identified\", \"Linear chain linkage at CD40 complex not directly demonstrated\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"The defining mechanistic question \\u2014 how HOIP generates linear chains \\u2014 was answered by showing a two-step RBR mechanism with a HECT-like thioester intermediate and an LDD that confers linear specificity.\",\n      \"evidence\": \"In vitro ubiquitination assays, active-site mutagenesis, cellular NF-\\u03baB reporter assays\",\n      \"pmids\": [\"22863777\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic basis of acceptor orientation not yet visualized\", \"Priming versus extension steps not distinguished\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Structural studies progressively explained linear chain specificity and catalysis, capturing donor- and acceptor-ubiquitin coordination and the active transfer conformation distinct from auto-inhibited RBRs.\",\n      \"evidence\": \"X-ray crystallography of apo, ubiquitin-bound, and HOIP RBR/E2~ubiquitin transfer complexes with mutagenesis\",\n      \"pmids\": [\"24141947\", \"26789245\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length LUBAC architecture not resolved\", \"Conformational transition kinetics not measured\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"How HOIP activity is restrained was addressed by defining deubiquitinase recruitment: direct PUB-domain binding to OTULIN (regulated by OTULIN Tyr56 phosphorylation) and SPATA2-bridged CYLD recruitment.\",\n      \"evidence\": \"Structural determination of the PUB-PIM interface, phospho-mimetic mutagenesis, reciprocal Co-IP, receptor-complex pull-down, SPATA2 KO necroptosis assays\",\n      \"pmids\": [\"24726327\", \"27545878\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative balance between OTULIN and CYLD restraint unresolved\", \"How phosphorylation is triggered in vivo not defined\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Genetic models established the physiological imperative for HOIP catalytic activity in preventing cytokine-induced death across endothelium, epidermis, and T cells, with TNFR1 epistasis defining the death ligand.\",\n      \"evidence\": \"Constitutive, Tie2-Cre, epidermis-specific conditional KO and catalytic knock-in mice with TNFR1-KO genetic rescue and CD127 rescue\",\n      \"pmids\": [\"25284787\", \"29728512\", \"27786304\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The specific substrate whose linear ubiquitination prevents complex-II death not fully resolved per tissue\", \"Necroptosis versus apoptosis contributions vary by context\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Receptor scope was extended by showing cIAP1-dependent HOIP recruitment to the TNFR2 complex, broadening the set of NF-\\u03baB pathways routed through linear ubiquitination.\",\n      \"evidence\": \"TNFR2 complex IP, cIAP antagonist treatment, M1/K63 linkage-specific blotting, NF-\\u03baB assays\",\n      \"pmids\": [\"29378181\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"TNFR2-complex substrate of M1 chains not identified\", \"Single study\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"How HOIP is switched off during apoptosis and which pathway-specific regulation it undergoes were addressed by mapping caspase cleavage sites, the K1056 auto-ubiquitination switch, and identifying FADD as a linear ubiquitination substrate.\",\n      \"evidence\": \"Caspase cleavage and site mutagenesis, K1056R mutagenesis, in vitro ubiquitination and NF-\\u03baB/apoptosis assays, NEMO/FADD linear ubiquitination assays\",\n      \"pmids\": [\"28189684\", \"27669734\", \"26578682\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological trigger of K1056 auto-ubiquitination in TLR4 signaling not defined\", \"Fate of cleavage fragments in vivo unclear\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Reconstitution chemistry refined the catalytic model by separating NEMO linear ubiquitylation into a priming event requiring factors beyond HOIP and a HOIP RBR-mediated extension step.\",\n      \"evidence\": \"Chemical ubiquitylation of NEMO, in vitro extension with HOIP RBR domain, ITC binding measurements\",\n      \"pmids\": [\"31942456\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Identity of the priming factor not established\", \"Single-lab in vitro reconstitution\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"The repertoire of HOIP substrates and pathways was expanded substantially, revealing both linear (GPx4, STING-associated) and non-canonical chain functions (NLRP3 K63 stabilization; K48 degradation of A20, PTEN, YAP, p53; ER\\u03b1/DAX-1 stabilization; ALYREF nuclear transport).\",\n      \"evidence\": \"Co-IP, linkage-specific and site-directed ubiquitination assays, knockdown/inhibitor rescue, in vivo disease and organoid models across ferroptosis, inflammasome, liver injury, mitophagy and chemoresistance\",\n      \"pmids\": [\"36279464\", \"37951199\", \"34416243\", \"34659546\", \"36581998\", \"33824292\", \"38615890\", \"24441041\", \"39915011\", \"39578541\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Most non-canonical substrates rest on single-lab Co-IP plus ubiquitination assays\", \"Chain linkage and direct-versus-indirect ubiquitination not uniformly confirmed\", \"Reconciliation of degradative versus stabilizing roles per substrate incomplete\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"The translational question of whether HOIP can be exploited therapeutically was answered by structure-based covalent inhibitors and by CRISPR/pharmacologic studies showing RNF31 ablation sensitizes tumors to TNF-dependent immune killing.\",\n      \"evidence\": \"Fragment-based covalent inhibitor crystallography and chemoproteomics; genome-wide CRISPR screens under NK/CD8+ T cell pressure, inhibitor treatment, organoid and orthotopic tumor models\",\n      \"pmids\": [\"30657686\", \"35688159\", \"35379808\", \"34467615\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo selectivity and off-target effects of inhibitors not fully defined\", \"Patient-level efficacy untested in the corpus\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how HOIP selects among its many reported substrates and switches between generating linear, K48, and K63 linkages on different targets, and which priming factors and contextual cues dictate these outcomes in vivo.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying mechanism explains linkage-type switching across substrates\", \"NEMO priming factor unidentified\", \"Many non-canonical substrate claims await independent validation\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 17, 21, 25]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [33]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [28]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [6, 33]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 7, 14, 28]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [18, 19, 20, 31]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [9, 15, 16, 17]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 21, 22, 25, 26]}\n    ],\n    \"complexes\": [\"LUBAC\"],\n    \"partners\": [\"RBCK1\", \"OTULIN\", \"SPATA2\", \"CYLD\", \"TRAF2\", \"A20\", \"NLRP3\", \"YAP\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}