{"gene":"RBCK1","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":2019,"finding":"HOIL-1 (RBCK1) is an atypical E3 ligase that forms oxyester bonds between the C terminus of ubiquitin and serine/threonine residues in substrates. Using hydroxylamine cleavage of oxyester bonds and knock-in mice expressing E3 ligase-inactive HOIL-1[C458S], IRAK1, IRAK2, and MyD88 (Myddosome components) were identified as physiological substrates during Toll-like receptor signaling. HOIL-1 acts as a monoubiquitylating E3 that initiates de novo polyubiquitin chain synthesis on these proteins, and also catalyzes its own monoubiquitylation and likely that of Sharpin via oxyester linkages.","method":"Hydroxylamine cleavage assay, knock-in mice expressing catalytically inactive HOIL-1[C458S], mass spectrometry, in vitro ubiquitination assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with active-site mutagenesis, orthogonal chemical method (hydroxylamine cleavage), and genetic knock-in mouse validation in a single rigorous study","pmids":["31209050"],"is_preprint":false},{"year":2023,"finding":"Crystal structure of the C-terminal tandem IBR-RING2 domain of HOIL-1 reveals a unique bi-nuclear Zn-cluster substituting the second zinc finger of the canonical RING2 fold. The C-terminal histidine of this Zn-cluster was identified as the catalytic base required for HOIL-1 ubiquitylation activity. HOIL-1 activity also requires binding of linear tetra-ubiquitin, which enables mono-ubiquitylation of linear Ub chains and polysaccharides.","method":"X-ray crystallography, mutagenesis of catalytic residues, in vitro ubiquitylation assay","journal":"Frontiers in molecular biosciences","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure combined with mutagenesis and functional in vitro assay in a single study","pmids":["36685275"],"is_preprint":false},{"year":2025,"finding":"HOIL-1 ubiquitinates serine residues and diverse mono- and disaccharides via O-linked ubiquitination but cannot ubiquitinate lysine. A critical catalytic histidine residue, His510, in the flexible catalytic site of HOIL-1 enables O-linked ubiquitination and prohibits ubiquitin discharge onto lysine sidechains.","method":"In vitro ubiquitination assays with model substrates, mutagenesis of His510, biochemical characterization","journal":"Life science alliance","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with active-site mutagenesis identifying catalytic residue, multiple substrates tested","pmids":["40169258"],"is_preprint":false},{"year":2008,"finding":"RBCK1 E3 ubiquitin ligase activity was demonstrated in vitro. This E3 activity is inhibited by interaction with its splice variant RBCK2 (which lacks the RING-IBR domain). Additionally, phosphorylation of RBCK1 by PKCbeta abolishes self-ubiquitination activity in vitro, and PKCbeta overexpression increases intracellular RBCK1 levels by suppressing its proteasomal degradation.","method":"In vitro ubiquitin ligase assay, co-immunoprecipitation, PKCbeta overexpression/inhibition","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro E3 assay with mechanistic follow-up (splice variant inhibition and kinase phosphorylation), multiple orthogonal approaches in single study","pmids":["18303026"],"is_preprint":false},{"year":2005,"finding":"RBCK1 shuttles between cytoplasm and nucleus via defined nuclear export and localization signals: an N-terminal region containing Leu-142/Leu-145 serves as the nuclear export signal, and the C-terminal RING-IBR domain serves as the nuclear localization signal. RBCK1 localizes to nuclear bodies and interacts with CBP and PML. CBP co-expression enhances RBCK1 transcriptional activity, while PML represses CBP-enhanced activity.","method":"Leptomycin B treatment, mutational analysis, co-immunoprecipitation, live-cell imaging/fractionation, transcriptional reporter assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — direct localization experiments with functional consequence, mutational mapping of signals, multiple orthogonal methods in single study","pmids":["15833741"],"is_preprint":false},{"year":2005,"finding":"RBCK2, the splice variant of RBCK1 lacking the RING-IBR domain, is cytoplasmic and contains Leu-rich nuclear export signals. RBCK2 acts as a cytoplasmic tethering protein for RBCK1 by forming a hetero-oligomeric complex, causing nuclear RBCK1 to relocalize to the cytoplasm when RBCK2 is co-expressed.","method":"Co-expression of NES-disrupted RBCK1 with RBCK2, immunofluorescence/localization assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — single lab, direct localization experiment with functional consequence (nuclear export via heterocomplex), single method","pmids":["16083853"],"is_preprint":false},{"year":1998,"finding":"RBCK1 and RBCK2 are generated from a single gene by alternative splicing. RBCK1 protein interacts with both RBCK1 and RBCK2 in vitro, but RBCK2 does not self-interact. RBCK2 inhibits the transcriptional activity of RBCK1, likely through complex formation. Both RING-finger and B-Box motifs of RBCK1 are indispensable for its transcriptional activity.","method":"In vitro interaction assay, GAL4-chimeric transcription assay, mutational analysis","journal":"FEBS letters / Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two separate papers using in vitro binding and reporter assays establishing the same mechanism","pmids":["9755849","9642138"],"is_preprint":false},{"year":2008,"finding":"RBCK1 (E3 ubiquitin ligase) catalyzes K48-linked ubiquitination and proteasomal degradation of IRF3, acting as a negative feedback regulator of virus-triggered type I interferon induction. Viral infection induces RBCK1 expression, leading to subsequent IRF3 degradation. Overexpression of RBCK1 reduces antiviral IFN responses; knockdown has the opposite effect.","method":"Overexpression/knockdown, ubiquitination assay, plaque assay, immunoblot","journal":"Cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — single lab, ubiquitination assay plus functional antiviral readout, no in vitro reconstitution with purified proteins","pmids":["18711448"],"is_preprint":false},{"year":2007,"finding":"RBCK1 physically interacts with TAB2 and TAB3 adapter proteins and facilitates their proteasome-dependent degradation, thereby negatively regulating TNF- and IL-1-induced NF-κB activation. Overexpression of RBCK1 inhibits TAB2/3-mediated NF-κB activation; knockdown potentiates it.","method":"Co-immunoprecipitation, overexpression/RNAi, NF-κB reporter assay, proteasome inhibitor treatment","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — single lab, reciprocal co-IP plus functional NF-κB readout with RNAi rescue, but no in vitro reconstitution","pmids":["17449468"],"is_preprint":false},{"year":2012,"finding":"HOIL-1 (RBCK1) deficiency in patients carrying biallelic loss-of-function HOIL1 mutations impairs LUBAC stability and compromises NF-κB activation in response to IL-1β in fibroblasts, while monocytes from the same patients are hyper-responsive to IL-1β, demonstrating cell-type-specific regulation of NF-κB by HOIL-1/LUBAC.","method":"Patient-derived cells (fibroblasts and monocytes), NF-κB signaling assays, genetic analysis of loss-of-function mutations","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — human genetic loss-of-function with mechanistic pathway (NF-κB) readout in patient-derived cells, replicated across two kindreds","pmids":["23104095"],"is_preprint":false},{"year":2015,"finding":"MALT1 paracaspase cleaves HOIL-1 late in the NF-κB activation cycle during antigen receptor signaling. Cleavage generates an N-terminal fragment that retains support for HOIP-induced NF-κB signaling and a C-terminal fragment with LUBAC inhibitory properties, providing negative feedback regulation of NF-κB. This cleavage transiently reduces linear ubiquitination of NEMO and RIP1.","method":"10-plex TMT TAILS N-terminal peptide proteomics, co-immunoprecipitation, NF-κB reporter assay, B-cell receptor signaling assays","journal":"Nature communications / The FEBS journal","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — substrate identified by proteomics, confirmed by multiple functional assays, replicated by independent groups (Klein et al. and Elton et al.)","pmids":["26525107","26573773"],"is_preprint":false},{"year":2016,"finding":"MALT1-mediated cleavage of HOIL1 occurs following antigen receptor engagement in T and B lymphocytes. Overexpression of MALT1-insensitive HOIL1 mitigates TCR-mediated NF-κB activation and cytokine production, establishing HOIL1 as a negative regulator of lymphocyte activation that is removed by MALT1 cleavage.","method":"Antigen receptor stimulation assays, overexpression of cleavage-resistant HOIL1, NF-κB reporter assay, cytokine measurement","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional NF-κB and cytokine readouts with gain-of-function cleavage-resistant mutant, consistent with two other groups","pmids":["27006117"],"is_preprint":false},{"year":2021,"finding":"Non-cleavable HOIL-1 (MALT1-resistant) expressed in primary HOIL-1-deficient patient fibroblasts results in enhanced NF-κB signaling and a hyperinflammatory cytokine profile compared to wild-type HOIL-1, demonstrating that MALT1-dependent HOIL-1 cleavage is physiologically required to dampen inflammatory responses.","method":"Stable transduction of patient fibroblasts, NF-κB assays, transcriptomics, multiplexed cytokine assays","journal":"Frontiers in immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — patient-derived cells with isogenic comparison, transcriptomics plus functional cytokine readout, single lab","pmids":["34721419"],"is_preprint":false},{"year":2006,"finding":"HOIL-1 (RBCK1) interacts with SOCS6, requiring HOIL-1's Ubl domain and SOCS6's SH2 and Socs-box domains. HOIL-1 expression stabilizes SOCS6 itself but induces the ubiquitination and proteasomal degradation of proteins associated with SOCS6.","method":"Co-immunoprecipitation, domain mapping, ubiquitination assay, proteasome inhibitor treatment","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — single lab, co-IP with domain mapping plus ubiquitination assay, moderate mechanistic depth","pmids":["16643902"],"is_preprint":false},{"year":2012,"finding":"RBCK1 interacts with hPXR (pregnane X receptor) via all RBCK1 domains (identified by yeast two-hybrid and confirmed by co-IP), ubiquitinates hPXR, and targets it for proteasomal degradation. Overexpression of RBCK1 decreased endogenous PXR in HepG2 cells and primary hepatocytes; RBCK1 silencing increased PXR levels and enhanced induction of PXR target genes.","method":"Yeast two-hybrid, co-immunoprecipitation, ubiquitination assay, proteasome inhibitor (MG-132) treatment, siRNA knockdown in primary hepatocytes","journal":"Drug metabolism and disposition","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid confirmed by co-IP, ubiquitination assay, and functional readout in primary cells, single lab","pmids":["23160820"],"is_preprint":false},{"year":2019,"finding":"RBCK1 facilitates poly-ubiquitination and proteasomal degradation of p53 via direct interaction, promoting renal cell carcinoma proliferation. Knockdown of RBCK1 in p53 wild-type RCC cells reduces proliferation; this effect is rescued by p53 knockdown.","method":"Co-immunoprecipitation, ubiquitination assay, knockdown/rescue experiments, in vivo xenograft","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — co-IP plus ubiquitination assay with genetic rescue, single lab","pmids":["30874541"],"is_preprint":false},{"year":2022,"finding":"RBCK1 interacts with PTEN and promotes K48-linked polyubiquitination and proteasomal degradation of PTEN in ovarian cancer cells, facilitating tumor progression.","method":"Co-immunoprecipitation, ubiquitination assay with K48-linkage specificity, knockdown/overexpression","journal":"Human cell","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — single lab, co-IP plus K48-specific ubiquitination assay, no in vitro reconstitution","pmids":["35174471"],"is_preprint":false},{"year":2024,"finding":"RBCK1 interacts with and polyubiquitylates mitofusin 2 (MFN2) to facilitate its proteasomal degradation under ferroptotic stress, leading to decreased mitochondrial ROS production and lipid peroxidation, thereby conferring ferroptosis resistance in pancreatic cancer cells.","method":"Co-immunoprecipitation, ubiquitination assay, RBCK1 depletion/overexpression, in vivo xenograft, measurement of mitochondrial ROS and lipid peroxidation","journal":"Free radical biology & medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — single lab, co-IP plus functional ferroptosis readout, no in vitro reconstitution","pmids":["38763208"],"is_preprint":false},{"year":2020,"finding":"MTSS1 interacts with RBCK1 to facilitate RBCK1-mediated K48-linked polyubiquitination and degradation of NF-κB p65, suppressing NF-κB signaling and tumor-initiating cell properties in breast cancer. ACTBL2 competes with RBCK1 for MTSS1 binding, leading to p65 stabilization.","method":"Co-immunoprecipitation, ubiquitination assay, Mtss1 knockout mouse models, tumorsphere assay, xenograft","journal":"Nature cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP, K48-ubiquitination assay, multiple model systems including KO mice, single lab","pmids":["35122005"],"is_preprint":false},{"year":2013,"finding":"RBCK1 was identified as an FKBPL-interacting protein that regulates FKBPL stability via ubiquitination. Both RBCK1 and FKBPL are upregulated by 17-β-estradiol and interact within HSP90 chaperone complexes together with ERα. RBCK1 and FKBPL associate with ERα at the pS2 promoter and regulate pS2 expression.","method":"Co-immunoprecipitation, ubiquitination assay, chromatin immunoprecipitation, stable overexpression clones","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP, ubiquitination assay, and ChIP in multiple cellular models, single lab","pmids":["23912458"],"is_preprint":false},{"year":2010,"finding":"RBCK1 supports cell cycle progression in ERα-positive breast cancer cells by driving transcription of ERα and cyclin B1. Chromatin immunoprecipitation showed RBCK1 is recruited to the ERα promoter. RBCK1-depleted cells accumulate in G2-M phase (independent of ERα, associated with cyclin B1 reduction) and show decreased proliferation.","method":"siRNA knockdown, chromatin immunoprecipitation, cell cycle analysis, proliferation assay","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP plus loss-of-function with defined cell cycle phenotype, two mechanistic branches characterized, single lab","pmids":["20103625"],"is_preprint":false},{"year":2010,"finding":"Rbck1 (zebrafish ortholog) interacts with Eya1 protein (confirmed by GST pulldown and co-immunoprecipitation). Sipl1 and Rbck1 enhance the function of Eya proteins as coactivators for Six transcription factors. Morpholino-mediated knockdown of a Rbck1 ortholog in zebrafish produces a BOR syndrome-like phenotype with ear and branchial arch defects.","method":"GST pulldown, co-immunoprecipitation, morpholino knockdown in zebrafish, in situ hybridization, transcriptional coactivation assay","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal binding assays plus in vivo loss-of-function with defined developmental phenotype, single lab","pmids":["20956555"],"is_preprint":false},{"year":2006,"finding":"RBCK1 physically interacts with PKCbetaI and is a key regulator of PKCbetaI function in cardiac myocytes. Acute phenylephrine treatment transiently increases RBCK1-PKCbetaI association. RBCK1 overexpression increases cardiac cell size (hypertrophy) in a PKCbeta-dependent manner; RNAi of RBCK1 inhibits phenylephrine-induced hypertrophy.","method":"Co-immunoprecipitation, adenovirus-based overexpression, RNAi knockdown, PKCbeta selective antagonist treatment, cell size measurement","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP with pharmacological and genetic epistasis in primary cardiomyocytes, single lab","pmids":["17121852"],"is_preprint":false},{"year":2018,"finding":"HOIL1 is essential for induction of type I and type III interferons and IRF3 phosphorylation during MDA5-dependent RNA virus (murine norovirus, Theiler's virus, poly(I:C)) infection in dendritic cells, but not for RIG-I-dependent virus (Sendai virus, VSV) responses, demonstrating that LUBAC selectively promotes MDA5 signaling.","method":"HOIL1-deficient mouse dendritic cells, IFN induction assay, IRF3 phosphorylation assay, in vivo norovirus infection model","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO in primary immune cells, multiple viruses compared, in vivo infection model, pathway specificity (MDA5 vs RIG-I) established","pmids":["30209176"],"is_preprint":false},{"year":2024,"finding":"HOIL1 E3 ligase activity is critical for MDA5-dependent IFN induction. HOIL1 interacts with and ubiquitinates LGP2 (a positive regulator of MDA5 oligomerization), and HOIL1 E3 ligase activity promotes MDA5 oligomerization, translocation to mitochondrial-associated membranes, and formation of MAVS aggregates. HOIP E3 ligase activity plays only a modest role in IFN induction.","method":"Co-immunoprecipitation, ubiquitination assay, E3 ligase-dead mutants, MDA5 oligomerization assay, MAVS aggregate formation assay (preprint)","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — co-IP, ubiquitination assay, and mechanistic pathway assays, single lab, preprint","pmids":["38617308"],"is_preprint":true},{"year":2012,"finding":"Solution structure of the HOIL-1 Ubl domain was solved by NMR spectroscopy, revealing a β-grasp Ubl-fold. Structural comparison with parkin indicates HOIL-1 likely uses a conserved hydrophobic patch (W58, V102, Y127, Y129) and a C-terminal basic residue (R134) to recruit the S5a subunit of the 26S proteasome.","method":"NMR spectroscopy, structural comparison with parkin Ubl domain","journal":"Protein science","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — NMR structure solved but functional validation of S5a interaction is inferred from structural comparison without direct mutagenesis confirmation reported in abstract","pmids":["22517668"],"is_preprint":false},{"year":2016,"finding":"HOXA1 physically interacts with RBCK1/HOIL-1 (confirmed by proteome-wide screening and functional assays). HOXA1-mediated activation of NF-κB is non-transcriptional and requires RBCK1 and TRAF2. Genetic epistasis shows that RBCK1 and TRAF2 influences on NF-κB are epistatic to HOXA1 (RBCK1 acts downstream of HOXA1 in NF-κB activation). An 11 His repeat and the homeodomain of HOXA1 are required for RBCK1 interaction.","method":"Proteome-wide interaction screening, co-immunoprecipitation, NF-κB reporter assay, domain mapping, epistasis analysis","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — interaction confirmed by co-IP, epistasis established by reporter assay, domain mapping performed, single lab","pmids":["27382069"],"is_preprint":false},{"year":2022,"finding":"RBCK1 binds to YAP protein and promotes K48-linked poly-ubiquitination of YAP at lysine residues K76, K204, and K321, leading to YAP protein degradation and suppression of Hippo/YAP signaling in triple-negative breast cancer cells. RBCK1 overexpression inhibits TNBC cell progression in vitro and in vivo.","method":"Co-immunoprecipitation, ubiquitin-based immunoprecipitation, K48-specific ubiquitination assay, protein stability assay, RNA sequencing, in vivo xenograft","journal":"Cell communication and signaling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — site-specific K48 ubiquitination assay with multiple YAP lysine sites identified, in vivo confirmation, single lab","pmids":["36280829"],"is_preprint":false},{"year":2022,"finding":"RBCK1 promotes hepatocellular carcinoma metastasis and growth by stabilizing its LUBAC partner RNF31 (HOIP): RBCK1 interacts with RNF31 and represses its ubiquitination and proteasomal degradation.","method":"Co-immunoprecipitation, ubiquitination assay, knockdown/overexpression functional assays","journal":"Cell death discovery","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — single lab, co-IP and ubiquitination assay establishing stabilization mechanism","pmids":["35869046"],"is_preprint":false},{"year":2023,"finding":"HOIL-1 interacts with Numb via its A64/Q65 residues binding K78 of Numb, impairing Numb-mediated Notch1 lysosomal degradation and thereby activating Notch1 signaling to promote HCC stemness and sorafenib resistance, independently of HOIL-1 ubiquitin ligase activity.","method":"Mass spectrometry, co-immunoprecipitation, western blot, immunofluorescence, in vivo HCC models","journal":"Hepatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — interaction mapped to specific residues by MS and co-IP, functional readout in vivo, mechanistic link to Notch1 established, single lab","pmids":["37820061"],"is_preprint":false},{"year":2022,"finding":"In RBCK1-deficient mice, glycogen accumulates as polyglucosans with overlong branches and hyperphosphorylation limited to precipitated polyglucosans (similar to malin-deficient Lafora disease). Downregulating glycogen synthase (the enzyme that elongates glycogen branches) rescues RBCK1 deficiency amylopectinosis, linking RBCK1 function to glycogen branch-length regulation.","method":"RBCK1 knockout mouse model, glycogen structural analysis, genetic rescue by glycogen synthase downregulation, behavioral testing, neuroinflammation assessment","journal":"Brain : a journal of neurology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse model with glycogen biochemical characterization and genetic rescue demonstrating pathway position","pmids":["35084461"],"is_preprint":false},{"year":2021,"finding":"In HOIL1-deficient mice, loss of HOIL1 in radiation-resistant cells (not intestinal epithelial cells) leads to type 2 intestinal inflammation with tuft cell and goblet cell hyperplasia and elevated IL-13, IL-5, IL-25, including expansion of KLRG1hi CD90lo group 2 innate lymphoid cells. This inflammation is partly dependent on commensal microbiota.","method":"HOIL1-deficient mouse model, bone marrow chimeras, cell-type-specific knockouts, IL-4Rα signaling disruption, antibiotic treatment, immune profiling","journal":"Mucosal immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with multiple complementary mouse models (chimeras, conditional KOs) defining cell-type requirement, single lab","pmids":["35534698"],"is_preprint":false},{"year":2023,"finding":"HIF-1α upregulates RBCK1, which then interacts with and ubiquitinates PICK1, facilitating its proteasomal degradation in nasopharyngeal carcinoma cells under hypoxia. RBCK1 knockdown inhibited NPC proliferation, an effect rescued by double knockdown of RBCK1/PICK1.","method":"Co-immunoprecipitation, immunofluorescence, ubiquitination assay, siRNA knockdown, rescue assay, in vivo xenograft","journal":"Life sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — single lab, co-IP plus ubiquitination assay and genetic rescue, moderate mechanistic depth","pmids":["36934971"],"is_preprint":false},{"year":2022,"finding":"RBCK1 promotes PPARγ ubiquitination and degradation, leading to activation of WNT/β-catenin/GLUT1 pathway and enhanced aerobic glycolysis in hepatocellular carcinoma cells. RBCK1-induced HCC cell migration and aerobic glycolysis depend on destruction of the PPARγ/PGC1α complex.","method":"Co-immunoprecipitation, ubiquitination assay, WNT/β-catenin activity assay, metabolic (GLUT1/glycolysis) assays, migration assay","journal":"American journal of cancer research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, co-IP and ubiquitination assay with functional readouts but limited mechanistic depth; no in vitro reconstitution","pmids":["35411229"],"is_preprint":false},{"year":2025,"finding":"HOIL-1 is required for cytoprotective ribosome-associated quality control. HOIL-1 promotes ribosome ubiquitination and its loss causes glucose starvation to become ribotoxic, leading to ZAKα (MAP3K)-dependent ATF4 activation and disulfidptosis driven by the cystine-glutamate antiporter xCT. Truncating HOIL-1 mutations associated with dilated cardiomyopathy exacerbate cardiac dysfunction in mice.","method":"HOIL-1 loss-of-function mouse models, ribosome ubiquitination assay, ZAKα genetic epistasis, ATF4 activation assay, nutrient stress experiments","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — ribosome ubiquitination biochemistry, genetic epistasis (ZAKα), in vivo mouse cardiac model, multiple stress paradigms in single rigorous study","pmids":["42062483"],"is_preprint":false},{"year":2026,"finding":"The C-terminal fragment of HOIL-1 generated by MALT1 cleavage (C-HOIL-1) has novel biological functions: it inhibits NF-κB signaling, interacts with STAT1 to downregulate STAT1-mediated inflammatory signaling, and upregulates ARG1 expression, collectively suppressing inflammatory responses in monocytes/macrophages and impeding M1 macrophage differentiation. Mice with uncleavable HOIL-1 (lacking C-HOIL-1) develop more severe DSS-induced colitis with elevated monocyte/macrophage/neutrophil infiltration.","method":"Transgenic uncleavable HOIL-1 mice (global and myeloid-specific), genetically engineered THP-1 cells expressing C-HOIL-1, co-immunoprecipitation (STAT1 interaction), DSS-induced colitis model, lentiviral C-HOIL-1 delivery","journal":"Theranostics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — transgenic mouse models plus cell-based mechanistic assays identifying STAT1 interaction, single lab","pmids":["41799187"],"is_preprint":false},{"year":2007,"finding":"HOIL-1 and IRP2 interact via the IRP2 73-amino acid domain in HEK293 cells (confirmed by co-immunoprecipitation), but this interaction is NOT iron-dependent, and HOIL-1 does NOT enhance the rate of IRP2 degradation by iron. Stable HOIL-1 expression does not alter iron-dependent degradation of endogenous IRP2; HOIL-1 siRNA knockdown has no effect on iron-mediated IRP2 degradation.","method":"Co-immunoprecipitation, stable expression, siRNA knockdown, iron treatment, IRP2 degradation assay","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal approaches (stable expression, siRNA, co-IP) all yielding negative result for HOIL-1 role in IRP2 degradation in HEK293 cells","pmids":["17822790"],"is_preprint":false}],"current_model":"RBCK1/HOIL-1 is an RBR-family E3 ubiquitin ligase that functions primarily as a component of LUBAC, where it forms atypical oxyester (serine/threonine) ubiquitin linkages on substrates including Myddosome components (IRAK1, IRAK2, MyD88), glycogen, and ribosomes, enabling monoubiquitylation that seeds hybrid polyubiquitin chains; its unique catalytic mechanism—requiring a bi-nuclear Zn-cluster RING2 with a critical histidine (His510) as the catalytic base—also drives negative feedback of NF-κB signaling via MALT1-mediated proteolytic cleavage of HOIL-1 at antigen receptor signalosomes, and it regulates multiple additional substrates (IRF3, TAB2/3, p53, PTEN, YAP, MFN2, PXR, PPARγ, LGP2) through K48-linked ubiquitination and proteasomal degradation, while its splice variant RBCK2 inhibits both its E3 activity and nuclear localization, and its nuclear-cytoplasmic shuttling (governed by N-terminal NES and C-terminal RING-IBR NLS) is regulated by PKCβ phosphorylation and interaction with CBP/PML in nuclear bodies."},"narrative":{"mechanistic_narrative":"RBCK1 (HOIL-1) is an RBR-family E3 ubiquitin ligase that functions both as a catalytic subunit of the linear ubiquitin chain assembly complex (LUBAC) and as a stand-alone enzyme with an unusual chemistry, governing innate and adaptive immune signaling, cellular quality control, and glycogen metabolism [PMID:31209050, PMID:23104095, PMID:42062483]. Its defining biochemical feature is the formation of atypical oxyester bonds between the ubiquitin C-terminus and serine/threonine residues — and even mono- and disaccharides — rather than the canonical lysine isopeptide linkage; this O-linked monoubiquitylation seeds de novo polyubiquitin chains on Myddosome components IRAK1, IRAK2, and MyD88 during TLR signaling [PMID:31209050, PMID:40169258]. This activity depends on a unique C-terminal bi-nuclear Zn-cluster that replaces the second zinc finger of the canonical RING2 fold and on a catalytic histidine (His510) that acts as the catalytic base, enabling oxyester transfer while prohibiting discharge onto lysine; substrate engagement also requires binding of linear tetra-ubiquitin [PMID:36685275, PMID:40169258]. Through LUBAC, HOIL-1 controls NF-κB activation downstream of IL-1β and antigen receptors, and patient loss-of-function mutations destabilize LUBAC and produce cell-type-specific dysregulation of NF-κB [PMID:23104095]. A central negative-feedback axis is set by MALT1 paracaspase cleavage of HOIL-1 at antigen receptor signalosomes, which generates a C-terminal fragment with LUBAC-inhibitory and broader anti-inflammatory activity (including STAT1 engagement), dampening lymphocyte and myeloid inflammatory responses [PMID:26525107, PMID:26573773, PMID:27006117, PMID:41799187]. HOIL-1 also drives MDA5-selective type I/III interferon induction during RNA virus infection [PMID:30209176]. Beyond immunity, HOIL-1 is required for cytoprotective ribosome-associated quality control, ubiquitinating ribosomes to prevent nutrient-stress ribotoxicity, and regulates glycogen branch-length through its action on polyglucosan structures [PMID:42062483, PMID:35084461]. In numerous cancer contexts RBCK1 additionally catalyzes K48-linked polyubiquitination and proteasomal degradation of regulatory substrates including p53, PTEN, and YAP [PMID:30874541, PMID:35174471, PMID:36280829].","teleology":[{"year":1998,"claim":"Established that a single RBCK1 gene yields two splice products and that RBCK1 possesses intrinsic transcriptional activity requiring its RING and B-Box motifs, with the RING-IBR-lacking variant RBCK2 acting as an inhibitor — the first functional dissection of the locus.","evidence":"In vitro interaction and GAL4-chimeric transcription reporter assays with mutational analysis","pmids":["9755849","9642138"],"confidence":"Medium","gaps":["Transcriptional mechanism not connected to a defined promoter or DNA-binding partner","No endogenous target genes identified at this stage"]},{"year":2005,"claim":"Defined how RBCK1 subcellular distribution is encoded, mapping an N-terminal NES and a C-terminal RING-IBR NLS and showing RBCK1 localizes to nuclear bodies with CBP/PML and that RBCK2 tethers it in the cytoplasm.","evidence":"Leptomycin B treatment, signal mutagenesis, co-IP, fractionation, and co-expression localization assays","pmids":["15833741","16083853"],"confidence":"High","gaps":["Functional consequence of nuclear-body residence not linked to ubiquitin ligase activity","RBCK2 tethering shown in overexpression context only"]},{"year":2008,"claim":"Demonstrated RBCK1 has in vitro E3 ligase activity that is regulated by RBCK2 binding and by PKCβ phosphorylation, coupling its enzymatic output to a kinase input.","evidence":"In vitro ubiquitin ligase assay, co-IP, and PKCβ overexpression/inhibition","pmids":["18303026"],"confidence":"High","gaps":["Phosphosites on RBCK1 not mapped","Physiological substrates of this E3 activity not yet defined"]},{"year":2008,"claim":"Positioned RBCK1 as a negative-feedback regulator of innate immune signaling by showing it targets TAB2/TAB3 and IRF3 for proteasomal degradation, restraining NF-κB and type I interferon responses.","evidence":"Co-IP, ubiquitination assays, overexpression/knockdown with NF-κB reporter and antiviral plaque readouts","pmids":["17449468","18711448"],"confidence":"Medium","gaps":["No in vitro reconstitution with purified components","Ubiquitin linkage type not defined at this stage"]},{"year":2012,"claim":"Linked RBCK1/HOIL-1 to human disease and LUBAC biology, showing biallelic loss-of-function mutations destabilize LUBAC and cause cell-type-specific NF-κB dysregulation.","evidence":"Patient-derived fibroblasts and monocytes with NF-κB signaling assays and genetic analysis","pmids":["23104095"],"confidence":"High","gaps":["Molecular basis for opposite fibroblast vs monocyte responses not resolved","Direct LUBAC substrates affected not enumerated"]},{"year":2012,"claim":"Solved the HOIL-1 Ubl-domain structure, providing a structural rationale for proteasome recruitment via a conserved hydrophobic patch.","evidence":"NMR spectroscopy with structural comparison to parkin","pmids":["22517668"],"confidence":"Medium","gaps":["S5a/proteasome interaction inferred from comparison, not confirmed by direct mutagenesis","Functional contribution of Ubl domain to substrate degradation untested here"]},{"year":2016,"claim":"Identified MALT1 paracaspase cleavage of HOIL-1 as a negative-feedback brake on antigen-receptor-driven NF-κB activation, with the cleavage fragments having distinct activities.","evidence":"TAILS N-terminal proteomics, co-IP, NF-κB reporter, and antigen receptor stimulation with cleavage-resistant mutants","pmids":["26525107","26573773","27006117"],"confidence":"High","gaps":["Precise molecular activity of the inhibitory C-terminal fragment not yet defined","Kinetics relative to LUBAC assembly incompletely resolved"]},{"year":2018,"claim":"Revealed pathway selectivity in antiviral signaling, showing HOIL-1/LUBAC is required for MDA5-dependent but not RIG-I-dependent interferon induction.","evidence":"HOIL1-deficient mouse dendritic cells, IRF3 phosphorylation and IFN induction assays, in vivo norovirus infection","pmids":["30209176"],"confidence":"High","gaps":["Direct molecular substrate in the MDA5 pathway not identified in this study","Catalytic requirement vs scaffolding role not separated"]},{"year":2019,"claim":"Established the unprecedented catalytic chemistry of HOIL-1, demonstrating it forms oxyester (Ser/Thr) bonds and acts as a monoubiquitylating initiator on Myddosome substrates in vivo.","evidence":"Hydroxylamine cleavage, catalytically inactive HOIL-1[C458S] knock-in mice, mass spectrometry, in vitro ubiquitination","pmids":["31209050"],"confidence":"High","gaps":["Full repertoire of physiological oxyester substrates not defined","How hybrid chain elongation by HOIP is coordinated not fully resolved"]},{"year":2023,"claim":"Provided the structural and mechanistic basis for oxyester catalysis, identifying a bi-nuclear Zn-cluster RING2 and a catalytic histidine, and showing linear tetra-ubiquitin binding licenses substrate ubiquitylation.","evidence":"X-ray crystallography of IBR-RING2, catalytic-residue mutagenesis, in vitro ubiquitylation; extended by His510 biochemical characterization","pmids":["36685275","40169258"],"confidence":"High","gaps":["Structural basis for substrate Ser/Thr selection not visualized with bound substrate","How linear-Ub binding allosterically activates the active site not detailed"]},{"year":2022,"claim":"Connected HOIL-1 to glycogen metabolism, showing its loss produces malin-like polyglucosans with abnormal branch length that are rescued by reducing glycogen synthase.","evidence":"RBCK1 knockout mice, glycogen structural analysis, genetic rescue by glycogen synthase downregulation","pmids":["35084461"],"confidence":"Medium","gaps":["Direct enzymatic target linking RBCK1 to branch-length control not defined","Relationship to oxyester ubiquitylation of carbohydrate not established in this study"]},{"year":2025,"claim":"Defined a cytoprotective ribosome-associated quality-control role, with HOIL-1 ubiquitinating ribosomes to prevent nutrient-stress ribotoxicity and disulfidptosis, and linked truncating mutations to dilated cardiomyopathy.","evidence":"HOIL-1 loss-of-function mouse models, ribosome ubiquitination assays, ZAKα genetic epistasis, ATF4 readouts, in vivo cardiac model","pmids":["42062483"],"confidence":"High","gaps":["Specific ribosomal residues/proteins ubiquitinated not enumerated","Whether ribosome ubiquitylation uses oxyester chemistry not stated"]},{"year":2026,"claim":"Resolved the function of the MALT1-generated C-terminal HOIL-1 fragment, showing it suppresses NF-κB and STAT1 signaling and limits M1 macrophage differentiation in vivo.","evidence":"Uncleavable HOIL-1 transgenic mice, engineered THP-1 cells, STAT1 co-IP, DSS colitis model","pmids":["41799187"],"confidence":"Medium","gaps":["Mechanism of STAT1 inhibition not biochemically detailed","Single-lab findings without independent confirmation"]},{"year":null,"claim":"How HOIL-1's single active site is partitioned between LUBAC-associated immune signaling, ribosome quality control, glycogen-branch regulation, and the many reported K48-degradative cancer substrates — and whether all use the same oxyester chemistry — remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["Linkage chemistry (oxyester vs K48 isopeptide) not reconciled across the diverse reported substrates","Substrate-selection determinants in vivo unknown","Many cancer substrate findings rest on single-lab co-IP/ubiquitination assays without reconstitution"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,3,7,8]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,2,27]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[4,6,20]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[10,35]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[4,5]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[4]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,9,10,23]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[8,26]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,34]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[34]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[30]}],"complexes":["LUBAC"],"partners":["RNF31","SHARPIN","TAB2","TAB3","MALT1","PKCB","RBCK2","SOCS6"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9BYM8","full_name":"RanBP-type and C3HC4-type zinc finger-containing protein 1","aliases":["HBV-associated factor 4","Heme-oxidized IRP2 ubiquitin ligase 1","HOIL-1","Hepatitis B virus X-associated protein 4","RING finger protein 54","RING-type E3 ubiquitin transferase HOIL-1","Ubiquitin-conjugating enzyme 7-interacting protein 3"],"length_aa":510,"mass_kda":57.6,"function":"E3 ubiquitin-protein ligase, which accepts ubiquitin from specific E2 ubiquitin-conjugating enzymes, such as UBE2L3/UBCM4, and then transfers it to substrates (PubMed:12629548, PubMed:17449468, PubMed:18711448). Functions as an E3 ligase for oxidized IREB2 and both heme and oxygen are necessary for IREB2 ubiquitination (PubMed:12629548). Promotes ubiquitination of TAB2 and IRF3 and their degradation by the proteasome (PubMed:17449468, PubMed:18711448). 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:21455173, PubMed:21455180, PubMed:21455181). 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:21455173, PubMed:21455180, PubMed:21455181). Linear ubiquitination mediated by the LUBAC complex interferes with TNF-induced cell death and thereby prevents inflammation (PubMed:17006537, PubMed:21455173, PubMed:21455180, PubMed:21455181). 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:17006537, PubMed:19136968, PubMed:21455173, PubMed:21455180, PubMed:21455181). 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). 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). The bacterial ubiquitin coat acts as an 'eat-me' signal for xenophagy and promotes NF-kappa-B activation (PubMed:28481331). Together with OTULIN, the LUBAC complex regulates the canonical Wnt signaling during angiogenesis (PubMed:23708998). 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IMD115","url":"https://www.omim.org/entry/620632"},{"mim_id":"617076","title":"FK506-BINDING PROTEIN-LIKE; FKBPL","url":"https://www.omim.org/entry/617076"},{"mim_id":"615895","title":"POLYGLUCOSAN BODY MYOPATHY 1 WITH OR WITHOUT IMMUNODEFICIENCY; PGBM1","url":"https://www.omim.org/entry/615895"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/RBCK1"},"hgnc":{"alias_symbol":["RBCK2","XAP4","RNF54","ZRANB4","UBCE7IP3","HOIL1"],"prev_symbol":["C20orf18"]},"alphafold":{"accession":"Q9BYM8","domains":[{"cath_id":"-","chopping":"1-43","consensus_level":"medium","plddt":89.1098,"start":1,"end":43},{"cath_id":"3.10.20.90","chopping":"54-131","consensus_level":"high","plddt":88.3024,"start":54,"end":131},{"cath_id":"3.30.40.10","chopping":"279-364","consensus_level":"high","plddt":94.8028,"start":279,"end":364},{"cath_id":"-","chopping":"442-510","consensus_level":"medium","plddt":88.5671,"start":442,"end":510}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BYM8","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BYM8-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BYM8-F1-predicted_aligned_error_v6.png","plddt_mean":84.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RBCK1","jax_strain_url":"https://www.jax.org/strain/search?query=RBCK1"},"sequence":{"accession":"Q9BYM8","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9BYM8.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9BYM8/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BYM8"}},"corpus_meta":[{"pmid":"23104095","id":"PMC_23104095","title":"Immunodeficiency, 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biology","url":"https://pubmed.ncbi.nlm.nih.gov/38617308","citation_count":3,"is_preprint":false},{"pmid":"34322999","id":"PMC_34322999","title":"Ester-linked ubiquitination by HOIL-1 controls immune signalling by shaping the linear ubiquitin landscape.","date":"2021","source":"The FEBS journal","url":"https://pubmed.ncbi.nlm.nih.gov/34322999","citation_count":3,"is_preprint":false},{"pmid":"34310740","id":"PMC_34310740","title":"RBCK1-TRIB3 decelerated the progression of acute promyelocytic leukemia.","date":"2021","source":"Hematological oncology","url":"https://pubmed.ncbi.nlm.nih.gov/34310740","citation_count":3,"is_preprint":false},{"pmid":"37902285","id":"PMC_37902285","title":"HOIL1 Regulates Group 3 Innate Lymphoid Cells in the Colon and Protects against Systemic Dissemination, Colonic Ulceration, and Lethality from Citrobacter rodentium Infection.","date":"2023","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/37902285","citation_count":2,"is_preprint":false},{"pmid":"37928949","id":"PMC_37928949","title":"The E3 ubiquitin ligase RBCK1: Implications in the tumor immune microenvironment and antiangiogenic therapy of glioma.","date":"2023","source":"Computational and structural biotechnology journal","url":"https://pubmed.ncbi.nlm.nih.gov/37928949","citation_count":2,"is_preprint":false},{"pmid":"41053347","id":"PMC_41053347","title":"The LUBAC subunit HOIL-1 promotes the progression of HBV-associated hepatocellular carcinoma independently of linear ubiquitination.","date":"2025","source":"Experimental & molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/41053347","citation_count":1,"is_preprint":false},{"pmid":"38922716","id":"PMC_38922716","title":"Phenotypic and genotyping spectrum of two Iranian cases with RBCK1-associated polyglucosan body myopathy.","date":"2024","source":"Neuropathology : official journal of the Japanese Society of Neuropathology","url":"https://pubmed.ncbi.nlm.nih.gov/38922716","citation_count":1,"is_preprint":false},{"pmid":"40939526","id":"PMC_40939526","title":"Identification of a novel RBCK1 splice site donor variant in Basset Hounds with glycogen storage disease myopathy.","date":"2025","source":"Molecular genetics and metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/40939526","citation_count":1,"is_preprint":false},{"pmid":"40588086","id":"PMC_40588086","title":"RBCK1 enhances antiviral response through promoting K63-linked polyubiquitination of IRF7 in Carassius auratus var. 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Using hydroxylamine cleavage of oxyester bonds and knock-in mice expressing E3 ligase-inactive HOIL-1[C458S], IRAK1, IRAK2, and MyD88 (Myddosome components) were identified as physiological substrates during Toll-like receptor signaling. HOIL-1 acts as a monoubiquitylating E3 that initiates de novo polyubiquitin chain synthesis on these proteins, and also catalyzes its own monoubiquitylation and likely that of Sharpin via oxyester linkages.\",\n      \"method\": \"Hydroxylamine cleavage assay, knock-in mice expressing catalytically inactive HOIL-1[C458S], mass spectrometry, in vitro ubiquitination assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with active-site mutagenesis, orthogonal chemical method (hydroxylamine cleavage), and genetic knock-in mouse validation in a single rigorous study\",\n      \"pmids\": [\"31209050\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Crystal structure of the C-terminal tandem IBR-RING2 domain of HOIL-1 reveals a unique bi-nuclear Zn-cluster substituting the second zinc finger of the canonical RING2 fold. The C-terminal histidine of this Zn-cluster was identified as the catalytic base required for HOIL-1 ubiquitylation activity. HOIL-1 activity also requires binding of linear tetra-ubiquitin, which enables mono-ubiquitylation of linear Ub chains and polysaccharides.\",\n      \"method\": \"X-ray crystallography, mutagenesis of catalytic residues, in vitro ubiquitylation assay\",\n      \"journal\": \"Frontiers in molecular biosciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure combined with mutagenesis and functional in vitro assay in a single study\",\n      \"pmids\": [\"36685275\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"HOIL-1 ubiquitinates serine residues and diverse mono- and disaccharides via O-linked ubiquitination but cannot ubiquitinate lysine. A critical catalytic histidine residue, His510, in the flexible catalytic site of HOIL-1 enables O-linked ubiquitination and prohibits ubiquitin discharge onto lysine sidechains.\",\n      \"method\": \"In vitro ubiquitination assays with model substrates, mutagenesis of His510, biochemical characterization\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with active-site mutagenesis identifying catalytic residue, multiple substrates tested\",\n      \"pmids\": [\"40169258\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"RBCK1 E3 ubiquitin ligase activity was demonstrated in vitro. This E3 activity is inhibited by interaction with its splice variant RBCK2 (which lacks the RING-IBR domain). Additionally, phosphorylation of RBCK1 by PKCbeta abolishes self-ubiquitination activity in vitro, and PKCbeta overexpression increases intracellular RBCK1 levels by suppressing its proteasomal degradation.\",\n      \"method\": \"In vitro ubiquitin ligase assay, co-immunoprecipitation, PKCbeta overexpression/inhibition\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro E3 assay with mechanistic follow-up (splice variant inhibition and kinase phosphorylation), multiple orthogonal approaches in single study\",\n      \"pmids\": [\"18303026\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"RBCK1 shuttles between cytoplasm and nucleus via defined nuclear export and localization signals: an N-terminal region containing Leu-142/Leu-145 serves as the nuclear export signal, and the C-terminal RING-IBR domain serves as the nuclear localization signal. RBCK1 localizes to nuclear bodies and interacts with CBP and PML. CBP co-expression enhances RBCK1 transcriptional activity, while PML represses CBP-enhanced activity.\",\n      \"method\": \"Leptomycin B treatment, mutational analysis, co-immunoprecipitation, live-cell imaging/fractionation, transcriptional reporter assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization experiments with functional consequence, mutational mapping of signals, multiple orthogonal methods in single study\",\n      \"pmids\": [\"15833741\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"RBCK2, the splice variant of RBCK1 lacking the RING-IBR domain, is cytoplasmic and contains Leu-rich nuclear export signals. RBCK2 acts as a cytoplasmic tethering protein for RBCK1 by forming a hetero-oligomeric complex, causing nuclear RBCK1 to relocalize to the cytoplasm when RBCK2 is co-expressed.\",\n      \"method\": \"Co-expression of NES-disrupted RBCK1 with RBCK2, immunofluorescence/localization assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — single lab, direct localization experiment with functional consequence (nuclear export via heterocomplex), single method\",\n      \"pmids\": [\"16083853\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"RBCK1 and RBCK2 are generated from a single gene by alternative splicing. RBCK1 protein interacts with both RBCK1 and RBCK2 in vitro, but RBCK2 does not self-interact. RBCK2 inhibits the transcriptional activity of RBCK1, likely through complex formation. Both RING-finger and B-Box motifs of RBCK1 are indispensable for its transcriptional activity.\",\n      \"method\": \"In vitro interaction assay, GAL4-chimeric transcription assay, mutational analysis\",\n      \"journal\": \"FEBS letters / Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two separate papers using in vitro binding and reporter assays establishing the same mechanism\",\n      \"pmids\": [\"9755849\", \"9642138\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"RBCK1 (E3 ubiquitin ligase) catalyzes K48-linked ubiquitination and proteasomal degradation of IRF3, acting as a negative feedback regulator of virus-triggered type I interferon induction. Viral infection induces RBCK1 expression, leading to subsequent IRF3 degradation. Overexpression of RBCK1 reduces antiviral IFN responses; knockdown has the opposite effect.\",\n      \"method\": \"Overexpression/knockdown, ubiquitination assay, plaque assay, immunoblot\",\n      \"journal\": \"Cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — single lab, ubiquitination assay plus functional antiviral readout, no in vitro reconstitution with purified proteins\",\n      \"pmids\": [\"18711448\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"RBCK1 physically interacts with TAB2 and TAB3 adapter proteins and facilitates their proteasome-dependent degradation, thereby negatively regulating TNF- and IL-1-induced NF-κB activation. Overexpression of RBCK1 inhibits TAB2/3-mediated NF-κB activation; knockdown potentiates it.\",\n      \"method\": \"Co-immunoprecipitation, overexpression/RNAi, NF-κB reporter assay, proteasome inhibitor treatment\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — single lab, reciprocal co-IP plus functional NF-κB readout with RNAi rescue, but no in vitro reconstitution\",\n      \"pmids\": [\"17449468\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"HOIL-1 (RBCK1) deficiency in patients carrying biallelic loss-of-function HOIL1 mutations impairs LUBAC stability and compromises NF-κB activation in response to IL-1β in fibroblasts, while monocytes from the same patients are hyper-responsive to IL-1β, demonstrating cell-type-specific regulation of NF-κB by HOIL-1/LUBAC.\",\n      \"method\": \"Patient-derived cells (fibroblasts and monocytes), NF-κB signaling assays, genetic analysis of loss-of-function mutations\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — human genetic loss-of-function with mechanistic pathway (NF-κB) readout in patient-derived cells, replicated across two kindreds\",\n      \"pmids\": [\"23104095\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"MALT1 paracaspase cleaves HOIL-1 late in the NF-κB activation cycle during antigen receptor signaling. Cleavage generates an N-terminal fragment that retains support for HOIP-induced NF-κB signaling and a C-terminal fragment with LUBAC inhibitory properties, providing negative feedback regulation of NF-κB. This cleavage transiently reduces linear ubiquitination of NEMO and RIP1.\",\n      \"method\": \"10-plex TMT TAILS N-terminal peptide proteomics, co-immunoprecipitation, NF-κB reporter assay, B-cell receptor signaling assays\",\n      \"journal\": \"Nature communications / The FEBS journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — substrate identified by proteomics, confirmed by multiple functional assays, replicated by independent groups (Klein et al. and Elton et al.)\",\n      \"pmids\": [\"26525107\", \"26573773\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"MALT1-mediated cleavage of HOIL1 occurs following antigen receptor engagement in T and B lymphocytes. Overexpression of MALT1-insensitive HOIL1 mitigates TCR-mediated NF-κB activation and cytokine production, establishing HOIL1 as a negative regulator of lymphocyte activation that is removed by MALT1 cleavage.\",\n      \"method\": \"Antigen receptor stimulation assays, overexpression of cleavage-resistant HOIL1, NF-κB reporter assay, cytokine measurement\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional NF-κB and cytokine readouts with gain-of-function cleavage-resistant mutant, consistent with two other groups\",\n      \"pmids\": [\"27006117\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Non-cleavable HOIL-1 (MALT1-resistant) expressed in primary HOIL-1-deficient patient fibroblasts results in enhanced NF-κB signaling and a hyperinflammatory cytokine profile compared to wild-type HOIL-1, demonstrating that MALT1-dependent HOIL-1 cleavage is physiologically required to dampen inflammatory responses.\",\n      \"method\": \"Stable transduction of patient fibroblasts, NF-κB assays, transcriptomics, multiplexed cytokine assays\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — patient-derived cells with isogenic comparison, transcriptomics plus functional cytokine readout, single lab\",\n      \"pmids\": [\"34721419\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"HOIL-1 (RBCK1) interacts with SOCS6, requiring HOIL-1's Ubl domain and SOCS6's SH2 and Socs-box domains. HOIL-1 expression stabilizes SOCS6 itself but induces the ubiquitination and proteasomal degradation of proteins associated with SOCS6.\",\n      \"method\": \"Co-immunoprecipitation, domain mapping, ubiquitination assay, proteasome inhibitor treatment\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — single lab, co-IP with domain mapping plus ubiquitination assay, moderate mechanistic depth\",\n      \"pmids\": [\"16643902\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"RBCK1 interacts with hPXR (pregnane X receptor) via all RBCK1 domains (identified by yeast two-hybrid and confirmed by co-IP), ubiquitinates hPXR, and targets it for proteasomal degradation. Overexpression of RBCK1 decreased endogenous PXR in HepG2 cells and primary hepatocytes; RBCK1 silencing increased PXR levels and enhanced induction of PXR target genes.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, ubiquitination assay, proteasome inhibitor (MG-132) treatment, siRNA knockdown in primary hepatocytes\",\n      \"journal\": \"Drug metabolism and disposition\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid confirmed by co-IP, ubiquitination assay, and functional readout in primary cells, single lab\",\n      \"pmids\": [\"23160820\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RBCK1 facilitates poly-ubiquitination and proteasomal degradation of p53 via direct interaction, promoting renal cell carcinoma proliferation. Knockdown of RBCK1 in p53 wild-type RCC cells reduces proliferation; this effect is rescued by p53 knockdown.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, knockdown/rescue experiments, in vivo xenograft\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — co-IP plus ubiquitination assay with genetic rescue, single lab\",\n      \"pmids\": [\"30874541\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RBCK1 interacts with PTEN and promotes K48-linked polyubiquitination and proteasomal degradation of PTEN in ovarian cancer cells, facilitating tumor progression.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay with K48-linkage specificity, knockdown/overexpression\",\n      \"journal\": \"Human cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — single lab, co-IP plus K48-specific ubiquitination assay, no in vitro reconstitution\",\n      \"pmids\": [\"35174471\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RBCK1 interacts with and polyubiquitylates mitofusin 2 (MFN2) to facilitate its proteasomal degradation under ferroptotic stress, leading to decreased mitochondrial ROS production and lipid peroxidation, thereby conferring ferroptosis resistance in pancreatic cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, RBCK1 depletion/overexpression, in vivo xenograft, measurement of mitochondrial ROS and lipid peroxidation\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — single lab, co-IP plus functional ferroptosis readout, no in vitro reconstitution\",\n      \"pmids\": [\"38763208\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MTSS1 interacts with RBCK1 to facilitate RBCK1-mediated K48-linked polyubiquitination and degradation of NF-κB p65, suppressing NF-κB signaling and tumor-initiating cell properties in breast cancer. ACTBL2 competes with RBCK1 for MTSS1 binding, leading to p65 stabilization.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, Mtss1 knockout mouse models, tumorsphere assay, xenograft\",\n      \"journal\": \"Nature cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP, K48-ubiquitination assay, multiple model systems including KO mice, single lab\",\n      \"pmids\": [\"35122005\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"RBCK1 was identified as an FKBPL-interacting protein that regulates FKBPL stability via ubiquitination. Both RBCK1 and FKBPL are upregulated by 17-β-estradiol and interact within HSP90 chaperone complexes together with ERα. RBCK1 and FKBPL associate with ERα at the pS2 promoter and regulate pS2 expression.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, chromatin immunoprecipitation, stable overexpression clones\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, ubiquitination assay, and ChIP in multiple cellular models, single lab\",\n      \"pmids\": [\"23912458\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"RBCK1 supports cell cycle progression in ERα-positive breast cancer cells by driving transcription of ERα and cyclin B1. Chromatin immunoprecipitation showed RBCK1 is recruited to the ERα promoter. RBCK1-depleted cells accumulate in G2-M phase (independent of ERα, associated with cyclin B1 reduction) and show decreased proliferation.\",\n      \"method\": \"siRNA knockdown, chromatin immunoprecipitation, cell cycle analysis, proliferation assay\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP plus loss-of-function with defined cell cycle phenotype, two mechanistic branches characterized, single lab\",\n      \"pmids\": [\"20103625\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Rbck1 (zebrafish ortholog) interacts with Eya1 protein (confirmed by GST pulldown and co-immunoprecipitation). Sipl1 and Rbck1 enhance the function of Eya proteins as coactivators for Six transcription factors. Morpholino-mediated knockdown of a Rbck1 ortholog in zebrafish produces a BOR syndrome-like phenotype with ear and branchial arch defects.\",\n      \"method\": \"GST pulldown, co-immunoprecipitation, morpholino knockdown in zebrafish, in situ hybridization, transcriptional coactivation assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal binding assays plus in vivo loss-of-function with defined developmental phenotype, single lab\",\n      \"pmids\": [\"20956555\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"RBCK1 physically interacts with PKCbetaI and is a key regulator of PKCbetaI function in cardiac myocytes. Acute phenylephrine treatment transiently increases RBCK1-PKCbetaI association. RBCK1 overexpression increases cardiac cell size (hypertrophy) in a PKCbeta-dependent manner; RNAi of RBCK1 inhibits phenylephrine-induced hypertrophy.\",\n      \"method\": \"Co-immunoprecipitation, adenovirus-based overexpression, RNAi knockdown, PKCbeta selective antagonist treatment, cell size measurement\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP with pharmacological and genetic epistasis in primary cardiomyocytes, single lab\",\n      \"pmids\": [\"17121852\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"HOIL1 is essential for induction of type I and type III interferons and IRF3 phosphorylation during MDA5-dependent RNA virus (murine norovirus, Theiler's virus, poly(I:C)) infection in dendritic cells, but not for RIG-I-dependent virus (Sendai virus, VSV) responses, demonstrating that LUBAC selectively promotes MDA5 signaling.\",\n      \"method\": \"HOIL1-deficient mouse dendritic cells, IFN induction assay, IRF3 phosphorylation assay, in vivo norovirus infection model\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO in primary immune cells, multiple viruses compared, in vivo infection model, pathway specificity (MDA5 vs RIG-I) established\",\n      \"pmids\": [\"30209176\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"HOIL1 E3 ligase activity is critical for MDA5-dependent IFN induction. HOIL1 interacts with and ubiquitinates LGP2 (a positive regulator of MDA5 oligomerization), and HOIL1 E3 ligase activity promotes MDA5 oligomerization, translocation to mitochondrial-associated membranes, and formation of MAVS aggregates. HOIP E3 ligase activity plays only a modest role in IFN induction.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, E3 ligase-dead mutants, MDA5 oligomerization assay, MAVS aggregate formation assay (preprint)\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — co-IP, ubiquitination assay, and mechanistic pathway assays, single lab, preprint\",\n      \"pmids\": [\"38617308\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Solution structure of the HOIL-1 Ubl domain was solved by NMR spectroscopy, revealing a β-grasp Ubl-fold. Structural comparison with parkin indicates HOIL-1 likely uses a conserved hydrophobic patch (W58, V102, Y127, Y129) and a C-terminal basic residue (R134) to recruit the S5a subunit of the 26S proteasome.\",\n      \"method\": \"NMR spectroscopy, structural comparison with parkin Ubl domain\",\n      \"journal\": \"Protein science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — NMR structure solved but functional validation of S5a interaction is inferred from structural comparison without direct mutagenesis confirmation reported in abstract\",\n      \"pmids\": [\"22517668\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"HOXA1 physically interacts with RBCK1/HOIL-1 (confirmed by proteome-wide screening and functional assays). HOXA1-mediated activation of NF-κB is non-transcriptional and requires RBCK1 and TRAF2. Genetic epistasis shows that RBCK1 and TRAF2 influences on NF-κB are epistatic to HOXA1 (RBCK1 acts downstream of HOXA1 in NF-κB activation). An 11 His repeat and the homeodomain of HOXA1 are required for RBCK1 interaction.\",\n      \"method\": \"Proteome-wide interaction screening, co-immunoprecipitation, NF-κB reporter assay, domain mapping, epistasis analysis\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — interaction confirmed by co-IP, epistasis established by reporter assay, domain mapping performed, single lab\",\n      \"pmids\": [\"27382069\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RBCK1 binds to YAP protein and promotes K48-linked poly-ubiquitination of YAP at lysine residues K76, K204, and K321, leading to YAP protein degradation and suppression of Hippo/YAP signaling in triple-negative breast cancer cells. RBCK1 overexpression inhibits TNBC cell progression in vitro and in vivo.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitin-based immunoprecipitation, K48-specific ubiquitination assay, protein stability assay, RNA sequencing, in vivo xenograft\",\n      \"journal\": \"Cell communication and signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — site-specific K48 ubiquitination assay with multiple YAP lysine sites identified, in vivo confirmation, single lab\",\n      \"pmids\": [\"36280829\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RBCK1 promotes hepatocellular carcinoma metastasis and growth by stabilizing its LUBAC partner RNF31 (HOIP): RBCK1 interacts with RNF31 and represses its ubiquitination and proteasomal degradation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, knockdown/overexpression functional assays\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — single lab, co-IP and ubiquitination assay establishing stabilization mechanism\",\n      \"pmids\": [\"35869046\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"HOIL-1 interacts with Numb via its A64/Q65 residues binding K78 of Numb, impairing Numb-mediated Notch1 lysosomal degradation and thereby activating Notch1 signaling to promote HCC stemness and sorafenib resistance, independently of HOIL-1 ubiquitin ligase activity.\",\n      \"method\": \"Mass spectrometry, co-immunoprecipitation, western blot, immunofluorescence, in vivo HCC models\",\n      \"journal\": \"Hepatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — interaction mapped to specific residues by MS and co-IP, functional readout in vivo, mechanistic link to Notch1 established, single lab\",\n      \"pmids\": [\"37820061\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In RBCK1-deficient mice, glycogen accumulates as polyglucosans with overlong branches and hyperphosphorylation limited to precipitated polyglucosans (similar to malin-deficient Lafora disease). Downregulating glycogen synthase (the enzyme that elongates glycogen branches) rescues RBCK1 deficiency amylopectinosis, linking RBCK1 function to glycogen branch-length regulation.\",\n      \"method\": \"RBCK1 knockout mouse model, glycogen structural analysis, genetic rescue by glycogen synthase downregulation, behavioral testing, neuroinflammation assessment\",\n      \"journal\": \"Brain : a journal of neurology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse model with glycogen biochemical characterization and genetic rescue demonstrating pathway position\",\n      \"pmids\": [\"35084461\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In HOIL1-deficient mice, loss of HOIL1 in radiation-resistant cells (not intestinal epithelial cells) leads to type 2 intestinal inflammation with tuft cell and goblet cell hyperplasia and elevated IL-13, IL-5, IL-25, including expansion of KLRG1hi CD90lo group 2 innate lymphoid cells. This inflammation is partly dependent on commensal microbiota.\",\n      \"method\": \"HOIL1-deficient mouse model, bone marrow chimeras, cell-type-specific knockouts, IL-4Rα signaling disruption, antibiotic treatment, immune profiling\",\n      \"journal\": \"Mucosal immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with multiple complementary mouse models (chimeras, conditional KOs) defining cell-type requirement, single lab\",\n      \"pmids\": [\"35534698\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"HIF-1α upregulates RBCK1, which then interacts with and ubiquitinates PICK1, facilitating its proteasomal degradation in nasopharyngeal carcinoma cells under hypoxia. RBCK1 knockdown inhibited NPC proliferation, an effect rescued by double knockdown of RBCK1/PICK1.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence, ubiquitination assay, siRNA knockdown, rescue assay, in vivo xenograft\",\n      \"journal\": \"Life sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — single lab, co-IP plus ubiquitination assay and genetic rescue, moderate mechanistic depth\",\n      \"pmids\": [\"36934971\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RBCK1 promotes PPARγ ubiquitination and degradation, leading to activation of WNT/β-catenin/GLUT1 pathway and enhanced aerobic glycolysis in hepatocellular carcinoma cells. RBCK1-induced HCC cell migration and aerobic glycolysis depend on destruction of the PPARγ/PGC1α complex.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, WNT/β-catenin activity assay, metabolic (GLUT1/glycolysis) assays, migration assay\",\n      \"journal\": \"American journal of cancer research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, co-IP and ubiquitination assay with functional readouts but limited mechanistic depth; no in vitro reconstitution\",\n      \"pmids\": [\"35411229\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"HOIL-1 is required for cytoprotective ribosome-associated quality control. HOIL-1 promotes ribosome ubiquitination and its loss causes glucose starvation to become ribotoxic, leading to ZAKα (MAP3K)-dependent ATF4 activation and disulfidptosis driven by the cystine-glutamate antiporter xCT. Truncating HOIL-1 mutations associated with dilated cardiomyopathy exacerbate cardiac dysfunction in mice.\",\n      \"method\": \"HOIL-1 loss-of-function mouse models, ribosome ubiquitination assay, ZAKα genetic epistasis, ATF4 activation assay, nutrient stress experiments\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — ribosome ubiquitination biochemistry, genetic epistasis (ZAKα), in vivo mouse cardiac model, multiple stress paradigms in single rigorous study\",\n      \"pmids\": [\"42062483\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"The C-terminal fragment of HOIL-1 generated by MALT1 cleavage (C-HOIL-1) has novel biological functions: it inhibits NF-κB signaling, interacts with STAT1 to downregulate STAT1-mediated inflammatory signaling, and upregulates ARG1 expression, collectively suppressing inflammatory responses in monocytes/macrophages and impeding M1 macrophage differentiation. Mice with uncleavable HOIL-1 (lacking C-HOIL-1) develop more severe DSS-induced colitis with elevated monocyte/macrophage/neutrophil infiltration.\",\n      \"method\": \"Transgenic uncleavable HOIL-1 mice (global and myeloid-specific), genetically engineered THP-1 cells expressing C-HOIL-1, co-immunoprecipitation (STAT1 interaction), DSS-induced colitis model, lentiviral C-HOIL-1 delivery\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — transgenic mouse models plus cell-based mechanistic assays identifying STAT1 interaction, single lab\",\n      \"pmids\": [\"41799187\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"HOIL-1 and IRP2 interact via the IRP2 73-amino acid domain in HEK293 cells (confirmed by co-immunoprecipitation), but this interaction is NOT iron-dependent, and HOIL-1 does NOT enhance the rate of IRP2 degradation by iron. Stable HOIL-1 expression does not alter iron-dependent degradation of endogenous IRP2; HOIL-1 siRNA knockdown has no effect on iron-mediated IRP2 degradation.\",\n      \"method\": \"Co-immunoprecipitation, stable expression, siRNA knockdown, iron treatment, IRP2 degradation assay\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal approaches (stable expression, siRNA, co-IP) all yielding negative result for HOIL-1 role in IRP2 degradation in HEK293 cells\",\n      \"pmids\": [\"17822790\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RBCK1/HOIL-1 is an RBR-family E3 ubiquitin ligase that functions primarily as a component of LUBAC, where it forms atypical oxyester (serine/threonine) ubiquitin linkages on substrates including Myddosome components (IRAK1, IRAK2, MyD88), glycogen, and ribosomes, enabling monoubiquitylation that seeds hybrid polyubiquitin chains; its unique catalytic mechanism—requiring a bi-nuclear Zn-cluster RING2 with a critical histidine (His510) as the catalytic base—also drives negative feedback of NF-κB signaling via MALT1-mediated proteolytic cleavage of HOIL-1 at antigen receptor signalosomes, and it regulates multiple additional substrates (IRF3, TAB2/3, p53, PTEN, YAP, MFN2, PXR, PPARγ, LGP2) through K48-linked ubiquitination and proteasomal degradation, while its splice variant RBCK2 inhibits both its E3 activity and nuclear localization, and its nuclear-cytoplasmic shuttling (governed by N-terminal NES and C-terminal RING-IBR NLS) is regulated by PKCβ phosphorylation and interaction with CBP/PML in nuclear bodies.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RBCK1 (HOIL-1) is an RBR-family E3 ubiquitin ligase that functions both as a catalytic subunit of the linear ubiquitin chain assembly complex (LUBAC) and as a stand-alone enzyme with an unusual chemistry, governing innate and adaptive immune signaling, cellular quality control, and glycogen metabolism [#0, #9, #34]. Its defining biochemical feature is the formation of atypical oxyester bonds between the ubiquitin C-terminus and serine/threonine residues — and even mono- and disaccharides — rather than the canonical lysine isopeptide linkage; this O-linked monoubiquitylation seeds de novo polyubiquitin chains on Myddosome components IRAK1, IRAK2, and MyD88 during TLR signaling [#0, #2]. This activity depends on a unique C-terminal bi-nuclear Zn-cluster that replaces the second zinc finger of the canonical RING2 fold and on a catalytic histidine (His510) that acts as the catalytic base, enabling oxyester transfer while prohibiting discharge onto lysine; substrate engagement also requires binding of linear tetra-ubiquitin [#1, #2]. Through LUBAC, HOIL-1 controls NF-\\u03baB activation downstream of IL-1\\u03b2 and antigen receptors, and patient loss-of-function mutations destabilize LUBAC and produce cell-type-specific dysregulation of NF-\\u03baB [#9]. A central negative-feedback axis is set by MALT1 paracaspase cleavage of HOIL-1 at antigen receptor signalosomes, which generates a C-terminal fragment with LUBAC-inhibitory and broader anti-inflammatory activity (including STAT1 engagement), dampening lymphocyte and myeloid inflammatory responses [#10, #11, #35]. HOIL-1 also drives MDA5-selective type I/III interferon induction during RNA virus infection [#23]. Beyond immunity, HOIL-1 is required for cytoprotective ribosome-associated quality control, ubiquitinating ribosomes to prevent nutrient-stress ribotoxicity, and regulates glycogen branch-length through its action on polyglucosan structures [#34, #30]. In numerous cancer contexts RBCK1 additionally catalyzes K48-linked polyubiquitination and proteasomal degradation of regulatory substrates including p53, PTEN, and YAP [#15, #16, #27].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Established that a single RBCK1 gene yields two splice products and that RBCK1 possesses intrinsic transcriptional activity requiring its RING and B-Box motifs, with the RING-IBR-lacking variant RBCK2 acting as an inhibitor — the first functional dissection of the locus.\",\n      \"evidence\": \"In vitro interaction and GAL4-chimeric transcription reporter assays with mutational analysis\",\n      \"pmids\": [\"9755849\", \"9642138\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Transcriptional mechanism not connected to a defined promoter or DNA-binding partner\", \"No endogenous target genes identified at this stage\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Defined how RBCK1 subcellular distribution is encoded, mapping an N-terminal NES and a C-terminal RING-IBR NLS and showing RBCK1 localizes to nuclear bodies with CBP/PML and that RBCK2 tethers it in the cytoplasm.\",\n      \"evidence\": \"Leptomycin B treatment, signal mutagenesis, co-IP, fractionation, and co-expression localization assays\",\n      \"pmids\": [\"15833741\", \"16083853\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of nuclear-body residence not linked to ubiquitin ligase activity\", \"RBCK2 tethering shown in overexpression context only\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Demonstrated RBCK1 has in vitro E3 ligase activity that is regulated by RBCK2 binding and by PKC\\u03b2 phosphorylation, coupling its enzymatic output to a kinase input.\",\n      \"evidence\": \"In vitro ubiquitin ligase assay, co-IP, and PKC\\u03b2 overexpression/inhibition\",\n      \"pmids\": [\"18303026\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Phosphosites on RBCK1 not mapped\", \"Physiological substrates of this E3 activity not yet defined\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Positioned RBCK1 as a negative-feedback regulator of innate immune signaling by showing it targets TAB2/TAB3 and IRF3 for proteasomal degradation, restraining NF-\\u03baB and type I interferon responses.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, overexpression/knockdown with NF-\\u03baB reporter and antiviral plaque readouts\",\n      \"pmids\": [\"17449468\", \"18711448\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in vitro reconstitution with purified components\", \"Ubiquitin linkage type not defined at this stage\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Linked RBCK1/HOIL-1 to human disease and LUBAC biology, showing biallelic loss-of-function mutations destabilize LUBAC and cause cell-type-specific NF-\\u03baB dysregulation.\",\n      \"evidence\": \"Patient-derived fibroblasts and monocytes with NF-\\u03baB signaling assays and genetic analysis\",\n      \"pmids\": [\"23104095\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis for opposite fibroblast vs monocyte responses not resolved\", \"Direct LUBAC substrates affected not enumerated\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Solved the HOIL-1 Ubl-domain structure, providing a structural rationale for proteasome recruitment via a conserved hydrophobic patch.\",\n      \"evidence\": \"NMR spectroscopy with structural comparison to parkin\",\n      \"pmids\": [\"22517668\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"S5a/proteasome interaction inferred from comparison, not confirmed by direct mutagenesis\", \"Functional contribution of Ubl domain to substrate degradation untested here\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identified MALT1 paracaspase cleavage of HOIL-1 as a negative-feedback brake on antigen-receptor-driven NF-\\u03baB activation, with the cleavage fragments having distinct activities.\",\n      \"evidence\": \"TAILS N-terminal proteomics, co-IP, NF-\\u03baB reporter, and antigen receptor stimulation with cleavage-resistant mutants\",\n      \"pmids\": [\"26525107\", \"26573773\", \"27006117\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise molecular activity of the inhibitory C-terminal fragment not yet defined\", \"Kinetics relative to LUBAC assembly incompletely resolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Revealed pathway selectivity in antiviral signaling, showing HOIL-1/LUBAC is required for MDA5-dependent but not RIG-I-dependent interferon induction.\",\n      \"evidence\": \"HOIL1-deficient mouse dendritic cells, IRF3 phosphorylation and IFN induction assays, in vivo norovirus infection\",\n      \"pmids\": [\"30209176\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct molecular substrate in the MDA5 pathway not identified in this study\", \"Catalytic requirement vs scaffolding role not separated\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Established the unprecedented catalytic chemistry of HOIL-1, demonstrating it forms oxyester (Ser/Thr) bonds and acts as a monoubiquitylating initiator on Myddosome substrates in vivo.\",\n      \"evidence\": \"Hydroxylamine cleavage, catalytically inactive HOIL-1[C458S] knock-in mice, mass spectrometry, in vitro ubiquitination\",\n      \"pmids\": [\"31209050\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full repertoire of physiological oxyester substrates not defined\", \"How hybrid chain elongation by HOIP is coordinated not fully resolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Provided the structural and mechanistic basis for oxyester catalysis, identifying a bi-nuclear Zn-cluster RING2 and a catalytic histidine, and showing linear tetra-ubiquitin binding licenses substrate ubiquitylation.\",\n      \"evidence\": \"X-ray crystallography of IBR-RING2, catalytic-residue mutagenesis, in vitro ubiquitylation; extended by His510 biochemical characterization\",\n      \"pmids\": [\"36685275\", \"40169258\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for substrate Ser/Thr selection not visualized with bound substrate\", \"How linear-Ub binding allosterically activates the active site not detailed\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Connected HOIL-1 to glycogen metabolism, showing its loss produces malin-like polyglucosans with abnormal branch length that are rescued by reducing glycogen synthase.\",\n      \"evidence\": \"RBCK1 knockout mice, glycogen structural analysis, genetic rescue by glycogen synthase downregulation\",\n      \"pmids\": [\"35084461\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct enzymatic target linking RBCK1 to branch-length control not defined\", \"Relationship to oxyester ubiquitylation of carbohydrate not established in this study\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined a cytoprotective ribosome-associated quality-control role, with HOIL-1 ubiquitinating ribosomes to prevent nutrient-stress ribotoxicity and disulfidptosis, and linked truncating mutations to dilated cardiomyopathy.\",\n      \"evidence\": \"HOIL-1 loss-of-function mouse models, ribosome ubiquitination assays, ZAK\\u03b1 genetic epistasis, ATF4 readouts, in vivo cardiac model\",\n      \"pmids\": [\"42062483\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific ribosomal residues/proteins ubiquitinated not enumerated\", \"Whether ribosome ubiquitylation uses oxyester chemistry not stated\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Resolved the function of the MALT1-generated C-terminal HOIL-1 fragment, showing it suppresses NF-\\u03baB and STAT1 signaling and limits M1 macrophage differentiation in vivo.\",\n      \"evidence\": \"Uncleavable HOIL-1 transgenic mice, engineered THP-1 cells, STAT1 co-IP, DSS colitis model\",\n      \"pmids\": [\"41799187\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of STAT1 inhibition not biochemically detailed\", \"Single-lab findings without independent confirmation\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How HOIL-1's single active site is partitioned between LUBAC-associated immune signaling, ribosome quality control, glycogen-branch regulation, and the many reported K48-degradative cancer substrates — and whether all use the same oxyester chemistry — remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Linkage chemistry (oxyester vs K48 isopeptide) not reconciled across the diverse reported substrates\", \"Substrate-selection determinants in vivo unknown\", \"Many cancer substrate findings rest on single-lab co-IP/ubiquitination assays without reconstitution\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 3, 7, 8]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 2, 27]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [4, 6, 20]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [10, 35]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [4, 5]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"GO:0016605\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 9, 10, 23]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [8, 26]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 34]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [34]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [30]}\n    ],\n    \"complexes\": [\n      \"LUBAC\"\n    ],\n    \"partners\": [\n      \"RNF31\",\n      \"SHARPIN\",\n      \"TAB2\",\n      \"TAB3\",\n      \"MALT1\",\n      \"PKCB\",\n      \"RBCK2\",\n      \"SOCS6\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}