{"gene":"BIRC2","run_date":"2026-06-09T22:02:44","timeline":{"discoveries":[{"year":1996,"finding":"c-IAP1 is recruited to the TNF-R1 signaling complex via TRADD-mediated association with TRAF2, establishing its presence in TNF-R1 signaling independently of TNF-R2.","method":"Co-immunoprecipitation, TNF-dependent recruitment assay in cells","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, TNF-dependent recruitment demonstrated, replicated across multiple subsequent studies","pmids":["8943045"],"is_preprint":false},{"year":1997,"finding":"c-IAP1 and c-IAP2 bind specifically to caspases-3 and -7 via their BIR domains and inhibit their enzymatic activity in vitro (estimated Ki ≤0.1 µM), blocking caspase-3 processing in cell-free and intact cell systems.","method":"In vitro binding assay, in vitro caspase activity assay, cell-free cytochrome-c system, gene transfection overexpression","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with purified recombinant proteins, Ki measurement, domain mapping, replicated in cell-free and intact-cell systems","pmids":["9384571"],"is_preprint":false},{"year":1998,"finding":"NF-κB transcriptional activation induces expression of c-IAP1 and c-IAP2 (along with TRAF1 and TRAF2), and these proteins cooperatively suppress TNF-alpha-induced caspase-8 activation; c-IAP1/2 alone are sufficient to suppress etoposide-induced apoptosis.","method":"NF-κB inhibition/activation, gene transfection, caspase-8 activation assay, apoptosis assays","journal":"Science (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic and overexpression approaches, multiple stimuli, replicated broadly","pmids":["9733516"],"is_preprint":false},{"year":2000,"finding":"c-IAP1 is cleaved during apoptosis by caspase-3 to produce C-terminal fragments containing the RING domain; these fragments are proapoptotic, and the RING domain negatively regulates the antiapoptotic function of the N-terminal BIR domain.","method":"In vitro cleavage with purified caspase-3, transfection of deletion mutants, apoptosis assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with purified caspase-3, domain mutagenesis/deletion, functional readout in cells","pmids":["11106668"],"is_preprint":false},{"year":2002,"finding":"c-IAP1 functions as an E3 ubiquitin ligase that ubiquitinates TRAF2 (but not TRAF1 despite binding both) downstream of TNF-RII engagement, leading to proteasomal degradation of TRAF2; E3-defective c-IAP1 mutant blocks this degradation and inhibits apoptosis.","method":"In vitro ubiquitination assay, co-immunoprecipitation, E3-defective mutant expression, proteasome inhibition, primary cell studies","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro ubiquitination with purified components, active-site mutagenesis, primary cell validation","pmids":["11907583"],"is_preprint":false},{"year":2003,"finding":"cIAP1 is irreversibly cleaved (inactivated) during p53-dependent apoptosis by the serine protease HTRA2, which interacts directly with cIAP1; serine protease inhibitors that block cIAP1 cleavage inhibit p53-dependent apoptosis.","method":"Co-immunoprecipitation, serine protease inhibitor treatment, apoptosis assay, p53 induction","journal":"Genes & development","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP of endogenous proteins, pharmacological inhibition, single lab","pmids":["12569127"],"is_preprint":false},{"year":2004,"finding":"c-IAP1 and c-IAP2 ubiquitinate RIP (RIP1) in vitro; expression of c-IAP1/2 decreases steady-state RIP levels in a proteasome-dependent manner, requiring the RING domain of c-IAP2.","method":"In vitro ubiquitination assay, proteasome inhibitor rescue, deletion mutant analysis","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro ubiquitination reconstitution, domain mutagenesis, single lab","pmids":["15147886"],"is_preprint":false},{"year":2004,"finding":"c-IAP1 translation is driven by an internal ribosome entry site (IRES) in its 5'-UTR, and IRES-mediated translation is stimulated by pro-apoptotic stimuli (etoposide, sodium arsenite) that inhibit cap-dependent translation.","method":"Dicistronic RNA constructs, in vitro translation, transfection in multiple cell lines, hairpin insertion, cap-independent translation assay","journal":"RNA (New York, N.Y.)","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — multiple orthogonal translation assays, multiple cell types, single lab","pmids":["14970392"],"is_preprint":false},{"year":2004,"finding":"cIAP1 and cIAP2 are nuclear-cytoplasmic shuttling proteins; their nuclear export is CRM1-dependent, and a leucine-rich NES in the BIR2-BIR3 linker region mediates nuclear export of cIAP1. TRAF2 retains cIAP1/2 in the cytoplasm and prevents nuclear translocation; TNFα treatment reduces TRAF2-mediated cytoplasmic retention.","method":"Leptomycin B treatment, site-directed mutagenesis of NES, co-expression with TRAF2, immunofluorescence, subcellular fractionation","journal":"Experimental cell research","confidence":"High","confidence_rationale":"Tier 2 / Moderate — NES mutagenesis, leptomycin B, TRAF2 co-expression, multiple orthogonal localization methods","pmids":["15265700"],"is_preprint":false},{"year":2004,"finding":"In hematopoietic cells undergoing differentiation, c-IAP1 translocates from the nucleus to the Golgi apparatus via a nuclear export signal (NES) located in the CARD domain, in a leptomycin B-sensitive, CRM1-dependent mechanism.","method":"Immunofluorescence, leptomycin B treatment, site-directed mutagenesis of CARD NES, phorbol ester differentiation model","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Moderate — NES mutagenesis, CRM1 inhibition, multiple cell lines and primary monocytes","pmids":["15187025"],"is_preprint":false},{"year":2005,"finding":"cIAP1 and cIAP2 BIR2 and BIR3 domains bind caspases-7 and -9 but do NOT inhibit caspase activity due to critical substitutions in caspase-inhibitory contact residues; substituting these residues with XIAP equivalents converts cIAP BIRs into tight caspase inhibitors.","method":"In vitro binding assay, fluorogenic caspase activity assay, domain mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution, active-site mutagenesis, negative result for inhibition rigorously established","pmids":["16339151"],"is_preprint":false},{"year":2005,"finding":"c-IAP1 mediates posttranscriptional downregulation of c-IAP2 via E3 ubiquitin ligase-dependent ubiquitination and proteasomal degradation, potentiated by TRAF2 as an adaptor; c-IAP1-deficient mice show elevated c-IAP2 protein without increased c-IAP2 mRNA.","method":"c-IAP1 knockout mice, wild-type vs. E3-defective c-IAP1 transfection, Western blot, RT-PCR","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout, E3-dead mutant complementation, mRNA vs. protein distinction, in vivo model","pmids":["15798218"],"is_preprint":false},{"year":2005,"finding":"cIAP1 localizes predominantly to the nucleus (unlike cytoplasmic XIAP); apoptotic stimuli induce nuclear export of cIAP1 in a caspase-dependent manner. During mitosis, cIAP1 is released to cytosol and reassociates with the midbody; it interacts with Survivin during mitosis. Overexpression of cIAP1 causes G2-M accumulation, cytokinesis defects, and polyploidy.","method":"Immunofluorescence microscopy, subcellular fractionation, caspase inhibitor treatment, stable overexpression, cell cycle analysis","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple localization methods, functional overexpression phenotype, single lab","pmids":["15665297"],"is_preprint":false},{"year":2005,"finding":"Upon TNF-R2 signaling, the c-IAP1/TRAF2 complex translocates to a Triton X-100-insoluble perinuclear ER compartment containing the E2 enzyme Ubc6; Ubc6 serves as a cognate E2 for c-IAP1's E3 activity and is required for TNF-R2-dependent TRAF2 ubiquitination and degradation.","method":"Subcellular fractionation, confocal microscopy, co-immunoprecipitation, in vitro ubiquitination with Ubc6, dominant-negative Ubc6 mutant","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro E2/E3 reconstitution, dominant-negative mutant, confocal co-localization, fractionation","pmids":["15861135"],"is_preprint":false},{"year":2004,"finding":"Smac/DIABLO selectively promotes rapid degradation of c-IAP1 and c-IAP2 (but not XIAP or Livin) by stimulating their auto-ubiquitination via its N-terminal IAP-binding motif binding to the BIR domains; ubiquitin-conjugating enzymes UbcH5a and UbcH6 are required for this ubiquitination.","method":"In vitro auto-ubiquitination assay, deletion mutants, N-terminal Smac peptide, proteasome inhibition, Western blot","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro ubiquitination, domain deletion analysis, peptide sufficiency test, E2 identification","pmids":["14960576"],"is_preprint":false},{"year":2007,"finding":"c-IAP1 functions as the E3 ubiquitin ligase for ASK1 downstream of TNFR2 signaling, leading to ASK1 ubiquitination and proteasomal degradation; c-IAP1 knockout primary B cells lack TNFR2-induced TRAF2 and ASK1 degradation and show prolonged p38 and JNK activation.","method":"Primary c-IAP1 knockout B cells, ubiquitination assay, proteasome inhibitor, kinase activity assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — primary knockout cells, E3 identification, kinase prolongation phenotype, in vivo validation","pmids":["17220297"],"is_preprint":false},{"year":2007,"finding":"Birc2 (cIAP1) positively regulates formation of the TNF receptor complex I in endothelial cells, promoting NF-κB activation and maintaining vascular integrity; loss of Birc2 in zebrafish causes caspase-8-dependent apoptosis and vessel regression.","method":"Zebrafish forward genetic screen, null mutant analysis, genetic epistasis with NF-κB pathway, caspase-8 assay","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo null mutant, genetic epistasis, multiple molecular and cellular readouts","pmids":["17934460"],"is_preprint":false},{"year":2007,"finding":"c-IAP1 acts as an E3 ubiquitin ligase for Mad1 (MAX dimerization protein 1), promoting its proteasomal degradation, which cooperates with Myc to promote cell proliferation.","method":"In vitro ubiquitination assay, proteasome inhibitor, overexpression/knockdown proliferation assay","journal":"Molecular cell","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro ubiquitination, proteasomal degradation rescue, functional proliferation readout, single lab","pmids":["18082613"],"is_preprint":false},{"year":2008,"finding":"cIAP1 and cIAP2 promote cancer cell survival by functioning as E3 ubiquitin ligases that maintain constitutive K63-linked ubiquitination of RIP1; ubiquitinated RIP1 associates with pro-survival TAK1, while deubiquitinated RIP1 binds caspase-8 and induces apoptosis.","method":"IAP antagonist AEG40730 treatment, in vitro ubiquitination with purified components, co-immunoprecipitation of RIP1-TAK1 and RIP1-caspase-8 complexes","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with purified components, multiple orthogonal methods, mechanistic switch demonstrated","pmids":["18570872"],"is_preprint":false},{"year":2008,"finding":"cIAP1 and cIAP2 are required for TNFα-stimulated RIP1 K63-linked polyubiquitination and downstream NF-κB activation; in vitro reconstitution with purified c-IAP1 and UbcH5a demonstrates direct K63-linked chain polymerization on RIP1.","method":"Genetic knockout cells, siRNA knockdown, IAP antagonist, in vitro ubiquitination reconstitution with purified cIAP1+UbcH5a","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with purified components, genetic ablation validated with pharmacological approach, replicated across labs","pmids":["18621737"],"is_preprint":false},{"year":2008,"finding":"Both cIAP1 and cIAP2 are required (redundantly) for TNFα-induced RIP1 polyubiquitination and NF-κB activation; combined loss of both cIAPs attenuates IKKβ phosphorylation and sensitizes cells to TNFα-mediated apoptosis.","method":"Combined genetic deletion and siRNA knockdown, TNF receptor complex immunoprecipitation, RIP1 ubiquitination assay, NF-κB activation assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — combined genetic/siRNA approaches, primary cells, receptor complex pull-down, replicated across labs","pmids":["18697935"],"is_preprint":false},{"year":2008,"finding":"Noncanonical NF-κB activation is suppressed by a regulatory complex in which TRAF2 recruits cIAP1/2 and TRAF3 recruits NIK; cIAP1 and cIAP2 mediate proteasomal degradation of NIK within this complex, and combined inhibition of both cIAPs is required for noncanonical NF-κB activation.","method":"Complex assembly assay, cIAP inhibition (genetic and pharmacological), NIK degradation assay, primary B lymphocyte survival/proliferation assay","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — complex reconstitution, genetic and pharmacological inhibition, primary cell and in vivo validation","pmids":["18997794"],"is_preprint":false},{"year":2008,"finding":"TWEAK/FN14 signaling promotes lysosomal (not proteasomal) degradation of the cIAP1-TRAF2 complex in a cIAP1-dependent manner, leading to noncanonical NF-κB activation and sensitization to TNFα-induced death.","method":"Lysosomal inhibitor vs. proteasomal inhibitor treatment, co-immunoprecipitation of FN14-cIAP1-TRAF2 complex, cIAP1 overexpression rescue","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — pathway dissection with inhibitors, complex co-IP, rescue experiment, single lab","pmids":["18606850"],"is_preprint":false},{"year":2008,"finding":"Small molecules (ME-BS class) directly interact with the BIR3 domain of cIAP1, promote RING domain-dependent auto-ubiquitylation, and facilitate proteasomal degradation of cIAP1 selectively (not XIAP or cIAP2), sensitizing cancer cells to apoptosis.","method":"Direct binding assay (BIR3 domain), auto-ubiquitylation assay, proteasome inhibitor rescue, Western blot, apoptosis assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — direct binding and in vitro ubiquitylation, domain mapping, single lab","pmids":["18230607"],"is_preprint":false},{"year":2008,"finding":"The CARD domain of cIAP1 contains a functional NES mediating CRM1-dependent nuclear export; the RING domain of cIAP1 degrades RING-bearing IAPs (cIAP1, cIAP2, XIAP, Livin) by ubiquitin-dependent and ubiquitin-independent proteasomal pathways.","method":"CARD NES mutagenesis, leptomycin B treatment, ubiquitination-deficient E1 mutant cells, cIAP1 RING domain transfection, proteasome inhibitor","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — mutagenesis, E1-deficient cells, multiple IAP substrates tested, single lab","pmids":["18434593"],"is_preprint":false},{"year":2008,"finding":"During monocyte-to-macrophage differentiation, cIAP1 (after translocating from nucleus to cytoplasm) mediates proteasomal degradation of TRAF2 via its E3 ligase activity; TRAF2 is initially required for NF-κB activation during differentiation, and its subsequent cIAP1-mediated degradation is required for full macrophage function including cytokine secretion.","method":"cIAP1 inhibitor, TRAF2 siRNA knockdown, TRAF2 overexpression, NF-κB assay, cytokine measurement","journal":"Blood","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function and gain-of-function, functional cytokine readout, pathway ordered, single lab","pmids":["18827186"],"is_preprint":false},{"year":2009,"finding":"cIAP1 binds caspase-3 and caspase-7 at distinct steps of their processing via different mechanisms: it binds mature caspase-7 via its exposed IAP-binding motif AKPD, and binds partially processed caspase-3 via a prodomain-dependent non-classical mechanism. cIAP1 ubiquitinates both caspases via UbcH5 subfamily E2s (and UbcH8 for caspase-3), leading to their proteasomal degradation without directly inhibiting their enzymatic activity.","method":"In vitro binding assay, fluorogenic substrate assay, ubiquitination assay, chimeric caspase-3 with AKPD motif, proteasome inhibitor, UbcH5/UbcH8 identification","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution, domain mapping, E2 identification, chimeric substrate, multiple orthogonal methods","pmids":["19258326"],"is_preprint":false},{"year":2009,"finding":"cIAP1 mediates NEMO monoubiquitination in response to genotoxic stress as part of NF-κB activation; cIAP1, cIAP2, and XIAP act cooperatively and non-redundantly at distinct steps: XIAP activates TAK1, cIAP1 ubiquitinates NEMO, and cIAP2 acts downstream of NEMO ubiquitination.","method":"Genotoxic agents (camptothecin, etoposide, doxorubicin), siRNA knockdown of individual IAPs, NEMO ubiquitination assay, NF-κB activation assay","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — individual siRNA knockdowns, pathway ordering by epistasis, single lab","pmids":["19223549"],"is_preprint":false},{"year":2009,"finding":"A UBA (ubiquitin-associated) domain in cIAP1 located between the BIR domains and the CARD/RING domains binds mono-ubiquitin and K48- and K63-linked polyubiquitin chains; UBA domain mutations abrogate ubiquitin binding and decrease IAP antagonist-stimulated proteasomal degradation of cIAP1.","method":"Surface plasmon resonance, isothermal titration calorimetry, NMR analysis of UBA-ubiquitin interaction, UBA domain point mutagenesis, proteasome degradation assay","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — NMR structure of domain-ubiquitin interaction, SPR/ITC binding measurements, mutagenesis, functional degradation readout","pmids":["18939944"],"is_preprint":false},{"year":2009,"finding":"cIAP1 and cIAP2 are required for NOD1- and NOD2-mediated innate immune signaling; they function as E3 ubiquitin ligases for RIP2 ubiquitination, and their deficiency impairs cytokine/chemokine production in macrophages and colonocytes in vitro and in vivo.","method":"Birc2−/− and Birc3−/− mouse macrophages, RNAi in colonocytes, RIP2 ubiquitination assay, in vivo NOD agonist challenge, colitis model","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout, E3 ligase assay, in vivo challenge, multiple cell types","pmids":["19464198"],"is_preprint":false},{"year":2010,"finding":"c-IAP1 and UbcH5 family E2 enzymes promote K11-linked polyubiquitination of RIP1 in vitro and in vivo within the TNFR1 signaling complex; TNFα-stimulated NF-κB activation involves endogenous K11-linked ubiquitination of RIP1 in a c-IAP1- and UbcH5-dependent manner. NEMO efficiently binds K11-linked ubiquitin chains.","method":"Directed yeast two-hybrid screen for E2 partners, in vitro ubiquitination with purified components, mass spectrometry of ubiquitin linkage, UbcH5 siRNA knockdown, TNFR1 complex immunoprecipitation","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with purified proteins, MS-based ubiquitin linkage determination, genetic validation in cells","pmids":["21113135"],"is_preprint":false},{"year":2010,"finding":"cIAP1 promotes cell survival against TNF-induced necrosis by maintaining RIP1 ubiquitination, which prevents RIP1 kinase activation, RIP1/RIP3 necrosome formation, and ROS accumulation; depletion of cIAP1 (but not cIAP2) is specifically responsible for sensitization to TNF-induced necrosis.","method":"RNAi (individual IAP knockdown), IAP antagonist BV6, RIP1 kinase inhibitor, RIP3 knockdown, CYLD knockdown, ROS measurement, cell death assay","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 2 / Strong — individual knockdowns identify cIAP1 specifically, epistasis with CYLD/RIP1/RIP3, ROS as mechanistic readout","pmids":["21052097"],"is_preprint":false},{"year":2010,"finding":"RIPK1 promotes cell survival in TNF-stimulated cells by stabilizing TRAF2 and cIAP1; in RIPK1-deficient cells, TNF stimulation causes rapid proteasomal degradation of TRAF2 and cIAP1, leading to NIK accumulation, non-canonical NF-κB activation, reduction of cFLIPL, and caspase-8 activation.","method":"RIPK1−/− cells, TNF stimulation time course, proteasome inhibitor, Western blot for TRAF2/cIAP1/NIK/cFLIPL","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic knockout, ordered pathway, proteasome rescue, single lab","pmids":["21339290"],"is_preprint":false},{"year":2010,"finding":"The RING domain of cIAP1 requires dimerization and E2 binding for IAP antagonist-induced auto-degradation and for TNF-induced NF-κB activation and prevention of non-canonical NF-κB; RING mutants unable to dimerize or bind E2 fail these functions.","method":"cIAP-deleted cells reconstituted with RING point mutants (dimerization-defective, E2-binding-defective), TNF stimulation, IAP antagonist treatment, NF-κB assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — structure-function mutagenesis in reconstituted knockout cells, multiple pathway readouts","pmids":["20356846"],"is_preprint":false},{"year":2011,"finding":"The CARD domain of cIAP1 autoinhibits its E3 ligase activity by preventing RING dimerization, E2 binding, and E2 activation; CARD-mediated autoregulation suppresses cell proliferation and migration, maximally suppresses caspase-8-dependent apoptosis, and prevents vascular degeneration in vivo.","method":"Crystal structure of CARD domain, RING dimerization assay, E2 activation assay, CARD deletion/mutation, cell proliferation/migration assay, zebrafish vascular assay","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure, in vitro E3 activity assays, mutagenesis, in vivo zebrafish validation","pmids":["21549626"],"is_preprint":false},{"year":2011,"finding":"cIAP1 and cIAP2 are direct E3 ubiquitin ligases for RIP1, RIP2, RIP3, and RIP4; cIAP1 conjugates diverse ubiquitin chain types including linear chains to these substrates. K63-linked ubiquitination of RIP4 on Lys51 and Lys145 by cIAP1 is required for NF-κB activation.","method":"Co-immunoprecipitation (direct binding), in vitro ubiquitination assay with purified components, RIP4 K51R/K145R mutagenesis, NF-κB reporter assay","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro ubiquitination reconstitution, substrate mapping, NF-κB functional readout, single lab","pmids":["21931591"],"is_preprint":false},{"year":2011,"finding":"cIAP1 and cIAP2 are required for efficient caspase-1 activation by the inflammasome; they interact with caspase-1-containing complexes via TRAF2 and mediate non-degradative K63-linked polyubiquitination of caspase-1; deficiency in Birc2 or Birc3 impairs caspase-1 activation and inflammasome responses in vivo.","method":"Birc2−/− and Birc3−/− mouse macrophages, co-immunoprecipitation, K63-ubiquitination assay, in vivo peritonitis model, caspase-1 activity assay","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout, co-IP, ubiquitin linkage assay, in vivo validation","pmids":["22195745"],"is_preprint":false},{"year":2011,"finding":"Smac mimetic-induced degradation of cIAP1 requires its binding to TRAF2; degradation of cIAP2 requires the presence of cIAP1 and cIAP2 RING finger dimerization and E2 binding. cIAP2-MALT1 oncofusion (lacking the RING) is resistant to Smac mimetics.","method":"TRAF2-binding mutants, cIAP1/2 double-knockout cells, RING mutants, cIAP2-MALT1 expression, Western blot","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — structure-function mutagenesis, knockout cells, multiple conditions, single lab","pmids":["21331077"],"is_preprint":false},{"year":2011,"finding":"USP19 deubiquitinase interacts with c-IAP1 and c-IAP2, and stabilizes c-IAP levels primarily through deubiquitinase-independent mechanisms in vivo; knockdown of USP19 decreases c-IAP levels and enhances TNFα-induced caspase activation and apoptosis in a c-IAP1/2-dependent manner.","method":"Co-immunoprecipitation, in vitro deubiquitination assay, USP19 knockdown, overexpression, caspase activity assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, in vitro DUB assay, knockdown/overexpression with functional readout, single lab","pmids":["21849505"],"is_preprint":false},{"year":2011,"finding":"Nuclear cIAP1 directly interacts with the DNA-binding domain of E2F1 transcription factor, increases E2F1 transcriptional activity on CCNE and CCNA promoters, and is recruited to E2F1 binding sites by chromatin immunoprecipitation; cIAP1 silencing inhibits cyclin E/A expression and cell proliferation.","method":"Co-immunoprecipitation, E2F1 transcription reporter assay, chromatin immunoprecipitation (ChIP), cIAP1 siRNA knockdown, cell proliferation assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ChIP, functional transcription assay, siRNA rescue, single lab","pmids":["21653699"],"is_preprint":false},{"year":2012,"finding":"cIAP1 limits macrophage necroptosis by suppressing RIP3 (and to a lesser extent RIP1) expression via post-transcriptional mechanisms, preventing necrosome formation; specific cIAP1 knockout causes elevated macrophage cell death and compromised control of Listeria monocytogenes.","method":"SMAC mimetic treatment, RNAi individual knockdown, RIP1 kinase inhibitor, RIP3 knockdown, single cIAP1 or cIAP2 knockout macrophages, in vivo Listeria model","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — specific cIAP1 knockout identifies distinct role, in vivo infection model, post-transcriptional mechanism, single lab","pmids":["22576661"],"is_preprint":false},{"year":2012,"finding":"Loss of cIAP1 activates the noncanonical NF-κB pathway (via NIK/p100/RelB), which promotes myoblast fusion; TWEAK at low concentrations activates this pathway to increase myoblast fusion without causing atrophy.","method":"cIAP1-null cells, siRNA knockdown of p100/RelB/IKKα/NIK, TWEAK treatment, myoblast fusion quantification","journal":"Science signaling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic loss-of-function, pathway component knockdown epistasis, quantitative fusion assay, single lab","pmids":["23074266"],"is_preprint":false},{"year":2013,"finding":"OTUB1 deubiquitinase associates with c-IAP1, disassembles K48-linked polyubiquitin chains from c-IAP1 in vitro and in vivo within the TWEAK receptor signaling complex, thereby stabilizing c-IAP1; OTUB1 knockdown promotes c-IAP1 degradation, caspase activation, and reduces TWEAK-induced NF-κB and MAPK signaling.","method":"Co-immunoprecipitation, in vitro deubiquitination assay (K48-linkage specific), OTUB1 siRNA, cell death assay, NF-κB/MAPK reporter, zebrafish OTUB1 suppression","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro DUB assay with linkage specificity, co-IP, genetic knockdown with multiple pathway readouts, in vivo zebrafish validation","pmids":["23524849"],"is_preprint":false},{"year":2015,"finding":"BIRC2 (cIAP1) functions as a negative regulator of noncanonical NF-κB-dependent HIV-1 LTR transcription; depletion of BIRC2 by Smac mimetics activates HIV-1 transcription and reverses latency, synergizing with HDACi panobinostat in patient-derived CD4+ T cells.","method":"RNAi screen, BIRC2 siRNA knockdown, Smac mimetic treatment, HIV-1 LTR reporter, latency reversal assay in JLat cells and primary CD4+ T cells from ART-suppressed patients","journal":"Cell host & microbe","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — targeted RNAi screen confirmed by knockdown and pharmacological depletion, primary patient cells, functional LTR readout, single lab","pmids":["26355217"],"is_preprint":false},{"year":2017,"finding":"cIAP1 E3 ubiquitin ligase promotes K63-linked polyubiquitination of E2F1 on lysine residues 161/164, which stabilizes E2F1 and increases its transcriptional activity; this modification is required for E2F1 binding to CCNE, TP73 and APAF1 promoters and for E2F1 stabilization in response to DNA damage (etoposide) and during S phase.","method":"In vitro ubiquitination assay, K161R/K164R E2F1 mutagenesis, ChIP on CCNE/TP73/APAF1 promoters, cell cycle synchronization, DNA damage assay","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro ubiquitination, site-directed mutagenesis, ChIP, multiple conditions, single lab","pmids":["28542143"],"is_preprint":false},{"year":2017,"finding":"TLR-MyD88 signaling causes proteasomal degradation of cIAP1 and TRAF2 by inducing TNF and TNFR2 signaling; myeloid-specific cIAP1 loss in XIAP-deficient cells promotes TLR-induced RIPK3-caspase-8 activation and IL-1β production; TNFR2 deletion limits cIAP1-TRAF2 degradation and cell death.","method":"Myeloid-specific cIAP1 conditional knockout, TNFR2 deletion, TLR ligand stimulation, caspase-8 activation assay, IL-1β measurement, Western blot for cIAP1/TRAF2","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional knockout, genetic epistasis with TNFR2 and XIAP, multiple death pathway readouts","pmids":["28723569"],"is_preprint":false},{"year":2018,"finding":"S-nitrosylation of cIAP1 on cysteines 571 and 574 by NO donor GTN inhibits its E3 ubiquitin ligase activity, reducing K63-linked ubiquitination of RIP1 and triggering assembly of a death complex, converting TNFα from a pro-survival to a pro-death signal.","method":"S-nitrosylation site identification (Cys571/574), E3 ligase activity assay after S-nitrosylation, RIP1 ubiquitination assay, death complex co-immunoprecipitation, cell death assay","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — site-specific PTM identification, E3 activity assay, mechanistic death complex formation, single lab","pmids":["29431638"],"is_preprint":false},{"year":2018,"finding":"cIAP1 is recruited to the TNFR2 signaling complex and mediates K63-linked polyubiquitination; cIAP1 recruitment to TNFR2 is required for HOIP (LUBAC) recruitment and 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, ubiquitin linkage analysis, HOIP recruitment assay, NF-κB activation assay","journal":"Biochemical pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — receptor complex IP, pharmacological cIAP depletion, ubiquitin linkage analysis, NF-κB functional readout, single lab","pmids":["29378181"],"is_preprint":false},{"year":2019,"finding":"Combined loss of cIAP1 and cIAP2 in adult mice causes rapid intestinal and hepatic cell death involving caspase-8 and caspase-3 cleavage; deletion of Casp8 and Ripk3 together (but not Ripk3 alone) prevents cell death and prolongs survival, indicating the primary function is suppression of caspase-8-dependent death. Residual inflammation is reduced by NIK inhibition.","method":"Conditional cIap1/2 double knockout in adult mice, Casp8/Ripk3/Mlkl genetic deletion epistasis, cleaved caspase immunostaining, NIK inhibition","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo conditional double knockout, multiple genetic epistasis tests, specific cell death pathway ordered","pmids":["31141691"],"is_preprint":false},{"year":2020,"finding":"Structural and biophysical studies of cIAP1 BIR3 domain in ternary complexes with BTK and heterobifunctional degraders (PROTACs) reveal that increased ternary complex stability or rigidity does not necessarily correlate with increased degradation efficiency.","method":"Biochemical binding assays, biophysical measurements, crystal structure of ternary complex (BTK-degrader-cIAP1)","journal":"Nature chemical biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure of ternary complex, multiple biophysical methods, structure-activity correlation","pmids":["33199914"],"is_preprint":false},{"year":2021,"finding":"OTUB1 prevents K48-linked polyubiquitination and degradation of c-IAP1 in hepatocytes; OTUB1 deficiency leads to c-IAP1 degradation, reduced K63-linked polyubiquitination of RIPK1, increased RIPK1 phosphorylation and necrosome formation, and lethal necroptosis in bacterial and sterile liver inflammation.","method":"Hepatocyte-specific OTUB1 knockout mice, OTUB1-deficient HepG2 cells, RIPK1 inhibitor necrostatin-1s, MLKL knockout epistasis, K48/K63 ubiquitin linkage assays, Listeria/DGal-TNF challenge","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 2 / Strong — tissue-specific knockout, genetic epistasis in vivo, ubiquitin linkage assays, RIPK1 phosphorylation, replicated in human cells","pmids":["33712742"],"is_preprint":false},{"year":2022,"finding":"cIAP1-targeting degraders (SNIPERs/PROTACs) require the K63-specific E2 enzyme UBE2N for efficient target protein degradation; UBE2N-catalyzed K63-linked chains facilitate assembly of branched K48/K63 and K11/K48 ubiquitin chains on substrates, recruiting p97/VCP, UCH37, and the proteasome for degradation.","method":"UBE2N siRNA/knockout, in vitro ubiquitination with E2 panel, mass spectrometry of ubiquitin chain linkages, p97/UCH37/proteasome interaction assays","journal":"Nature chemical biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution, MS-based ubiquitin linkage mapping, genetic validation, mechanistic complex assembly demonstrated","pmids":["36316570"],"is_preprint":false},{"year":2008,"finding":"HSP90β acts as a chaperone for c-IAP1; HSP90β inhibition or siRNA depletion induces c-IAP1 auto-ubiquitination and proteasomal degradation, and prevents cell differentiation; specific depletion of c-IAP1 alone is sufficient to inhibit cell differentiation.","method":"HSP90 inhibitor treatment, HSP90α/β-specific siRNA, co-immunoprecipitation, auto-ubiquitination assay, proteasome inhibitor rescue, differentiation assay","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — specific HSP90β isoform identified by siRNA, co-IP, ubiquitination assay, functional differentiation readout, single lab","pmids":["18239673"],"is_preprint":false},{"year":2014,"finding":"cIAP1 conjugates predominantly K63-linked ubiquitin chains to MEKK2 and MEKK3, which impedes their interaction with MEK5 in a trimeric complex, leading to ERK5 inactivation; loss of cIAP1 causes hyperactivation of ERK5 and promotes skeletal myoblast differentiation.","method":"Co-immunoprecipitation, in vitro ubiquitination assay, K63-ubiquitin linkage analysis, MEKK2/3-MEK5 interaction assay, ERK5 activation assay, myoblast differentiation model","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro ubiquitination reconstitution, K63 linkage identified, trimeric complex disruption mechanism, functional differentiation readout","pmids":["24975362"],"is_preprint":false},{"year":2006,"finding":"F-box protein Fbxo7 interacts with cIAP1 in human cells and promotes cIAP1 ubiquitination; they co-localize in cytoplasm, nucleus, and Golgi-like structures when co-expressed.","method":"Yeast two-hybrid screen, co-immunoprecipitation, co-localization by microscopy, ubiquitination assay","journal":"Biochemical and biophysical research communications","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single co-IP and ubiquitination assay, yeast two-hybrid identified interaction, single lab","pmids":["16510124"],"is_preprint":false},{"year":2011,"finding":"cIAP1 promotes SNIPER-4-mediated ubiquitination and proteasomal degradation of CRABP-II when crosslinked to cIAP1 by a hybrid small molecule; this establishes cIAP1 as a recruitable E3 ligase for targeted protein degradation of neo-substrates.","method":"SNIPER hybrid molecule, Western blot of CRABP-II degradation, proteasome inhibitor rescue, ubiquitination assay","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — chemical biology reconstitution of E3-substrate crosslinking, mechanistic proteasome rescue, single lab","pmids":["21414315"],"is_preprint":false},{"year":2003,"finding":"cIAP1 is a component of the endogenous LIGHT·LTβR complex, associating with TRAF2, TRAF3, and Smac at the lymphotoxin-beta receptor; its presence with Smac reveals a mechanism for LIGHT/LTβR-induced apoptosis.","method":"Affinity purification of endogenous LIGHT·LTβR complex, mass spectrometry identification, co-immunoprecipitation confirmation in U937 and HEK293 cells","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — endogenous complex purification with MS, co-IP confirmation in two cell lines, single lab","pmids":["12571250"],"is_preprint":false}],"current_model":"BIRC2/cIAP1 is a RING domain E3 ubiquitin ligase that promotes cell survival by constitutively maintaining K11- and K63-linked polyubiquitination of RIP1 (and other RIP kinases) within TNF receptor signaling complexes, thereby enabling TAK1 recruitment and NF-κB activation while preventing RIP1 from forming pro-death complexes with caspase-8; it also targets NIK for proteasomal degradation to suppress non-canonical NF-κB, ubiquitinates TRAF2 and ASK1 to terminate TNF signaling, ubiquitinates caspase-3/7 (promoting their degradation without direct inhibition), ubiquitinates RIP2 for NOD signaling, and ubiquitinates E2F1 (K63-linked, stabilizing) and Mad1 (K48-linked, degradative) to regulate transcription; its E3 activity is autoinhibited by CARD domain-mediated prevention of RING dimerization, stabilized by HSP90β and deubiquitinases OTUB1 and USP19, and activated by Smac/DIABLO or IAP antagonists which trigger auto-ubiquitination and proteasomal degradation of cIAP1 itself via branched K63/K48-linked ubiquitin chains assembled with UBE2N."},"narrative":{"mechanistic_narrative":"BIRC2/cIAP1 is a RING-domain E3 ubiquitin ligase that functions as a central post-translational rheostat of TNF-receptor-superfamily signaling, determining whether cells survive or die [PMID:18570872, PMID:31141691]. Within the TNF-R1 signaling complex, to which it is recruited through TRADD-mediated association with TRAF2 [PMID:8943045], cIAP1 (redundantly with cIAP2) constitutively conjugates K63-, K11-, and linear ubiquitin chains onto RIP1, generating the platform that recruits TAK1/IKK to drive canonical NF-κB activation while preventing RIP1 from nucleating pro-death complexes with caspase-8 or RIP3-dependent necrosomes [PMID:18570872, PMID:18621737, PMID:21113135, PMID:21052097]. This ligase activity, executed with UbcH5-family E2 enzymes, extends across the RIP-kinase family (RIP1–RIP4) and to RIP2 in NOD1/NOD2 innate immune signaling and caspase-1 in inflammasome activation [PMID:19258326, PMID:19464198, PMID:21931591, PMID:22195745]. cIAP1 also sets the threshold for non-canonical NF-κB by targeting NIK for proteasomal degradation within a TRAF2–TRAF3 complex, and terminates TNF-R2 signaling by ubiquitinating TRAF2 and ASK1 [PMID:18997794, PMID:11907583, PMID:17220297]. In vivo, combined cIAP1/2 loss causes caspase-8-dependent intestinal and hepatic death, establishing suppression of caspase-8 apoptosis as the core survival function [PMID:31141691]. Although its BIR domains bind caspases-3 and -7, cIAP1 promotes their proteasomal degradation rather than directly inhibiting their activity [PMID:16339151, PMID:19258326]. The enzyme is autoinhibited by a CARD domain that blocks RING dimerization and E2 activation [PMID:21549626], stabilized by the chaperone HSP90β and the deubiquitinases OTUB1 and USP19 [PMID:18239673, PMID:23524849, PMID:33712742, PMID:21849505], and selectively eliminated by Smac/DIABLO or synthetic IAP antagonists that bind its BIR3 domain and trigger RING-dependent auto-ubiquitination and proteasomal degradation [PMID:14960576, PMID:18230607, PMID:20356846]. Beyond cytoplasmic signaling, nuclear cIAP1 binds E2F1 and stabilizes it through K63-linked ubiquitination to promote cell-cycle gene transcription [PMID:21653699, PMID:28542143].","teleology":[{"year":1996,"claim":"Established that cIAP1 is physically positioned within TNF receptor signaling, identifying how it gains access to the death-and-survival machinery via the TRADD-TRAF2 adaptor module.","evidence":"Co-immunoprecipitation and TNF-dependent recruitment assays in cells","pmids":["8943045"],"confidence":"High","gaps":["Did not define a catalytic activity or downstream substrate at the receptor complex"]},{"year":1997,"claim":"First assigned cIAP1 an anti-apoptotic biochemical activity by showing BIR-domain binding to caspases-3/-7, initially interpreted as direct enzymatic inhibition.","evidence":"In vitro binding and caspase activity assays with recombinant proteins, cell-free cytochrome-c system","pmids":["9384571"],"confidence":"High","gaps":["The inferred direct inhibition was later overturned for cIAP BIRs","Did not address the RING/E3 contribution"]},{"year":2002,"claim":"Reframed cIAP1 as an E3 ubiquitin ligase rather than a stoichiometric inhibitor, by demonstrating it ubiquitinates TRAF2 for proteasomal degradation downstream of TNF-R2.","evidence":"In vitro ubiquitination with purified components, E3-defective mutant, proteasome inhibition in primary cells","pmids":["11907583"],"confidence":"High","gaps":["Did not identify the full substrate repertoire","E2 partner and chain linkage unresolved at this stage"]},{"year":2008,"claim":"Resolved the core survival mechanism: cIAP1/2 maintain constitutive ubiquitination of RIP1 that recruits TAK1 for NF-κB while blocking caspase-8 association, defining a molecular survival/death switch.","evidence":"IAP antagonist treatment, in vitro reconstitution with purified cIAP1+UbcH5a, RIP1-TAK1/RIP1-caspase-8 co-IPs, genetic and siRNA ablation","pmids":["18570872","18621737","18697935"],"confidence":"High","gaps":["Did not resolve which ubiquitin linkages dominate in vivo","Redundancy with cIAP2 left the unique cIAP1 contribution unclear"]},{"year":2008,"claim":"Defined cIAP1 as the threshold-setter for non-canonical NF-κB by showing it degrades NIK within a TRAF2/TRAF3 complex, linking IAP loss to constitutive RelB activation.","evidence":"Complex reconstitution, genetic and pharmacological cIAP inhibition, NIK degradation and primary B cell assays","pmids":["18997794"],"confidence":"High","gaps":["Did not establish chain linkage on NIK","Stoichiometry within the TRAF2/TRAF3 complex undefined"]},{"year":2008,"claim":"Identified the mechanism by which IAP antagonists kill: Smac/DIABLO binds BIR domains to stimulate RING-dependent cIAP1 auto-ubiquitination and selective degradation.","evidence":"In vitro auto-ubiquitination, Smac N-terminal peptide sufficiency, E2 identification, proteasome inhibition","pmids":["14960576","18230607"],"confidence":"Medium","gaps":["Did not fully define the conformational activation step","Selectivity over XIAP/cIAP2 mechanism incomplete"]},{"year":2009,"claim":"Overturned the direct-caspase-inhibitor model by showing cIAP1 binds and ubiquitinates caspases-3/-7 for degradation without inhibiting their catalytic activity.","evidence":"In vitro binding, fluorogenic substrate assays, ubiquitination with UbcH5/UbcH8, chimeric caspase-3, domain mapping","pmids":["19258326","16339151"],"confidence":"High","gaps":["In vivo contribution of caspase degradation versus RIP1 control not separated"]},{"year":2009,"claim":"Extended cIAP1 function into innate immunity by identifying RIP2 ubiquitination as required for NOD1/NOD2 signaling.","evidence":"Birc2/Birc3 knockout macrophages, RIP2 ubiquitination assay, in vivo NOD agonist challenge and colitis model","pmids":["19464198"],"confidence":"High","gaps":["Relative contribution of cIAP1 versus cIAP2 to RIP2 modification not isolated"]},{"year":2010,"claim":"Mapped the ubiquitin chain architecture on RIP1, showing cIAP1/UbcH5 assemble K11-linked chains read by NEMO, diversifying the chain code at the receptor complex.","evidence":"Yeast two-hybrid E2 screen, in vitro reconstitution, mass spectrometry of linkages, TNFR1 complex IP","pmids":["21113135"],"confidence":"High","gaps":["Relative in vivo abundance of K11 versus K63 chains not quantified"]},{"year":2011,"claim":"Explained how cIAP1 E3 activity is restrained, with a crystal structure showing the CARD domain autoinhibits by preventing RING dimerization and E2 activation.","evidence":"CARD crystal structure, RING dimerization and E2 activation assays, mutagenesis, zebrafish vascular readout","pmids":["21549626","20356846"],"confidence":"High","gaps":["Physiological trigger that relieves CARD autoinhibition not defined"]},{"year":2011,"claim":"Identified a nuclear, transcription-regulatory role: cIAP1 binds the E2F1 DNA-binding domain at cell-cycle gene promoters to enhance proliferation.","evidence":"Co-IP, E2F1 reporter, ChIP on CCNE/CCNA, siRNA knockdown proliferation assay","pmids":["21653699"],"confidence":"Medium","gaps":["Single lab","Did not initially establish a ubiquitin-based mechanism for E2F1 regulation"]},{"year":2013,"claim":"Established a stabilizing regulatory layer, showing OTUB1 removes K48 chains from cIAP1 within the TWEAK receptor complex to protect it from degradation.","evidence":"Co-IP, linkage-specific in vitro DUB assay, OTUB1 knockdown with pathway readouts, zebrafish validation","pmids":["23524849","33712742"],"confidence":"High","gaps":["Balance between OTUB1, USP19 and HSP90β in setting steady-state cIAP1 levels not integrated"]},{"year":2017,"claim":"Resolved the molecular basis of cIAP1-driven transcription by showing K63-linked ubiquitination of E2F1 stabilizes it and licenses promoter binding during S phase and DNA damage.","evidence":"In vitro ubiquitination, K161R/K164R mutagenesis, ChIP on CCNE/TP73/APAF1, cell-cycle and DNA damage assays","pmids":["28542143"],"confidence":"Medium","gaps":["Single lab","How nuclear versus cytoplasmic cIAP1 pools are partitioned to this function unclear"]},{"year":2019,"claim":"Defined the dominant in vivo physiological function by showing combined cIAP1/2 loss kills intestinal and hepatic tissue via caspase-8, rescued by Casp8/Ripk3 co-deletion.","evidence":"Conditional cIap1/2 double knockout mice, Casp8/Ripk3/Mlkl epistasis, cleaved caspase staining, NIK inhibition","pmids":["31141691"],"confidence":"High","gaps":["Did not separate cIAP1-specific from cIAP2-specific roles in vivo"]},{"year":2022,"claim":"Detailed the degradation chemistry exploited by cIAP1-recruiting degraders, showing UBE2N-seeded K63 chains nucleate branched K48/K63 chains that recruit p97 and the proteasome.","evidence":"UBE2N knockout, in vitro E2 panel ubiquitination, MS linkage mapping, p97/UCH37/proteasome interaction assays","pmids":["36316570"],"confidence":"High","gaps":["Whether this branched-chain logic governs cIAP1's endogenous substrates not established"]},{"year":null,"claim":"How the many cIAP1 activities—receptor-complex RIP ubiquitination, NIK degradation, caspase turnover, and nuclear E2F1/Mad1 regulation—are coordinated across subcellular compartments and which are physiologically dominant in specific tissues remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model linking compartmental localization to substrate selection","Tissue-specific substrate hierarchy undefined","Most non-RIP1 substrates rest on single-lab evidence"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[4,11,15,18,19,29,35,53]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[4,18,26,30,35,53]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[10,26,34]},{"term_id":"GO:0031386","term_label":"protein tag activity","supporting_discovery_ids":[18,30,35]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[8,12,18,25]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[8,9,12,39]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[9]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[13]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,47]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,18,19,20,21,47]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[18,31,48,31]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[29,36,45]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[4,11,26,51]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[39,44,12]}],"complexes":["TNF-R1 signaling complex","TNF-R2 / TRAF2 complex","TRAF2-TRAF3-NIK non-canonical NF-κB regulatory complex","LIGHT·LTβR complex"],"partners":["TRAF2","RIPK1","TRAF3","NIK","OTUB1","USP19","E2F1","UBE2N"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q13490","full_name":"Baculoviral IAP repeat-containing protein 2","aliases":["Cellular inhibitor of apoptosis 1","C-IAP1","IAP homolog B","Inhibitor of apoptosis protein 2","hIAP-2","hIAP2","RING finger protein 48","RING-type E3 ubiquitin transferase BIRC2","TNFR2-TRAF-signaling complex protein 2"],"length_aa":618,"mass_kda":69.9,"function":"Multi-functional protein which regulates not only caspases and apoptosis, but also modulates inflammatory signaling and immunity, mitogenic kinase signaling, and cell proliferation, as well as cell invasion and metastasis. Acts as an E3 ubiquitin-protein ligase regulating NF-kappa-B signaling and regulates both canonical and non-canonical NF-kappa-B signaling by acting in opposite directions: acts as a positive regulator of the canonical pathway and suppresses constitutive activation of non-canonical NF-kappa-B signaling. The target proteins for its E3 ubiquitin-protein ligase activity include: RIPK1, RIPK2, RIPK3, RIPK4, CASP3, CASP7, CASP8, TRAF2, DIABLO/SMAC, MAP3K14/NIK, MAP3K5/ASK1, IKBKG/NEMO, IKBKE and MXD1/MAD1. Can also function as an E3 ubiquitin-protein ligase of the NEDD8 conjugation pathway, targeting effector caspases for neddylation and inactivation. Acts as an important regulator of innate immune signaling via regulation of Toll-like receptors (TLRs), Nodlike receptors (NLRs) and RIG-I like receptors (RLRs), collectively referred to as pattern recognition receptors (PRRs). Protects cells from spontaneous formation of the ripoptosome, a large multi-protein complex that has the capability to kill cancer cells in a caspase-dependent and caspase-independent manner. Suppresses ripoptosome formation by ubiquitinating RIPK1 and CASP8. Can stimulate the transcriptional activity of E2F1. Plays a role in the modulation of the cell cycle","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q13490/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/BIRC2","classification":"Not Classified","n_dependent_lines":174,"n_total_lines":1208,"dependency_fraction":0.14403973509933773},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"EIF4A1","stoichiometry":0.2},{"gene":"HNRNPH1","stoichiometry":0.2},{"gene":"RBM14","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/BIRC2","total_profiled":1310},"omim":[{"mim_id":"614989","title":"EPS8-LIKE PROTEIN 3; EPS8L3","url":"https://www.omim.org/entry/614989"},{"mim_id":"614988","title":"EPS8-LIKE PROTEIN 2; EPS8L2","url":"https://www.omim.org/entry/614988"},{"mim_id":"614987","title":"EPS8-LIKE PROTEIN 1; EPS8L1","url":"https://www.omim.org/entry/614987"},{"mim_id":"614215","title":"ACTIVATING SIGNAL COINTEGRATOR 1 COMPLEX, SUBUNIT 1; ASCC1","url":"https://www.omim.org/entry/614215"},{"mim_id":"606965","title":"FAS-ACTIVATED SERINE/THREONINE KINASE; FASTK","url":"https://www.omim.org/entry/606965"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoli fibrillar center","reliability":"Supported"},{"location":"Cytosol","reliability":"Supported"},{"location":"Nucleoplasm","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/BIRC2"},"hgnc":{"alias_symbol":["cIAP1","hiap-2","MIHB","RNF48","c-IAP1"],"prev_symbol":["API1"]},"alphafold":{"accession":"Q13490","domains":[{"cath_id":"1.10.1170.10","chopping":"45-115","consensus_level":"high","plddt":87.1494,"start":45,"end":115},{"cath_id":"1.10.1170.10","chopping":"178-256","consensus_level":"high","plddt":86.5459,"start":178,"end":256},{"cath_id":"1.10.1170.10","chopping":"263-353","consensus_level":"medium","plddt":91.294,"start":263,"end":353},{"cath_id":"1.10.8.10","chopping":"386-449","consensus_level":"medium","plddt":86.1889,"start":386,"end":449},{"cath_id":"1.10.533.10","chopping":"455-613","consensus_level":"medium","plddt":90.7069,"start":455,"end":613}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13490","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q13490-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q13490-F1-predicted_aligned_error_v6.png","plddt_mean":76.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=BIRC2","jax_strain_url":"https://www.jax.org/strain/search?query=BIRC2"},"sequence":{"accession":"Q13490","fasta_url":"https://rest.uniprot.org/uniprotkb/Q13490.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q13490/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13490"}},"corpus_meta":[{"pmid":"9733516","id":"PMC_9733516","title":"NF-kappaB antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation.","date":"1998","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/9733516","citation_count":2440,"is_preprint":false},{"pmid":"9384571","id":"PMC_9384571","title":"The c-IAP-1 and c-IAP-2 proteins are direct inhibitors of specific caspases.","date":"1997","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/9384571","citation_count":1060,"is_preprint":false},{"pmid":"18570872","id":"PMC_18570872","title":"cIAP1 and cIAP2 facilitate cancer cell survival by functioning as E3 ligases that promote RIP1 ubiquitination.","date":"2008","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/18570872","citation_count":933,"is_preprint":false},{"pmid":"18997794","id":"PMC_18997794","title":"Noncanonical NF-kappaB activation requires coordinated assembly of a regulatory complex of the adaptors cIAP1, cIAP2, TRAF2 and TRAF3 and the kinase 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Treatment.","date":"2021","source":"Cancers","url":"https://pubmed.ncbi.nlm.nih.gov/34298797","citation_count":33,"is_preprint":false},{"pmid":"11071269","id":"PMC_11071269","title":"p21WAF1 prevents down-modulation of the apoptotic inhibitor protein c-IAP1 and inhibits leukemic apoptosis.","date":"2000","source":"Molecular medicine (Cambridge, Mass.)","url":"https://pubmed.ncbi.nlm.nih.gov/11071269","citation_count":33,"is_preprint":false},{"pmid":"24521431","id":"PMC_24521431","title":"Potent and selective small-molecule inhibitors of cIAP1/2 proteins reveal that the binding of Smac mimetics to XIAP BIR3 is not required for their effective induction of cell death in tumor cells.","date":"2014","source":"ACS chemical biology","url":"https://pubmed.ncbi.nlm.nih.gov/24521431","citation_count":33,"is_preprint":false},{"pmid":"26011589","id":"PMC_26011589","title":"IRF-1 inhibits NF-κB activity, suppresses TRAF2 and cIAP1 and induces breast cancer cell specific growth inhibition.","date":"2015","source":"Cancer biology & therapy","url":"https://pubmed.ncbi.nlm.nih.gov/26011589","citation_count":31,"is_preprint":false},{"pmid":"20458734","id":"PMC_20458734","title":"Lipopolysaccharide induces autophagy through BIRC2 in human umbilical vein endothelial cells.","date":"2010","source":"Journal of cellular physiology","url":"https://pubmed.ncbi.nlm.nih.gov/20458734","citation_count":30,"is_preprint":false},{"pmid":"26094954","id":"PMC_26094954","title":"Oncogenic activity of BIRC2 and BIRC3 mutants independent of nuclear factor-κB-activating potential.","date":"2015","source":"Cancer science","url":"https://pubmed.ncbi.nlm.nih.gov/26094954","citation_count":29,"is_preprint":false},{"pmid":"36471347","id":"PMC_36471347","title":"TRIM56 promotes malignant progression of glioblastoma by stabilizing cIAP1 protein.","date":"2022","source":"Journal of experimental & clinical cancer research : CR","url":"https://pubmed.ncbi.nlm.nih.gov/36471347","citation_count":29,"is_preprint":false},{"pmid":"19416853","id":"PMC_19416853","title":"Identification of ARIA regulating endothelial apoptosis and angiogenesis by modulating proteasomal degradation of cIAP-1 and cIAP-2.","date":"2009","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/19416853","citation_count":28,"is_preprint":false},{"pmid":"33526051","id":"PMC_33526051","title":"Shikonin promotes ubiquitination and degradation of cIAP1/2-mediated apoptosis and necrosis in triple negative breast cancer cells.","date":"2021","source":"Chinese medicine","url":"https://pubmed.ncbi.nlm.nih.gov/33526051","citation_count":27,"is_preprint":false},{"pmid":"31126323","id":"PMC_31126323","title":"Targeted ablation of the cellular inhibitor of apoptosis 1 (cIAP1) attenuates denervation-induced skeletal muscle atrophy.","date":"2019","source":"Skeletal muscle","url":"https://pubmed.ncbi.nlm.nih.gov/31126323","citation_count":27,"is_preprint":false},{"pmid":"24975362","id":"PMC_24975362","title":"Ubiquitin-dependent regulation of MEKK2/3-MEK5-ERK5 signaling module by XIAP and cIAP1.","date":"2014","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/24975362","citation_count":27,"is_preprint":false},{"pmid":"17074018","id":"PMC_17074018","title":"Chenodeoxycholic acid and taurochenodexycholic acid induce anti-apoptotic cIAP-1 expression in human hepatocytes.","date":"2006","source":"Journal of gastroenterology and hepatology","url":"https://pubmed.ncbi.nlm.nih.gov/17074018","citation_count":26,"is_preprint":false},{"pmid":"28542143","id":"PMC_28542143","title":"DNA damage and S phase-dependent E2F1 stabilization requires the cIAP1 E3-ubiquitin ligase and is associated with K63-poly-ubiquitination on lysine 161/164 residues.","date":"2017","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/28542143","citation_count":25,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":53038,"output_tokens":14016,"usd":0.184677,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":25811,"output_tokens":5245,"usd":0.13009,"stage2_stop_reason":"end_turn"},"total_usd":0.314767,"stage1_batch_id":"msgbatch_0179Z2pxRYBsXi2xADneafRw","stage2_batch_id":"msgbatch_01LaY9ZaVdgjWArcZo9eqQE5","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1996,\n      \"finding\": \"c-IAP1 is recruited to the TNF-R1 signaling complex via TRADD-mediated association with TRAF2, establishing its presence in TNF-R1 signaling independently of TNF-R2.\",\n      \"method\": \"Co-immunoprecipitation, TNF-dependent recruitment assay in cells\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, TNF-dependent recruitment demonstrated, replicated across multiple subsequent studies\",\n      \"pmids\": [\"8943045\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"c-IAP1 and c-IAP2 bind specifically to caspases-3 and -7 via their BIR domains and inhibit their enzymatic activity in vitro (estimated Ki ≤0.1 µM), blocking caspase-3 processing in cell-free and intact cell systems.\",\n      \"method\": \"In vitro binding assay, in vitro caspase activity assay, cell-free cytochrome-c system, gene transfection overexpression\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with purified recombinant proteins, Ki measurement, domain mapping, replicated in cell-free and intact-cell systems\",\n      \"pmids\": [\"9384571\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"NF-κB transcriptional activation induces expression of c-IAP1 and c-IAP2 (along with TRAF1 and TRAF2), and these proteins cooperatively suppress TNF-alpha-induced caspase-8 activation; c-IAP1/2 alone are sufficient to suppress etoposide-induced apoptosis.\",\n      \"method\": \"NF-κB inhibition/activation, gene transfection, caspase-8 activation assay, apoptosis assays\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic and overexpression approaches, multiple stimuli, replicated broadly\",\n      \"pmids\": [\"9733516\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"c-IAP1 is cleaved during apoptosis by caspase-3 to produce C-terminal fragments containing the RING domain; these fragments are proapoptotic, and the RING domain negatively regulates the antiapoptotic function of the N-terminal BIR domain.\",\n      \"method\": \"In vitro cleavage with purified caspase-3, transfection of deletion mutants, apoptosis assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with purified caspase-3, domain mutagenesis/deletion, functional readout in cells\",\n      \"pmids\": [\"11106668\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"c-IAP1 functions as an E3 ubiquitin ligase that ubiquitinates TRAF2 (but not TRAF1 despite binding both) downstream of TNF-RII engagement, leading to proteasomal degradation of TRAF2; E3-defective c-IAP1 mutant blocks this degradation and inhibits apoptosis.\",\n      \"method\": \"In vitro ubiquitination assay, co-immunoprecipitation, E3-defective mutant expression, proteasome inhibition, primary cell studies\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro ubiquitination with purified components, active-site mutagenesis, primary cell validation\",\n      \"pmids\": [\"11907583\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"cIAP1 is irreversibly cleaved (inactivated) during p53-dependent apoptosis by the serine protease HTRA2, which interacts directly with cIAP1; serine protease inhibitors that block cIAP1 cleavage inhibit p53-dependent apoptosis.\",\n      \"method\": \"Co-immunoprecipitation, serine protease inhibitor treatment, apoptosis assay, p53 induction\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP of endogenous proteins, pharmacological inhibition, single lab\",\n      \"pmids\": [\"12569127\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"c-IAP1 and c-IAP2 ubiquitinate RIP (RIP1) in vitro; expression of c-IAP1/2 decreases steady-state RIP levels in a proteasome-dependent manner, requiring the RING domain of c-IAP2.\",\n      \"method\": \"In vitro ubiquitination assay, proteasome inhibitor rescue, deletion mutant analysis\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro ubiquitination reconstitution, domain mutagenesis, single lab\",\n      \"pmids\": [\"15147886\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"c-IAP1 translation is driven by an internal ribosome entry site (IRES) in its 5'-UTR, and IRES-mediated translation is stimulated by pro-apoptotic stimuli (etoposide, sodium arsenite) that inhibit cap-dependent translation.\",\n      \"method\": \"Dicistronic RNA constructs, in vitro translation, transfection in multiple cell lines, hairpin insertion, cap-independent translation assay\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — multiple orthogonal translation assays, multiple cell types, single lab\",\n      \"pmids\": [\"14970392\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"cIAP1 and cIAP2 are nuclear-cytoplasmic shuttling proteins; their nuclear export is CRM1-dependent, and a leucine-rich NES in the BIR2-BIR3 linker region mediates nuclear export of cIAP1. TRAF2 retains cIAP1/2 in the cytoplasm and prevents nuclear translocation; TNFα treatment reduces TRAF2-mediated cytoplasmic retention.\",\n      \"method\": \"Leptomycin B treatment, site-directed mutagenesis of NES, co-expression with TRAF2, immunofluorescence, subcellular fractionation\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — NES mutagenesis, leptomycin B, TRAF2 co-expression, multiple orthogonal localization methods\",\n      \"pmids\": [\"15265700\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"In hematopoietic cells undergoing differentiation, c-IAP1 translocates from the nucleus to the Golgi apparatus via a nuclear export signal (NES) located in the CARD domain, in a leptomycin B-sensitive, CRM1-dependent mechanism.\",\n      \"method\": \"Immunofluorescence, leptomycin B treatment, site-directed mutagenesis of CARD NES, phorbol ester differentiation model\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — NES mutagenesis, CRM1 inhibition, multiple cell lines and primary monocytes\",\n      \"pmids\": [\"15187025\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"cIAP1 and cIAP2 BIR2 and BIR3 domains bind caspases-7 and -9 but do NOT inhibit caspase activity due to critical substitutions in caspase-inhibitory contact residues; substituting these residues with XIAP equivalents converts cIAP BIRs into tight caspase inhibitors.\",\n      \"method\": \"In vitro binding assay, fluorogenic caspase activity assay, domain mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution, active-site mutagenesis, negative result for inhibition rigorously established\",\n      \"pmids\": [\"16339151\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"c-IAP1 mediates posttranscriptional downregulation of c-IAP2 via E3 ubiquitin ligase-dependent ubiquitination and proteasomal degradation, potentiated by TRAF2 as an adaptor; c-IAP1-deficient mice show elevated c-IAP2 protein without increased c-IAP2 mRNA.\",\n      \"method\": \"c-IAP1 knockout mice, wild-type vs. E3-defective c-IAP1 transfection, Western blot, RT-PCR\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout, E3-dead mutant complementation, mRNA vs. protein distinction, in vivo model\",\n      \"pmids\": [\"15798218\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"cIAP1 localizes predominantly to the nucleus (unlike cytoplasmic XIAP); apoptotic stimuli induce nuclear export of cIAP1 in a caspase-dependent manner. During mitosis, cIAP1 is released to cytosol and reassociates with the midbody; it interacts with Survivin during mitosis. Overexpression of cIAP1 causes G2-M accumulation, cytokinesis defects, and polyploidy.\",\n      \"method\": \"Immunofluorescence microscopy, subcellular fractionation, caspase inhibitor treatment, stable overexpression, cell cycle analysis\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple localization methods, functional overexpression phenotype, single lab\",\n      \"pmids\": [\"15665297\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Upon TNF-R2 signaling, the c-IAP1/TRAF2 complex translocates to a Triton X-100-insoluble perinuclear ER compartment containing the E2 enzyme Ubc6; Ubc6 serves as a cognate E2 for c-IAP1's E3 activity and is required for TNF-R2-dependent TRAF2 ubiquitination and degradation.\",\n      \"method\": \"Subcellular fractionation, confocal microscopy, co-immunoprecipitation, in vitro ubiquitination with Ubc6, dominant-negative Ubc6 mutant\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro E2/E3 reconstitution, dominant-negative mutant, confocal co-localization, fractionation\",\n      \"pmids\": [\"15861135\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Smac/DIABLO selectively promotes rapid degradation of c-IAP1 and c-IAP2 (but not XIAP or Livin) by stimulating their auto-ubiquitination via its N-terminal IAP-binding motif binding to the BIR domains; ubiquitin-conjugating enzymes UbcH5a and UbcH6 are required for this ubiquitination.\",\n      \"method\": \"In vitro auto-ubiquitination assay, deletion mutants, N-terminal Smac peptide, proteasome inhibition, Western blot\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro ubiquitination, domain deletion analysis, peptide sufficiency test, E2 identification\",\n      \"pmids\": [\"14960576\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"c-IAP1 functions as the E3 ubiquitin ligase for ASK1 downstream of TNFR2 signaling, leading to ASK1 ubiquitination and proteasomal degradation; c-IAP1 knockout primary B cells lack TNFR2-induced TRAF2 and ASK1 degradation and show prolonged p38 and JNK activation.\",\n      \"method\": \"Primary c-IAP1 knockout B cells, ubiquitination assay, proteasome inhibitor, kinase activity assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — primary knockout cells, E3 identification, kinase prolongation phenotype, in vivo validation\",\n      \"pmids\": [\"17220297\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Birc2 (cIAP1) positively regulates formation of the TNF receptor complex I in endothelial cells, promoting NF-κB activation and maintaining vascular integrity; loss of Birc2 in zebrafish causes caspase-8-dependent apoptosis and vessel regression.\",\n      \"method\": \"Zebrafish forward genetic screen, null mutant analysis, genetic epistasis with NF-κB pathway, caspase-8 assay\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo null mutant, genetic epistasis, multiple molecular and cellular readouts\",\n      \"pmids\": [\"17934460\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"c-IAP1 acts as an E3 ubiquitin ligase for Mad1 (MAX dimerization protein 1), promoting its proteasomal degradation, which cooperates with Myc to promote cell proliferation.\",\n      \"method\": \"In vitro ubiquitination assay, proteasome inhibitor, overexpression/knockdown proliferation assay\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro ubiquitination, proteasomal degradation rescue, functional proliferation readout, single lab\",\n      \"pmids\": [\"18082613\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"cIAP1 and cIAP2 promote cancer cell survival by functioning as E3 ubiquitin ligases that maintain constitutive K63-linked ubiquitination of RIP1; ubiquitinated RIP1 associates with pro-survival TAK1, while deubiquitinated RIP1 binds caspase-8 and induces apoptosis.\",\n      \"method\": \"IAP antagonist AEG40730 treatment, in vitro ubiquitination with purified components, co-immunoprecipitation of RIP1-TAK1 and RIP1-caspase-8 complexes\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with purified components, multiple orthogonal methods, mechanistic switch demonstrated\",\n      \"pmids\": [\"18570872\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"cIAP1 and cIAP2 are required for TNFα-stimulated RIP1 K63-linked polyubiquitination and downstream NF-κB activation; in vitro reconstitution with purified c-IAP1 and UbcH5a demonstrates direct K63-linked chain polymerization on RIP1.\",\n      \"method\": \"Genetic knockout cells, siRNA knockdown, IAP antagonist, in vitro ubiquitination reconstitution with purified cIAP1+UbcH5a\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with purified components, genetic ablation validated with pharmacological approach, replicated across labs\",\n      \"pmids\": [\"18621737\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Both cIAP1 and cIAP2 are required (redundantly) for TNFα-induced RIP1 polyubiquitination and NF-κB activation; combined loss of both cIAPs attenuates IKKβ phosphorylation and sensitizes cells to TNFα-mediated apoptosis.\",\n      \"method\": \"Combined genetic deletion and siRNA knockdown, TNF receptor complex immunoprecipitation, RIP1 ubiquitination assay, NF-κB activation assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — combined genetic/siRNA approaches, primary cells, receptor complex pull-down, replicated across labs\",\n      \"pmids\": [\"18697935\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Noncanonical NF-κB activation is suppressed by a regulatory complex in which TRAF2 recruits cIAP1/2 and TRAF3 recruits NIK; cIAP1 and cIAP2 mediate proteasomal degradation of NIK within this complex, and combined inhibition of both cIAPs is required for noncanonical NF-κB activation.\",\n      \"method\": \"Complex assembly assay, cIAP inhibition (genetic and pharmacological), NIK degradation assay, primary B lymphocyte survival/proliferation assay\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — complex reconstitution, genetic and pharmacological inhibition, primary cell and in vivo validation\",\n      \"pmids\": [\"18997794\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"TWEAK/FN14 signaling promotes lysosomal (not proteasomal) degradation of the cIAP1-TRAF2 complex in a cIAP1-dependent manner, leading to noncanonical NF-κB activation and sensitization to TNFα-induced death.\",\n      \"method\": \"Lysosomal inhibitor vs. proteasomal inhibitor treatment, co-immunoprecipitation of FN14-cIAP1-TRAF2 complex, cIAP1 overexpression rescue\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pathway dissection with inhibitors, complex co-IP, rescue experiment, single lab\",\n      \"pmids\": [\"18606850\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Small molecules (ME-BS class) directly interact with the BIR3 domain of cIAP1, promote RING domain-dependent auto-ubiquitylation, and facilitate proteasomal degradation of cIAP1 selectively (not XIAP or cIAP2), sensitizing cancer cells to apoptosis.\",\n      \"method\": \"Direct binding assay (BIR3 domain), auto-ubiquitylation assay, proteasome inhibitor rescue, Western blot, apoptosis assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct binding and in vitro ubiquitylation, domain mapping, single lab\",\n      \"pmids\": [\"18230607\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The CARD domain of cIAP1 contains a functional NES mediating CRM1-dependent nuclear export; the RING domain of cIAP1 degrades RING-bearing IAPs (cIAP1, cIAP2, XIAP, Livin) by ubiquitin-dependent and ubiquitin-independent proteasomal pathways.\",\n      \"method\": \"CARD NES mutagenesis, leptomycin B treatment, ubiquitination-deficient E1 mutant cells, cIAP1 RING domain transfection, proteasome inhibitor\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis, E1-deficient cells, multiple IAP substrates tested, single lab\",\n      \"pmids\": [\"18434593\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"During monocyte-to-macrophage differentiation, cIAP1 (after translocating from nucleus to cytoplasm) mediates proteasomal degradation of TRAF2 via its E3 ligase activity; TRAF2 is initially required for NF-κB activation during differentiation, and its subsequent cIAP1-mediated degradation is required for full macrophage function including cytokine secretion.\",\n      \"method\": \"cIAP1 inhibitor, TRAF2 siRNA knockdown, TRAF2 overexpression, NF-κB assay, cytokine measurement\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function and gain-of-function, functional cytokine readout, pathway ordered, single lab\",\n      \"pmids\": [\"18827186\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"cIAP1 binds caspase-3 and caspase-7 at distinct steps of their processing via different mechanisms: it binds mature caspase-7 via its exposed IAP-binding motif AKPD, and binds partially processed caspase-3 via a prodomain-dependent non-classical mechanism. cIAP1 ubiquitinates both caspases via UbcH5 subfamily E2s (and UbcH8 for caspase-3), leading to their proteasomal degradation without directly inhibiting their enzymatic activity.\",\n      \"method\": \"In vitro binding assay, fluorogenic substrate assay, ubiquitination assay, chimeric caspase-3 with AKPD motif, proteasome inhibitor, UbcH5/UbcH8 identification\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution, domain mapping, E2 identification, chimeric substrate, multiple orthogonal methods\",\n      \"pmids\": [\"19258326\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"cIAP1 mediates NEMO monoubiquitination in response to genotoxic stress as part of NF-κB activation; cIAP1, cIAP2, and XIAP act cooperatively and non-redundantly at distinct steps: XIAP activates TAK1, cIAP1 ubiquitinates NEMO, and cIAP2 acts downstream of NEMO ubiquitination.\",\n      \"method\": \"Genotoxic agents (camptothecin, etoposide, doxorubicin), siRNA knockdown of individual IAPs, NEMO ubiquitination assay, NF-κB activation assay\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — individual siRNA knockdowns, pathway ordering by epistasis, single lab\",\n      \"pmids\": [\"19223549\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"A UBA (ubiquitin-associated) domain in cIAP1 located between the BIR domains and the CARD/RING domains binds mono-ubiquitin and K48- and K63-linked polyubiquitin chains; UBA domain mutations abrogate ubiquitin binding and decrease IAP antagonist-stimulated proteasomal degradation of cIAP1.\",\n      \"method\": \"Surface plasmon resonance, isothermal titration calorimetry, NMR analysis of UBA-ubiquitin interaction, UBA domain point mutagenesis, proteasome degradation assay\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — NMR structure of domain-ubiquitin interaction, SPR/ITC binding measurements, mutagenesis, functional degradation readout\",\n      \"pmids\": [\"18939944\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"cIAP1 and cIAP2 are required for NOD1- and NOD2-mediated innate immune signaling; they function as E3 ubiquitin ligases for RIP2 ubiquitination, and their deficiency impairs cytokine/chemokine production in macrophages and colonocytes in vitro and in vivo.\",\n      \"method\": \"Birc2−/− and Birc3−/− mouse macrophages, RNAi in colonocytes, RIP2 ubiquitination assay, in vivo NOD agonist challenge, colitis model\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout, E3 ligase assay, in vivo challenge, multiple cell types\",\n      \"pmids\": [\"19464198\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"c-IAP1 and UbcH5 family E2 enzymes promote K11-linked polyubiquitination of RIP1 in vitro and in vivo within the TNFR1 signaling complex; TNFα-stimulated NF-κB activation involves endogenous K11-linked ubiquitination of RIP1 in a c-IAP1- and UbcH5-dependent manner. NEMO efficiently binds K11-linked ubiquitin chains.\",\n      \"method\": \"Directed yeast two-hybrid screen for E2 partners, in vitro ubiquitination with purified components, mass spectrometry of ubiquitin linkage, UbcH5 siRNA knockdown, TNFR1 complex immunoprecipitation\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with purified proteins, MS-based ubiquitin linkage determination, genetic validation in cells\",\n      \"pmids\": [\"21113135\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"cIAP1 promotes cell survival against TNF-induced necrosis by maintaining RIP1 ubiquitination, which prevents RIP1 kinase activation, RIP1/RIP3 necrosome formation, and ROS accumulation; depletion of cIAP1 (but not cIAP2) is specifically responsible for sensitization to TNF-induced necrosis.\",\n      \"method\": \"RNAi (individual IAP knockdown), IAP antagonist BV6, RIP1 kinase inhibitor, RIP3 knockdown, CYLD knockdown, ROS measurement, cell death assay\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — individual knockdowns identify cIAP1 specifically, epistasis with CYLD/RIP1/RIP3, ROS as mechanistic readout\",\n      \"pmids\": [\"21052097\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"RIPK1 promotes cell survival in TNF-stimulated cells by stabilizing TRAF2 and cIAP1; in RIPK1-deficient cells, TNF stimulation causes rapid proteasomal degradation of TRAF2 and cIAP1, leading to NIK accumulation, non-canonical NF-κB activation, reduction of cFLIPL, and caspase-8 activation.\",\n      \"method\": \"RIPK1−/− cells, TNF stimulation time course, proteasome inhibitor, Western blot for TRAF2/cIAP1/NIK/cFLIPL\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockout, ordered pathway, proteasome rescue, single lab\",\n      \"pmids\": [\"21339290\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The RING domain of cIAP1 requires dimerization and E2 binding for IAP antagonist-induced auto-degradation and for TNF-induced NF-κB activation and prevention of non-canonical NF-κB; RING mutants unable to dimerize or bind E2 fail these functions.\",\n      \"method\": \"cIAP-deleted cells reconstituted with RING point mutants (dimerization-defective, E2-binding-defective), TNF stimulation, IAP antagonist treatment, NF-κB assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — structure-function mutagenesis in reconstituted knockout cells, multiple pathway readouts\",\n      \"pmids\": [\"20356846\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"The CARD domain of cIAP1 autoinhibits its E3 ligase activity by preventing RING dimerization, E2 binding, and E2 activation; CARD-mediated autoregulation suppresses cell proliferation and migration, maximally suppresses caspase-8-dependent apoptosis, and prevents vascular degeneration in vivo.\",\n      \"method\": \"Crystal structure of CARD domain, RING dimerization assay, E2 activation assay, CARD deletion/mutation, cell proliferation/migration assay, zebrafish vascular assay\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure, in vitro E3 activity assays, mutagenesis, in vivo zebrafish validation\",\n      \"pmids\": [\"21549626\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"cIAP1 and cIAP2 are direct E3 ubiquitin ligases for RIP1, RIP2, RIP3, and RIP4; cIAP1 conjugates diverse ubiquitin chain types including linear chains to these substrates. K63-linked ubiquitination of RIP4 on Lys51 and Lys145 by cIAP1 is required for NF-κB activation.\",\n      \"method\": \"Co-immunoprecipitation (direct binding), in vitro ubiquitination assay with purified components, RIP4 K51R/K145R mutagenesis, NF-κB reporter assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro ubiquitination reconstitution, substrate mapping, NF-κB functional readout, single lab\",\n      \"pmids\": [\"21931591\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"cIAP1 and cIAP2 are required for efficient caspase-1 activation by the inflammasome; they interact with caspase-1-containing complexes via TRAF2 and mediate non-degradative K63-linked polyubiquitination of caspase-1; deficiency in Birc2 or Birc3 impairs caspase-1 activation and inflammasome responses in vivo.\",\n      \"method\": \"Birc2−/− and Birc3−/− mouse macrophages, co-immunoprecipitation, K63-ubiquitination assay, in vivo peritonitis model, caspase-1 activity assay\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout, co-IP, ubiquitin linkage assay, in vivo validation\",\n      \"pmids\": [\"22195745\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Smac mimetic-induced degradation of cIAP1 requires its binding to TRAF2; degradation of cIAP2 requires the presence of cIAP1 and cIAP2 RING finger dimerization and E2 binding. cIAP2-MALT1 oncofusion (lacking the RING) is resistant to Smac mimetics.\",\n      \"method\": \"TRAF2-binding mutants, cIAP1/2 double-knockout cells, RING mutants, cIAP2-MALT1 expression, Western blot\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — structure-function mutagenesis, knockout cells, multiple conditions, single lab\",\n      \"pmids\": [\"21331077\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"USP19 deubiquitinase interacts with c-IAP1 and c-IAP2, and stabilizes c-IAP levels primarily through deubiquitinase-independent mechanisms in vivo; knockdown of USP19 decreases c-IAP levels and enhances TNFα-induced caspase activation and apoptosis in a c-IAP1/2-dependent manner.\",\n      \"method\": \"Co-immunoprecipitation, in vitro deubiquitination assay, USP19 knockdown, overexpression, caspase activity assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, in vitro DUB assay, knockdown/overexpression with functional readout, single lab\",\n      \"pmids\": [\"21849505\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Nuclear cIAP1 directly interacts with the DNA-binding domain of E2F1 transcription factor, increases E2F1 transcriptional activity on CCNE and CCNA promoters, and is recruited to E2F1 binding sites by chromatin immunoprecipitation; cIAP1 silencing inhibits cyclin E/A expression and cell proliferation.\",\n      \"method\": \"Co-immunoprecipitation, E2F1 transcription reporter assay, chromatin immunoprecipitation (ChIP), cIAP1 siRNA knockdown, cell proliferation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ChIP, functional transcription assay, siRNA rescue, single lab\",\n      \"pmids\": [\"21653699\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"cIAP1 limits macrophage necroptosis by suppressing RIP3 (and to a lesser extent RIP1) expression via post-transcriptional mechanisms, preventing necrosome formation; specific cIAP1 knockout causes elevated macrophage cell death and compromised control of Listeria monocytogenes.\",\n      \"method\": \"SMAC mimetic treatment, RNAi individual knockdown, RIP1 kinase inhibitor, RIP3 knockdown, single cIAP1 or cIAP2 knockout macrophages, in vivo Listeria model\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — specific cIAP1 knockout identifies distinct role, in vivo infection model, post-transcriptional mechanism, single lab\",\n      \"pmids\": [\"22576661\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Loss of cIAP1 activates the noncanonical NF-κB pathway (via NIK/p100/RelB), which promotes myoblast fusion; TWEAK at low concentrations activates this pathway to increase myoblast fusion without causing atrophy.\",\n      \"method\": \"cIAP1-null cells, siRNA knockdown of p100/RelB/IKKα/NIK, TWEAK treatment, myoblast fusion quantification\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic loss-of-function, pathway component knockdown epistasis, quantitative fusion assay, single lab\",\n      \"pmids\": [\"23074266\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"OTUB1 deubiquitinase associates with c-IAP1, disassembles K48-linked polyubiquitin chains from c-IAP1 in vitro and in vivo within the TWEAK receptor signaling complex, thereby stabilizing c-IAP1; OTUB1 knockdown promotes c-IAP1 degradation, caspase activation, and reduces TWEAK-induced NF-κB and MAPK signaling.\",\n      \"method\": \"Co-immunoprecipitation, in vitro deubiquitination assay (K48-linkage specific), OTUB1 siRNA, cell death assay, NF-κB/MAPK reporter, zebrafish OTUB1 suppression\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro DUB assay with linkage specificity, co-IP, genetic knockdown with multiple pathway readouts, in vivo zebrafish validation\",\n      \"pmids\": [\"23524849\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"BIRC2 (cIAP1) functions as a negative regulator of noncanonical NF-κB-dependent HIV-1 LTR transcription; depletion of BIRC2 by Smac mimetics activates HIV-1 transcription and reverses latency, synergizing with HDACi panobinostat in patient-derived CD4+ T cells.\",\n      \"method\": \"RNAi screen, BIRC2 siRNA knockdown, Smac mimetic treatment, HIV-1 LTR reporter, latency reversal assay in JLat cells and primary CD4+ T cells from ART-suppressed patients\",\n      \"journal\": \"Cell host & microbe\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — targeted RNAi screen confirmed by knockdown and pharmacological depletion, primary patient cells, functional LTR readout, single lab\",\n      \"pmids\": [\"26355217\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"cIAP1 E3 ubiquitin ligase promotes K63-linked polyubiquitination of E2F1 on lysine residues 161/164, which stabilizes E2F1 and increases its transcriptional activity; this modification is required for E2F1 binding to CCNE, TP73 and APAF1 promoters and for E2F1 stabilization in response to DNA damage (etoposide) and during S phase.\",\n      \"method\": \"In vitro ubiquitination assay, K161R/K164R E2F1 mutagenesis, ChIP on CCNE/TP73/APAF1 promoters, cell cycle synchronization, DNA damage assay\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro ubiquitination, site-directed mutagenesis, ChIP, multiple conditions, single lab\",\n      \"pmids\": [\"28542143\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TLR-MyD88 signaling causes proteasomal degradation of cIAP1 and TRAF2 by inducing TNF and TNFR2 signaling; myeloid-specific cIAP1 loss in XIAP-deficient cells promotes TLR-induced RIPK3-caspase-8 activation and IL-1β production; TNFR2 deletion limits cIAP1-TRAF2 degradation and cell death.\",\n      \"method\": \"Myeloid-specific cIAP1 conditional knockout, TNFR2 deletion, TLR ligand stimulation, caspase-8 activation assay, IL-1β measurement, Western blot for cIAP1/TRAF2\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional knockout, genetic epistasis with TNFR2 and XIAP, multiple death pathway readouts\",\n      \"pmids\": [\"28723569\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"S-nitrosylation of cIAP1 on cysteines 571 and 574 by NO donor GTN inhibits its E3 ubiquitin ligase activity, reducing K63-linked ubiquitination of RIP1 and triggering assembly of a death complex, converting TNFα from a pro-survival to a pro-death signal.\",\n      \"method\": \"S-nitrosylation site identification (Cys571/574), E3 ligase activity assay after S-nitrosylation, RIP1 ubiquitination assay, death complex co-immunoprecipitation, cell death assay\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — site-specific PTM identification, E3 activity assay, mechanistic death complex formation, single lab\",\n      \"pmids\": [\"29431638\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"cIAP1 is recruited to the TNFR2 signaling complex and mediates K63-linked polyubiquitination; cIAP1 recruitment to TNFR2 is required for HOIP (LUBAC) recruitment and 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, ubiquitin linkage analysis, HOIP recruitment assay, NF-κB activation assay\",\n      \"journal\": \"Biochemical pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — receptor complex IP, pharmacological cIAP depletion, ubiquitin linkage analysis, NF-κB functional readout, single lab\",\n      \"pmids\": [\"29378181\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Combined loss of cIAP1 and cIAP2 in adult mice causes rapid intestinal and hepatic cell death involving caspase-8 and caspase-3 cleavage; deletion of Casp8 and Ripk3 together (but not Ripk3 alone) prevents cell death and prolongs survival, indicating the primary function is suppression of caspase-8-dependent death. Residual inflammation is reduced by NIK inhibition.\",\n      \"method\": \"Conditional cIap1/2 double knockout in adult mice, Casp8/Ripk3/Mlkl genetic deletion epistasis, cleaved caspase immunostaining, NIK inhibition\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo conditional double knockout, multiple genetic epistasis tests, specific cell death pathway ordered\",\n      \"pmids\": [\"31141691\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Structural and biophysical studies of cIAP1 BIR3 domain in ternary complexes with BTK and heterobifunctional degraders (PROTACs) reveal that increased ternary complex stability or rigidity does not necessarily correlate with increased degradation efficiency.\",\n      \"method\": \"Biochemical binding assays, biophysical measurements, crystal structure of ternary complex (BTK-degrader-cIAP1)\",\n      \"journal\": \"Nature chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure of ternary complex, multiple biophysical methods, structure-activity correlation\",\n      \"pmids\": [\"33199914\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"OTUB1 prevents K48-linked polyubiquitination and degradation of c-IAP1 in hepatocytes; OTUB1 deficiency leads to c-IAP1 degradation, reduced K63-linked polyubiquitination of RIPK1, increased RIPK1 phosphorylation and necrosome formation, and lethal necroptosis in bacterial and sterile liver inflammation.\",\n      \"method\": \"Hepatocyte-specific OTUB1 knockout mice, OTUB1-deficient HepG2 cells, RIPK1 inhibitor necrostatin-1s, MLKL knockout epistasis, K48/K63 ubiquitin linkage assays, Listeria/DGal-TNF challenge\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — tissue-specific knockout, genetic epistasis in vivo, ubiquitin linkage assays, RIPK1 phosphorylation, replicated in human cells\",\n      \"pmids\": [\"33712742\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"cIAP1-targeting degraders (SNIPERs/PROTACs) require the K63-specific E2 enzyme UBE2N for efficient target protein degradation; UBE2N-catalyzed K63-linked chains facilitate assembly of branched K48/K63 and K11/K48 ubiquitin chains on substrates, recruiting p97/VCP, UCH37, and the proteasome for degradation.\",\n      \"method\": \"UBE2N siRNA/knockout, in vitro ubiquitination with E2 panel, mass spectrometry of ubiquitin chain linkages, p97/UCH37/proteasome interaction assays\",\n      \"journal\": \"Nature chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution, MS-based ubiquitin linkage mapping, genetic validation, mechanistic complex assembly demonstrated\",\n      \"pmids\": [\"36316570\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"HSP90β acts as a chaperone for c-IAP1; HSP90β inhibition or siRNA depletion induces c-IAP1 auto-ubiquitination and proteasomal degradation, and prevents cell differentiation; specific depletion of c-IAP1 alone is sufficient to inhibit cell differentiation.\",\n      \"method\": \"HSP90 inhibitor treatment, HSP90α/β-specific siRNA, co-immunoprecipitation, auto-ubiquitination assay, proteasome inhibitor rescue, differentiation assay\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — specific HSP90β isoform identified by siRNA, co-IP, ubiquitination assay, functional differentiation readout, single lab\",\n      \"pmids\": [\"18239673\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"cIAP1 conjugates predominantly K63-linked ubiquitin chains to MEKK2 and MEKK3, which impedes their interaction with MEK5 in a trimeric complex, leading to ERK5 inactivation; loss of cIAP1 causes hyperactivation of ERK5 and promotes skeletal myoblast differentiation.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ubiquitination assay, K63-ubiquitin linkage analysis, MEKK2/3-MEK5 interaction assay, ERK5 activation assay, myoblast differentiation model\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro ubiquitination reconstitution, K63 linkage identified, trimeric complex disruption mechanism, functional differentiation readout\",\n      \"pmids\": [\"24975362\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"F-box protein Fbxo7 interacts with cIAP1 in human cells and promotes cIAP1 ubiquitination; they co-localize in cytoplasm, nucleus, and Golgi-like structures when co-expressed.\",\n      \"method\": \"Yeast two-hybrid screen, co-immunoprecipitation, co-localization by microscopy, ubiquitination assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single co-IP and ubiquitination assay, yeast two-hybrid identified interaction, single lab\",\n      \"pmids\": [\"16510124\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"cIAP1 promotes SNIPER-4-mediated ubiquitination and proteasomal degradation of CRABP-II when crosslinked to cIAP1 by a hybrid small molecule; this establishes cIAP1 as a recruitable E3 ligase for targeted protein degradation of neo-substrates.\",\n      \"method\": \"SNIPER hybrid molecule, Western blot of CRABP-II degradation, proteasome inhibitor rescue, ubiquitination assay\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — chemical biology reconstitution of E3-substrate crosslinking, mechanistic proteasome rescue, single lab\",\n      \"pmids\": [\"21414315\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"cIAP1 is a component of the endogenous LIGHT·LTβR complex, associating with TRAF2, TRAF3, and Smac at the lymphotoxin-beta receptor; its presence with Smac reveals a mechanism for LIGHT/LTβR-induced apoptosis.\",\n      \"method\": \"Affinity purification of endogenous LIGHT·LTβR complex, mass spectrometry identification, co-immunoprecipitation confirmation in U937 and HEK293 cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — endogenous complex purification with MS, co-IP confirmation in two cell lines, single lab\",\n      \"pmids\": [\"12571250\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"BIRC2/cIAP1 is a RING domain E3 ubiquitin ligase that promotes cell survival by constitutively maintaining K11- and K63-linked polyubiquitination of RIP1 (and other RIP kinases) within TNF receptor signaling complexes, thereby enabling TAK1 recruitment and NF-κB activation while preventing RIP1 from forming pro-death complexes with caspase-8; it also targets NIK for proteasomal degradation to suppress non-canonical NF-κB, ubiquitinates TRAF2 and ASK1 to terminate TNF signaling, ubiquitinates caspase-3/7 (promoting their degradation without direct inhibition), ubiquitinates RIP2 for NOD signaling, and ubiquitinates E2F1 (K63-linked, stabilizing) and Mad1 (K48-linked, degradative) to regulate transcription; its E3 activity is autoinhibited by CARD domain-mediated prevention of RING dimerization, stabilized by HSP90β and deubiquitinases OTUB1 and USP19, and activated by Smac/DIABLO or IAP antagonists which trigger auto-ubiquitination and proteasomal degradation of cIAP1 itself via branched K63/K48-linked ubiquitin chains assembled with UBE2N.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"BIRC2/cIAP1 is a RING-domain E3 ubiquitin ligase that functions as a central post-translational rheostat of TNF-receptor-superfamily signaling, determining whether cells survive or die [#18, #48]. Within the TNF-R1 signaling complex, to which it is recruited through TRADD-mediated association with TRAF2 [#0], cIAP1 (redundantly with cIAP2) constitutively conjugates K63-, K11-, and linear ubiquitin chains onto RIP1, generating the platform that recruits TAK1/IKK to drive canonical NF-\\u03baB activation while preventing RIP1 from nucleating pro-death complexes with caspase-8 or RIP3-dependent necrosomes [#18, #19, #30, #31]. This ligase activity, executed with UbcH5-family E2 enzymes, extends across the RIP-kinase family (RIP1\\u2013RIP4) and to RIP2 in NOD1/NOD2 innate immune signaling and caspase-1 in inflammasome activation [#26, #29, #35, #36]. cIAP1 also sets the threshold for non-canonical NF-\\u03baB by targeting NIK for proteasomal degradation within a TRAF2\\u2013TRAF3 complex, and terminates TNF-R2 signaling by ubiquitinating TRAF2 and ASK1 [#21, #4, #15]. In vivo, combined cIAP1/2 loss causes caspase-8-dependent intestinal and hepatic death, establishing suppression of caspase-8 apoptosis as the core survival function [#48]. Although its BIR domains bind caspases-3 and -7, cIAP1 promotes their proteasomal degradation rather than directly inhibiting their activity [#10, #26]. The enzyme is autoinhibited by a CARD domain that blocks RING dimerization and E2 activation [#34], stabilized by the chaperone HSP90\\u03b2 and the deubiquitinases OTUB1 and USP19 [#52, #42, #50, #38], and selectively eliminated by Smac/DIABLO or synthetic IAP antagonists that bind its BIR3 domain and trigger RING-dependent auto-ubiquitination and proteasomal degradation [#14, #23, #33]. Beyond cytoplasmic signaling, nuclear cIAP1 binds E2F1 and stabilizes it through K63-linked ubiquitination to promote cell-cycle gene transcription [#39, #44].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Established that cIAP1 is physically positioned within TNF receptor signaling, identifying how it gains access to the death-and-survival machinery via the TRADD-TRAF2 adaptor module.\",\n      \"evidence\": \"Co-immunoprecipitation and TNF-dependent recruitment assays in cells\",\n      \"pmids\": [\"8943045\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define a catalytic activity or downstream substrate at the receptor complex\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"First assigned cIAP1 an anti-apoptotic biochemical activity by showing BIR-domain binding to caspases-3/-7, initially interpreted as direct enzymatic inhibition.\",\n      \"evidence\": \"In vitro binding and caspase activity assays with recombinant proteins, cell-free cytochrome-c system\",\n      \"pmids\": [\"9384571\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The inferred direct inhibition was later overturned for cIAP BIRs\", \"Did not address the RING/E3 contribution\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Reframed cIAP1 as an E3 ubiquitin ligase rather than a stoichiometric inhibitor, by demonstrating it ubiquitinates TRAF2 for proteasomal degradation downstream of TNF-R2.\",\n      \"evidence\": \"In vitro ubiquitination with purified components, E3-defective mutant, proteasome inhibition in primary cells\",\n      \"pmids\": [\"11907583\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the full substrate repertoire\", \"E2 partner and chain linkage unresolved at this stage\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Resolved the core survival mechanism: cIAP1/2 maintain constitutive ubiquitination of RIP1 that recruits TAK1 for NF-\\u03baB while blocking caspase-8 association, defining a molecular survival/death switch.\",\n      \"evidence\": \"IAP antagonist treatment, in vitro reconstitution with purified cIAP1+UbcH5a, RIP1-TAK1/RIP1-caspase-8 co-IPs, genetic and siRNA ablation\",\n      \"pmids\": [\"18570872\", \"18621737\", \"18697935\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve which ubiquitin linkages dominate in vivo\", \"Redundancy with cIAP2 left the unique cIAP1 contribution unclear\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Defined cIAP1 as the threshold-setter for non-canonical NF-\\u03baB by showing it degrades NIK within a TRAF2/TRAF3 complex, linking IAP loss to constitutive RelB activation.\",\n      \"evidence\": \"Complex reconstitution, genetic and pharmacological cIAP inhibition, NIK degradation and primary B cell assays\",\n      \"pmids\": [\"18997794\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish chain linkage on NIK\", \"Stoichiometry within the TRAF2/TRAF3 complex undefined\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identified the mechanism by which IAP antagonists kill: Smac/DIABLO binds BIR domains to stimulate RING-dependent cIAP1 auto-ubiquitination and selective degradation.\",\n      \"evidence\": \"In vitro auto-ubiquitination, Smac N-terminal peptide sufficiency, E2 identification, proteasome inhibition\",\n      \"pmids\": [\"14960576\", \"18230607\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not fully define the conformational activation step\", \"Selectivity over XIAP/cIAP2 mechanism incomplete\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Overturned the direct-caspase-inhibitor model by showing cIAP1 binds and ubiquitinates caspases-3/-7 for degradation without inhibiting their catalytic activity.\",\n      \"evidence\": \"In vitro binding, fluorogenic substrate assays, ubiquitination with UbcH5/UbcH8, chimeric caspase-3, domain mapping\",\n      \"pmids\": [\"19258326\", \"16339151\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo contribution of caspase degradation versus RIP1 control not separated\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Extended cIAP1 function into innate immunity by identifying RIP2 ubiquitination as required for NOD1/NOD2 signaling.\",\n      \"evidence\": \"Birc2/Birc3 knockout macrophages, RIP2 ubiquitination assay, in vivo NOD agonist challenge and colitis model\",\n      \"pmids\": [\"19464198\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contribution of cIAP1 versus cIAP2 to RIP2 modification not isolated\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Mapped the ubiquitin chain architecture on RIP1, showing cIAP1/UbcH5 assemble K11-linked chains read by NEMO, diversifying the chain code at the receptor complex.\",\n      \"evidence\": \"Yeast two-hybrid E2 screen, in vitro reconstitution, mass spectrometry of linkages, TNFR1 complex IP\",\n      \"pmids\": [\"21113135\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative in vivo abundance of K11 versus K63 chains not quantified\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Explained how cIAP1 E3 activity is restrained, with a crystal structure showing the CARD domain autoinhibits by preventing RING dimerization and E2 activation.\",\n      \"evidence\": \"CARD crystal structure, RING dimerization and E2 activation assays, mutagenesis, zebrafish vascular readout\",\n      \"pmids\": [\"21549626\", \"20356846\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological trigger that relieves CARD autoinhibition not defined\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Identified a nuclear, transcription-regulatory role: cIAP1 binds the E2F1 DNA-binding domain at cell-cycle gene promoters to enhance proliferation.\",\n      \"evidence\": \"Co-IP, E2F1 reporter, ChIP on CCNE/CCNA, siRNA knockdown proliferation assay\",\n      \"pmids\": [\"21653699\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Did not initially establish a ubiquitin-based mechanism for E2F1 regulation\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Established a stabilizing regulatory layer, showing OTUB1 removes K48 chains from cIAP1 within the TWEAK receptor complex to protect it from degradation.\",\n      \"evidence\": \"Co-IP, linkage-specific in vitro DUB assay, OTUB1 knockdown with pathway readouts, zebrafish validation\",\n      \"pmids\": [\"23524849\", \"33712742\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Balance between OTUB1, USP19 and HSP90\\u03b2 in setting steady-state cIAP1 levels not integrated\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Resolved the molecular basis of cIAP1-driven transcription by showing K63-linked ubiquitination of E2F1 stabilizes it and licenses promoter binding during S phase and DNA damage.\",\n      \"evidence\": \"In vitro ubiquitination, K161R/K164R mutagenesis, ChIP on CCNE/TP73/APAF1, cell-cycle and DNA damage assays\",\n      \"pmids\": [\"28542143\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"How nuclear versus cytoplasmic cIAP1 pools are partitioned to this function unclear\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defined the dominant in vivo physiological function by showing combined cIAP1/2 loss kills intestinal and hepatic tissue via caspase-8, rescued by Casp8/Ripk3 co-deletion.\",\n      \"evidence\": \"Conditional cIap1/2 double knockout mice, Casp8/Ripk3/Mlkl epistasis, cleaved caspase staining, NIK inhibition\",\n      \"pmids\": [\"31141691\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not separate cIAP1-specific from cIAP2-specific roles in vivo\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Detailed the degradation chemistry exploited by cIAP1-recruiting degraders, showing UBE2N-seeded K63 chains nucleate branched K48/K63 chains that recruit p97 and the proteasome.\",\n      \"evidence\": \"UBE2N knockout, in vitro E2 panel ubiquitination, MS linkage mapping, p97/UCH37/proteasome interaction assays\",\n      \"pmids\": [\"36316570\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this branched-chain logic governs cIAP1's endogenous substrates not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the many cIAP1 activities\\u2014receptor-complex RIP ubiquitination, NIK degradation, caspase turnover, and nuclear E2F1/Mad1 regulation\\u2014are coordinated across subcellular compartments and which are physiologically dominant in specific tissues remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model linking compartmental localization to substrate selection\", \"Tissue-specific substrate hierarchy undefined\", \"Most non-RIP1 substrates rest on single-lab evidence\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [4, 11, 15, 18, 19, 29, 35, 53]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [4, 18, 26, 30, 35, 53]},\n      {\"term_id\": \"GO:0061630\", \"supporting_discovery_ids\": [4, 19, 30]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [10, 26, 34]},\n      {\"term_id\": \"GO:0031386\", \"supporting_discovery_ids\": [18, 30, 35]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [8, 12, 18, 25]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [8, 9, 12, 39]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [9]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [13]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 47]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 18, 19, 20, 21, 47]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [18, 31, 48, 31]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [29, 36, 45]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [4, 11, 26, 51]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [39, 44, 12]}\n    ],\n    \"complexes\": [\n      \"TNF-R1 signaling complex\",\n      \"TNF-R2 / TRAF2 complex\",\n      \"TRAF2-TRAF3-NIK non-canonical NF-\\u03baB regulatory complex\",\n      \"LIGHT\\u00b7LT\\u03b2R complex\"\n    ],\n    \"partners\": [\n      \"TRAF2\",\n      \"RIPK1\",\n      \"TRAF3\",\n      \"NIK\",\n      \"OTUB1\",\n      \"USP19\",\n      \"E2F1\",\n      \"UBE2N\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}