{"gene":"BID","run_date":"2026-06-09T22:02:44","timeline":{"discoveries":[{"year":1998,"finding":"Caspase-8 cleaves BID, and the C-terminal fragment (tBid) translocates to mitochondria where it triggers cytochrome c release; immunodepletion of BID eliminated cytochrome c releasing activity; a BH3 domain mutation diminished activity; Bcl2 antagonized tBid activity","method":"Protein purification, peptide mass fingerprinting, in vitro caspase cleavage assay, immunodepletion, site-directed mutagenesis, cell-free cytochrome c release assay","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstitution in vitro with mutagenesis, immunodepletion controls, replicated across multiple subsequent labs","pmids":["9727491"],"is_preprint":false},{"year":1996,"finding":"BID is a BH3-only protein that heterodimerizes with both BAX and BCL-2 via its BH3 domain; an intact BH3 domain is required to bind the BH1 domain of BCL-2 or BAX; BH3 mutagenesis dissociated binding from death-promoting activity; BID expression induces ICE-like proteases and apoptosis","method":"Interactive (yeast two-hybrid) cloning, site-directed mutagenesis, co-immunoprecipitation, cell death assays","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — mutagenesis dissecting binding vs. function, replicated extensively","pmids":["8918887"],"is_preprint":false},{"year":2001,"finding":"BID is phosphorylated by casein kinase I (CKI) and casein kinase II (CKII); phosphorylated BID is insensitive to caspase-8 cleavage in vitro; inhibition of CKI/CKII accelerated Fas-mediated apoptosis and BID cleavage; a non-phosphorylatable BID mutant was more toxic than wild-type","method":"In vitro kinase assay, site-directed mutagenesis, pharmacological kinase inhibition, Fas-mediated apoptosis assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay plus mutagenesis plus cellular pharmacology, single lab but multiple orthogonal methods","pmids":["11583622"],"is_preprint":false},{"year":2005,"finding":"BID is phosphorylated by ATM kinase following DNA double-strand breaks on two ATM consensus sites; BID-deficient cells fail to accumulate in S phase after etoposide treatment; a non-phosphorylatable BID mutant did not restore S phase arrest and increased sensitivity to etoposide-induced apoptosis; BID partially localizes to the nucleus in healthy cells","method":"Phosphorylation mapping, ATM kinase assay, Bid-/- cell reconstitution with wild-type vs. phospho-mutant BID, cell cycle analysis, nuclear fractionation","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — two independent Cell papers (PMIDs 16122425 and 16122426) using phospho-mapping, mutagenesis, and KO reconstitution","pmids":["16122425","16122426"],"is_preprint":false},{"year":2005,"finding":"PACS-2 translocates BID to mitochondria in response to apoptotic inducers, initiating tBid formation, cytochrome c release, and caspase-3 activation; depletion of PACS-2 prevents BID-mediated apoptosis","method":"siRNA knockdown, subcellular fractionation, Western blot for tBid/cytochrome c/caspase-3, live-cell imaging of mitochondria-ER contacts","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal functional assays with siRNA and reconstitution, single lab with multiple readouts","pmids":["15692567"],"is_preprint":false},{"year":2010,"finding":"BID, BIM, and PUMA are essential activators of BAX and BAK; triple knockout of Bid/Bim/Puma phenocopies Bax/Bak double knockout (persistent interdigital webs, imperforate vaginas) and prevents BAX/BAK homo-oligomerization and cytochrome c release in neurons and T lymphocytes","method":"Triple-knockout mouse genetics, epistasis analysis, cytochrome c release assay, BAX/BAK oligomerization assay","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic epistasis with multiple cell types and multiple orthogonal readouts","pmids":["21127253"],"is_preprint":false},{"year":2013,"finding":"NMR solution structure of the human BID BH3–BAK complex identified the activation site at the canonical BH3-binding groove of BAK; BAK BH1 groove mutations prevented MOMP but not BID binding; BAK BH3 mutations allowed BID binding but blocked oligomerization; BID follows a 'hit-and-run' mechanism dissociating from the trigger site to allow BAK oligomerization; NOXA and BAD are predicted to clash with the trigger site and are not activators of BAK","method":"NMR structure determination, site-directed mutagenesis of BAK BH1 and BH3 domains, MOMP assay, binding assay","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — NMR structure plus mutagenesis plus functional MOMP validation in single study","pmids":["23604079"],"is_preprint":false},{"year":2013,"finding":"BID preferentially activates BAK while BIM preferentially activates BAX; cells lacking BAK are relatively resistant to agents requiring BID activation (topoisomerase inhibitors, TRAIL)","method":"BH3 profiling, BAK/BAX knockout cell lines, apoptosis assays, clinical correlation","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple KO cell lines with functional apoptosis assays and biochemical BH3 profiling","pmids":["24074954"],"is_preprint":false},{"year":2002,"finding":"Calpain cleaves BID between Gly70 and Arg71 in cisplatin-treated cells, yielding a 14-kDa fragment; calpain-cleaved BID induces cytochrome c release from isolated mitochondria; calpain inhibitors but not caspase or cathepsin L inhibitors prevented BID cleavage","method":"In vitro calpain cleavage of recombinant BID, cleavage site mapping, isolated mitochondria cytochrome c release assay, pharmacological inhibitor panel","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with recombinant proteins plus cleavage site mapping, single lab","pmids":["11940658"],"is_preprint":false},{"year":2000,"finding":"Granzyme B cleaves cytosolic BID to produce truncated BID, which is the sole cytosolic protein responsible for GrB-induced cytochrome c release in a cell-free system; GrB also acts directly on mitochondria to open the permeability transition pore independently of cytosolic proteins","method":"Cell-free in vitro system, immunodepletion, recombinant protein assays, mitochondrial membrane potential measurement","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with immunodepletion establishing BID as sole cytosolic mediator","pmids":["11114298"],"is_preprint":false},{"year":2004,"finding":"Bid-deficient cells from multiple tissues are resistant to granzyme B-induced cell death; cytochrome c remains in mitochondria of Bid-deficient cells treated with granzyme B; surviving Bid-deficient cells proliferate normally","method":"Bid-/- mouse-derived primary cells, granzyme B treatment, cytochrome c localization by fractionation, cell viability and proliferation assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO cells from multiple tissues with defined molecular readout (cytochrome c retention)","pmids":["15574417"],"is_preprint":false},{"year":2008,"finding":"Lysosomal cysteine cathepsins (B, L, S, K, H) cleave BID in vitro; cathepsin-mediated BID cleavage triggers the mitochondrial apoptosis pathway; E-64d (cysteine protease inhibitor) prevented BID cleavage and apoptosis","method":"In vitro protease cleavage assays with purified cathepsins and recombinant BID, pharmacological inhibitor studies, cell death assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with multiple purified enzymes plus cellular inhibitor validation","pmids":["18469004"],"is_preprint":false},{"year":2002,"finding":"BID is a p53 transcriptional target; the human and mouse BID genomic loci contain p53-binding DNA response elements that bind p53 and mediate p53-dependent transactivation; BID-null MEFs are more resistant to adriamycin and 5-fluorouracil","method":"Reporter gene assay with p53 response elements, chromatin immunoprecipitation-like binding assay, BID-null MEF apoptosis assays","journal":"Nature cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — p53 response element binding and reporter assay plus KO cellular phenotype, single lab","pmids":["12402042"],"is_preprint":false},{"year":2011,"finding":"BID interacts with NOD1, NOD2, and the IKK complex, impacting NF-κB and ERK signaling; BID-deficient colonocytes and macrophages are markedly defective in cytokine production in response to NOD activation; Bid-/- mice are unresponsive to NOD agonists in experimental colitis","method":"Genome-wide RNAi screen, co-immunoprecipitation of BID with NOD1/NOD2/IKK, Bid-/- macrophage cytokine assays, in vivo colitis model","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP plus KO in vivo and in vitro with multiple readouts, single lab but genome-wide screen validation","pmids":["21552281"],"is_preprint":false},{"year":2000,"finding":"Recombinant BID and Bax cause complete cytochrome c loss from isolated mitochondria in vitro while preserving inner membrane ultrastructure and protein import function; BID and BAX act only on the outer mitochondrial membrane; inner membrane lesions during apoptosis are secondary caspase-dependent events","method":"Isolated mitochondria in vitro assay, electron microscopy of ultrastructure, mitochondrial protein import assay, caspase inhibition experiments","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with recombinant proteins plus structural (EM) and functional validation, replicated","pmids":["10973993"],"is_preprint":false},{"year":2000,"finding":"BID-induced mitochondrial membrane permeabilization can be inhibited by PTPC inhibitors (cyclosporin A, bongkrekic acid); full-length BID preferentially permeabilizes membranes containing both ANT and BAX; tBid acts on membranes containing ANT alone, suggesting functional interaction with ANT (adenine nucleotide translocase)","method":"Intact cell microinjection, isolated mitochondria assay, proteoliposome reconstitution, planar bilayer electrophysiology","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with proteoliposomes and electrophysiology, single lab","pmids":["11175349"],"is_preprint":false},{"year":2002,"finding":"tBid requires interaction with BAX and the BAX C-terminal transmembrane domain to induce ionic channel formation in liposomes; cut BID alone cannot induce BAX oligomerization in liposomes; an additional mitochondrial factor is required for BAX oligomerization","method":"Liposome reconstitution, planar bilayer electrophysiology, gel filtration, recombinant protein interaction assays","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution in defined lipid system with electrophysiology, single lab","pmids":["11964155"],"is_preprint":false},{"year":2016,"finding":"Cleavage by caspase-8 (not effector caspases) is the primary event activating BID during TRAIL-induced apoptosis; additionally, association of tBid with the outer mitochondrial membrane via helices α6 and α7 is required for full apoptotic activity; a tBid mutant lacking these helices has diminished activity despite an intact BH3 domain","method":"CRISPR/Cas9 BID-KO and BID/BAX/BAK TKO cells, reconstitution with wild-type and caspase-resistant/BH3-defective/helix-deletion mutants, TRAIL apoptosis assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO with systematic reconstitution using multiple point mutants, clean causal assignment","pmids":["27053107"],"is_preprint":false},{"year":2011,"finding":"BID controls BAX activation in AIF-mediated caspase-independent necroptosis; BID is processed by calpains into tBid (non-cleavable BID-G70A or BID-Δ68-71 abolishes BAX activation); tBid localizes to mitochondria to facilitate BAX activation and programmed necrotic death","method":"Bid-/- and calpain-resistant BID mutant MEFs, BAX activation assay, necroptosis (MNNG) model, reintroduction of wild-type vs. mutant BID","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO cells with rescue by wild-type but not calpain-cleavage-resistant mutants, multiple readouts","pmids":["21738214"],"is_preprint":false},{"year":2012,"finding":"ATM-mediated BID phosphorylation maintains quiescence of haematopoietic stem cells (HSCs); loss of BID phosphorylation leads to escape from quiescence, HSC pool exhaustion, and reduced repopulating potential; BID phosphorylation protects HSCs from irradiation by regulating oxidative stress; loss of phosphorylation or ATM knockout increases mitochondrial BID correlating with increased mitochondrial ROS","method":"Knock-in phospho-mutant BID mice, HSC quiescence assays (BrdU), competitive reconstitution assays, ROS measurement, irradiation survival","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo knock-in phospho-mutant mice with multiple orthogonal functional readouts","pmids":["22446738"],"is_preprint":false},{"year":2011,"finding":"BID associates with RPA70 N-terminal basic cleft; BID stimulates association of RPA with ATR-ATRIP complex; disruption of BID-RPA interaction impairs ATR-ATRIP chromatin association, CHK1 activation, and recovery of DNA replication following hydroxyurea","method":"Co-immunoprecipitation, NMR mapping of BID-RPA70 interaction, BID mutants disrupting RPA binding, chromatin fractionation, CHK1 phosphorylation assay, DNA replication recovery assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — NMR-guided interaction mapping plus functional mutagenesis with multiple ATR pathway readouts","pmids":["21859891"],"is_preprint":false},{"year":2010,"finding":"BID functions at the level of the ATR damage sensor complex following replicative stress; BID is found in nuclear foci with RPA and associates with ATR/ATRIP/RPA complex; BID-deficient cells show reduced ATR/ATRIP chromatin loading, reduced CHK1 activation, and impaired recovery of DNA synthesis","method":"Nuclear foci colocalization, co-immunoprecipitation of BID with ATR/ATRIP/RPA, Bid-/- cell chromatin fractionation, CHK1 phosphorylation assay","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP plus KO phenotype with multiple ATR-pathway readouts","pmids":["21113148"],"is_preprint":false},{"year":2005,"finding":"Humanin (HN) peptide binds purified BID and tBid in vitro; HN blocks tBid-induced cytochrome c and SMAC release from isolated mitochondria; HN inhibits tBid-induced BAX and BAK oligomerization in mitochondrial membranes; inactive HN mutants fail to bind BID or block these activities","method":"In vitro binding assay with purified proteins, isolated mitochondria cytochrome c/SMAC release assay, chemical cross-linking/gel filtration for oligomerization, gene transfection","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with mutagenesis of HN, single lab","pmids":["15661737"],"is_preprint":false},{"year":2004,"finding":"Membrane-insertion analysis shows that the α6 helix of tBid inserts into membranes only as part of the α6-α7 hairpin (not individually), indicating synergistic hairpin insertion into mitochondrial membrane is required for function","method":"Glycosylation mapping of chimeric single-helix segments in a model membrane insertion system","journal":"Biochemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — direct membrane insertion assay with glycosylation reporter, single lab","pmids":["15323553"],"is_preprint":false},{"year":2021,"finding":"Stepwise activation of cBid at real mitochondrial membranes: upon mitochondrial binding, cBid reorganizes to expose the BH3 domain while maintaining overall structural integrity; membrane-bound cBid associates with MTCH2 and remains primed until interacting with BAX, which triggers cBid fragmentation, large conformational changes, and BAX-mediated MOMP","method":"Spin-label ESR spectroscopy, site-directed PEGylation at real mitochondrial membranes, co-immunoprecipitation with MTCH2","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 1 / Moderate — ESR and site-directed PEGylation at authentic mitochondrial membranes, mechanistically detailed, single lab","pmids":["33462413"],"is_preprint":false},{"year":2016,"finding":"In live cells, BID adopts an extended conformation at the mitochondrial outer membrane critical for its association with the membrane and for intermolecular contacts within BID oligomers; direct intermolecular FRET contacts between BID and BAX were observed in live cells, confirming BID as a component of the BID-BAX permeabilization network","method":"Confocal FRET microscopy in live cells, FRET efficiency measurement, conformational analysis","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — live-cell FRET with conformational analysis, single lab","pmids":["27763642"],"is_preprint":false},{"year":2003,"finding":"Three novel BID isoforms are generated by alternative splicing: BidS (lacks BH3 domain, inhibits tBid pro-apoptotic effects and Fas-mediated apoptosis), BidEL (induces apoptosis), and BidES (induces apoptosis but partially inhibits tBid); expression of these isoforms is regulated during granulocyte maturation and they differ in subcellular localization","method":"RT-PCR/gene structure analysis, endogenous protein detection, cell death assays, subcellular localization","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — endogenous isoform identification plus functional apoptosis assays, single lab","pmids":["14583606"],"is_preprint":false},{"year":2017,"finding":"BID translocates to mitochondria during erastin-induced ferroptosis; CRISPR/Cas9 Bid knockout preserves mitochondrial integrity and function, and mediates neuroprotection against ferroptosis and oxytosis; BID inhibitor BI-6c9 inhibited ferroptosis; ferroptosis inhibitors (ferrostatin-1, liproxstatin-1) prevented BID-dependent mitochondrial dysfunction","method":"CRISPR/Cas9 BID knockout, mitochondrial membrane potential measurement, ATP assay, mitochondrial fragmentation imaging, pharmacological inhibitors","journal":"Redox biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with multiple mitochondrial readouts, single lab","pmids":["28384611"],"is_preprint":false},{"year":2020,"finding":"Caspase-1 directly cleaves BID to generate tBid; in GSDMD-deficient cells, tBid-induced MOMP drives SMAC release to relieve IAP inhibition of caspase-3, enabling rapid secondary necrosis; caspase-1-driven activation of caspase-8/-9 and Bid cleavage act synergistically","method":"GSDMD-/- and GSDMD/BID double-KO cells, caspase activation assays, SMAC release measurement, caspase-1 cleavage assay","journal":"Life science alliance","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — double-KO genetic dissection with multiple caspase readouts, single lab","pmids":["32345661"],"is_preprint":false},{"year":2008,"finding":"BID translocation to mitochondria precedes AIF release; BID-mediated mitochondrial AIF release causes rapid nuclear translocation and caspase-independent neuronal death; BID siRNA and BID inhibitor prevented AIF nuclear translocation and cell death; tBid-induced death was inhibited by AIF siRNA, establishing AIF as the main downstream effector of BID in glutamate-induced neuronal death","method":"Fluorescence video microscopy, BID siRNA, small molecule BID inhibitor, AIF siRNA, caspase-3 inhibitor comparison","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — live-cell imaging plus siRNA epistasis, single lab","pmids":["18535584"],"is_preprint":false},{"year":2002,"finding":"BID sequence has significant similarity to plant lipid transfer proteins; isolated BID shows lipid transfer activity higher than plant LTPs; lysolipids alter BID association with mitochondria and stimulate BID-induced cytochrome c release; BID enhances incorporation of fluorescent lysolipids into mitochondria","method":"Sequence analysis, in vitro lipid transfer assay, isolated mitochondria cytochrome c release assay with exogenous lipids, fluorescent lipid incorporation assay","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro biochemical assays, single lab, no mutagenesis validation of lipid-transfer relevance","pmids":["11997142"],"is_preprint":false},{"year":2010,"finding":"Activity of protein kinase CK2 temporally uncouples caspase-8 activation from BID cleavage during TRAIL-induced apoptosis; CK2 inhibition (DRB) or dominant-negative CK2α largely eliminated the lag time between caspase-8 activation and BID cleavage in individual cells","method":"Single-cell FRET probes for caspase-8 (IETD) and full-length BID, CK2 inhibitor and dominant-negative expression, time-lapse microscopy","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — FRET-based single-cell kinetics with pharmacological and genetic CK2 manipulation, single lab","pmids":["20356928"],"is_preprint":false},{"year":2006,"finding":"PLZF transcription factor binds a high-affinity site upstream of the BID transcriptional start site (EMSA) and represses BID expression; PLZF-expressing Jurkat cells show retarded mitochondrial membrane potential loss and resistance to apoptosis","method":"Differential expression screen, EMSA for PLZF binding to BID promoter, inducible PLZF expression, annexin V/TUNEL apoptosis assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — EMSA plus functional transcriptional repression assay with inducible system, single lab","pmids":["14769944"],"is_preprint":false},{"year":2013,"finding":"Caspase-cleaved arrestin-2-(1-380) directly binds tBID and doubles tBid-induced cytochrome c release from isolated mitochondria; arrestin-2 pro-apoptotic action requires BID: it does not facilitate apoptosis in BID KO cells, and rescue of BID expression restores activity","method":"Co-immunoprecipitation of arrestin-2 fragment with tBID, isolated mitochondria cytochrome c release assay, BID-/- cells with reconstitution, caspase-3 activity assay","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding plus KO epistasis plus reconstitution, single lab","pmids":["24141717"],"is_preprint":false},{"year":2000,"finding":"In some cell types, tBID induces BAX insertion into mitochondria in vivo and in vitro; tBID relieves inhibition of the BAX transmembrane signal-anchor by the N-terminal domain; however, Bid-null MEFs support BAX insertion into mitochondria in response to TNFα or E1A without BID, demonstrating a parallel BID-independent pathway for BAX membrane insertion in which cytochrome c release is uncoupled from BAX insertion","method":"tBid reconstitution in vitro, Bid-null MEF apoptosis and BAX insertion assays, confocal BAX localization","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro reconstitution plus KO genetics showing both BID-dependent and independent routes","pmids":["11139284"],"is_preprint":false},{"year":2006,"finding":"Human granzyme B efficiently cleaves human or mouse BID, but mouse granzyme B is highly resistant to cleaving BID, indicating the BID pathway is not a major primary mediator of mouse granzyme B cytotoxicity; substrate specificity differences mapped to tetrapeptide recognition","method":"Substrate specificity profiling, in vitro cleavage assay with human vs. mouse granzyme B and BID substrates","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro biochemical specificity assay comparing species variants, single lab","pmids":["17179148"],"is_preprint":false},{"year":2004,"finding":"BID cleavage at Asp60 and/or Asp75 (caspase-8/granzyme B target residues) is required for mitochondrial permeabilization and apoptosis induced by etoposide and gamma-radiation in p53-mutant T leukemic cells; this activation is independent of death receptor or mitochondrial inducer caspase activation; caspase-2 knockdown did not prevent BID activation by DNA damage","method":"Dominant-negative caspase constructs, CrmA expression, siRNA against caspase-2, BID cleavage site mapping, mitochondrial permeabilization assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple genetic/pharmacological tools excluding known upstream activators, single lab","pmids":["15117953"],"is_preprint":false}],"current_model":"BID is a BH3-only BCL-2 family protein that functions as a molecular bridge between the death receptor extrinsic pathway and the mitochondrial intrinsic pathway: caspase-8 (and alternatively granzyme B, calpains, or cathepsins) cleaves full-length BID, and the resulting tBid translocates to the outer mitochondrial membrane via its α6-α7 hydrophobic hairpin helices where—in a stepwise process involving MTCH2 association followed by a 'hit-and-run' interaction at the canonical BH3-binding groove of BAK (or BAX)—it promotes BAK/BAX homo-oligomerization and MOMP, releasing cytochrome c, SMAC, and AIF to activate downstream caspase-dependent and caspase-independent death; phosphorylation of BID by casein kinases I/II protects it from caspase-8 cleavage, while ATM-mediated phosphorylation recruits nuclear BID to the ATR/ATRIP/RPA sensor complex to enforce S-phase checkpoint arrest and maintain haematopoietic stem cell quiescence, establishing BID as a dual-function integrator of apoptotic and DNA-damage-response signaling."},"narrative":{"mechanistic_narrative":"BID is a BH3-only BCL-2 family protein that functions as the principal molecular bridge coupling proteolytic death signals to mitochondrial outer-membrane permeabilization (MOMP) and apoptosis [PMID:9727491, PMID:8918887]. Cytosolic full-length BID is cleaved by caspase-8 to yield a C-terminal fragment (tBid) that translocates to mitochondria and triggers cytochrome c release; an intact BH3 domain and Bcl-2 antagonism define this as a canonical apoptotic switch [PMID:9727491, PMID:8918887]. Diverse proteases converge on BID to generate active tBid—granzyme B during cytotoxic-lymphocyte killing [PMID:11114298, PMID:15574417], calpains during cisplatin treatment and AIF-mediated necroptosis [PMID:11940658, PMID:21738214], lysosomal cathepsins [PMID:18469004], and caspase-1 during inflammatory secondary necrosis [PMID:32345661]—establishing BID as a shared effector node for extrinsic, lysosomal, and inflammatory death routes. Together with BIM and PUMA, BID is an essential direct activator of BAX and BAK, and BID-deficient or triple-knockout cells fail to oligomerize BAX/BAK or release cytochrome c [PMID:21127253, PMID:24074954]. At the membrane, tBid inserts via its synergistic α6–α7 hydrophobic hairpin [PMID:27053107, PMID:15323553], engages MTCH2 in a primed state, and then activates BAK at its canonical BH3-binding groove through a 'hit-and-run' mechanism that permits BAK homo-oligomerization and MOMP [PMID:23604079, PMID:33462413], with BID preferentially activating BAK over BAX [PMID:24074954]. BID activity acts strictly on the outer membrane to release cytochrome c, SMAC, and AIF, driving both caspase-dependent and caspase-independent death [PMID:10973993, PMID:18535584]. BID expression is induced as a p53 transcriptional target [PMID:12402042] and repressed by PLZF [PMID:14769944], while phosphorylation by casein kinases I/II protects it from caspase-8 cleavage and tempers death-receptor signaling [PMID:11583622, PMID:20356928]. Beyond apoptosis, ATM-dependent phosphorylation directs nuclear BID into the ATR/ATRIP/RPA replication-stress sensor complex—where it binds the RPA70 N-terminal cleft to support CHK1 activation and S-phase checkpoint recovery [PMID:16122425, PMID:16122426, PMID:21859891, PMID:21113148]—and this DNA-damage-response function maintains haematopoietic stem cell quiescence and limits mitochondrial oxidative stress [PMID:22446738]. BID also participates in NOD1/NOD2–IKK innate-immune signaling [PMID:21552281] and is required for mitochondrial dysfunction in ferroptosis [PMID:28384611], extending its role beyond classical cell death.","teleology":[{"year":1996,"claim":"Established BID as a BH3-only protein whose death-promoting activity is separable from its physical interactions, defining the structural basis of its function.","evidence":"Yeast two-hybrid cloning, BH3 mutagenesis, and co-immunoprecipitation showing BH3-dependent binding to BAX and BCL-2","pmids":["8918887"],"confidence":"High","gaps":["Did not define the upstream activator that processes BID","Mechanism of BAX/BCL-2 binding versus death promotion left unresolved"]},{"year":1998,"claim":"Answered how the extrinsic and intrinsic pathways connect by showing caspase-8 cleaves BID into tBid that triggers mitochondrial cytochrome c release.","evidence":"In vitro caspase cleavage, immunodepletion, BH3 mutagenesis, and cell-free cytochrome c release assay","pmids":["9727491"],"confidence":"High","gaps":["How tBid permeabilizes the membrane was not resolved","Whether other proteases activate BID was unknown"]},{"year":2000,"claim":"Defined the membrane target of BID by showing recombinant BID/BAX act only on the outer mitochondrial membrane, sparing inner-membrane function.","evidence":"Isolated mitochondria assays with EM ultrastructure and protein-import readouts; cell-free granzyme B reconstitution with immunodepletion","pmids":["10973993","11114298"],"confidence":"High","gaps":["Required mitochondrial co-factors for BAX oligomerization not identified","Channel-forming versus pore-inducing mechanism unresolved"]},{"year":2001,"claim":"Showed that casein kinase phosphorylation gates BID's susceptibility to caspase-8 cleavage, establishing post-translational control of the death switch.","evidence":"In vitro kinase assay, phospho-mutant mutagenesis, and pharmacological CKI/CKII inhibition in Fas apoptosis","pmids":["11583622"],"confidence":"High","gaps":["Kinetic coupling to caspase-8 in living cells not yet measured","Physiological signals controlling CKI/CKII activity on BID unknown"]},{"year":2002,"claim":"Broadened BID activation beyond caspases by identifying calpain cleavage and p53-dependent transcriptional induction as alternative routes to BID activity.","evidence":"In vitro calpain cleavage with site mapping and inhibitor panels; p53 response-element reporter and BID-null MEF chemoresistance","pmids":["11940658","12402042"],"confidence":"High","gaps":["p53 transactivation evidence is Medium-confidence and single-lab","Relative in vivo contribution of calpain versus caspase cleavage unclear"]},{"year":2004,"claim":"Demonstrated that DNA-damage-induced BID cleavage can occur independently of death-receptor or known mitochondrial caspases, hinting at a distinct stress-response role.","evidence":"Dominant-negative caspases, CrmA, caspase-2 siRNA, and cleavage-site mapping in p53-mutant leukemic cells; granzyme B species-specificity profiling","pmids":["15117953","17179148"],"confidence":"Medium","gaps":["Identity of the protease cleaving BID after DNA damage not defined","Mouse granzyme B resistance limits mouse-model interpretation"]},{"year":2005,"claim":"Revealed a non-apoptotic nuclear function: ATM phosphorylates BID after double-strand breaks to enforce S-phase arrest, separating BID's DNA-damage role from its death role.","evidence":"Phospho-mapping, ATM kinase assay, Bid-/- reconstitution with phospho-mutants, cell-cycle analysis, and nuclear fractionation; PACS-2-dependent mitochondrial translocation","pmids":["16122425","16122426","15692567"],"confidence":"High","gaps":["Molecular target of nuclear BID at the checkpoint not yet identified","PACS-2 translocation evidence is Medium-confidence single-lab"]},{"year":2008,"claim":"Extended BID's protease repertoire to lysosomal cathepsins and established BID as the link between mitochondria and caspase-independent AIF-driven neuronal death.","evidence":"In vitro cathepsin cleavage with E-64d; live imaging and siRNA epistasis placing AIF downstream of BID","pmids":["18469004","18535584"],"confidence":"Medium","gaps":["Cathepsin cleavage sites in cells not mapped","AIF-pathway evidence rests on siRNA in a single neuronal system"]},{"year":2010,"claim":"Defined BID as a genetically essential direct activator of BAX/BAK and placed it at the ATR damage-sensor complex during replicative stress.","evidence":"Triple-knockout mouse epistasis with oligomerization assays; nuclear foci colocalization and Co-IP of BID with ATR/ATRIP/RPA in Bid-/- cells; single-cell CK2 kinetics","pmids":["21127253","21113148","20356928"],"confidence":"High","gaps":["Direct activator role does not exclude redundancy with BIM/PUMA in specific tissues","Precise step of ATR-complex assembly stimulated by BID not yet defined"]},{"year":2011,"claim":"Mapped BID's nuclear interaction surface to RPA70 and uncovered roles in NOD innate immunity and calpain-driven necroptosis, expanding its functional range.","evidence":"NMR-guided BID-RPA70 mapping with functional mutants; genome-wide RNAi and reciprocal Co-IP with NOD1/NOD2/IKK plus in vivo colitis; calpain-resistant BID mutant rescue in necroptosis","pmids":["21859891","21552281","21738214"],"confidence":"High","gaps":["How a single protein partitions between mitochondrial, nuclear, and IKK pools is unresolved","Whether NOD/IKK function requires BID cleavage is unclear"]},{"year":2012,"claim":"Showed that ATM-BID phosphorylation maintains haematopoietic stem cell quiescence and limits mitochondrial ROS, giving the DNA-damage function an in vivo physiological output.","evidence":"Knock-in phospho-mutant mice with quiescence, competitive reconstitution, ROS, and irradiation-survival assays","pmids":["22446738"],"confidence":"High","gaps":["Mechanism linking BID phosphorylation to mitochondrial ROS control not defined","Connection between nuclear checkpoint role and stem-cell phenotype incompletely mapped"]},{"year":2013,"claim":"Provided the structural and selectivity logic of BID-mediated activation: a 'hit-and-run' engagement of the BAK BH3 groove and a preference for BAK over BAX.","evidence":"NMR structure of BID BH3–BAK with BH1/BH3 mutagenesis and MOMP assays; BH3 profiling across BAK/BAX-knockout lines","pmids":["23604079","24074954"],"confidence":"High","gaps":["Structural basis of BAK-over-BAX preference not fully explained","Transient nature of the trigger interaction limits direct visualization"]},{"year":2016,"claim":"Confirmed caspase-8 as the primary physiological activator during TRAIL apoptosis and showed the α6–α7 helices anchor tBid to mitochondria for full activity.","evidence":"CRISPR BID-KO and BID/BAX/BAK TKO reconstitution with caspase-resistant, BH3-defective, and helix-deletion mutants; live-cell FRET of BID-BAX contacts","pmids":["27053107","27763642"],"confidence":"High","gaps":["Role of effector caspases in BID activation excluded only in the TRAIL context","Live-cell FRET (Medium) needs corroboration of oligomer stoichiometry"]},{"year":2020,"claim":"Linked BID to inflammatory cell death by showing caspase-1 cleaves BID to drive SMAC release and secondary necrosis when GSDMD is absent.","evidence":"GSDMD-/- and GSDMD/BID double-knockout cells with caspase and SMAC-release assays","pmids":["32345661"],"confidence":"Medium","gaps":["Single-lab study without in vivo validation","Physiological settings where caspase-1–BID axis dominates not defined"]},{"year":2021,"claim":"Resolved the stepwise membrane activation pathway, showing membrane-bound cBid exposes its BH3 domain, associates with MTCH2, and remains primed until BAX-triggered fragmentation.","evidence":"Spin-label ESR, site-directed PEGylation at authentic mitochondrial membranes, and Co-IP with MTCH2","pmids":["33462413"],"confidence":"High","gaps":["Functional consequence of MTCH2 association in vivo not established","How the primed state is released specifically by BAX versus BAK unclear"]},{"year":null,"claim":"How a single BID protein is partitioned and regulated across its mitochondrial death, nuclear checkpoint, innate-immune, and ferroptosis functions—and what determines which output dominates in a given cell—remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying model coordinating cytosolic, mitochondrial, and nuclear BID pools","Determinants of protease and partner selection in different stress contexts not defined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[30]},{"term_id":"GO:0140313","term_label":"molecular sequestering activity","supporting_discovery_ids":[1,5,6]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,5,6,7]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[20,21]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[0,14,24]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[9,0]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[3,20,21]}],"pathway":[{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[0,5,17,18,28]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[3,20,21]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[9,10,13]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[19,27]}],"complexes":["ATR/ATRIP/RPA replication-stress sensor complex"],"partners":["BAK","BAX","BCL-2","MTCH2","RPA70","PACS-2","NOD2","AIF"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P55957","full_name":"BH3-interacting domain death agonist","aliases":["p22 BID","BID"],"length_aa":195,"mass_kda":22.0,"function":"Induces caspases and apoptosis (PubMed:14583606). Counters the protective effect of BCL2 (By similarity) Induces caspase activation and apoptosis (PubMed:15661737, PubMed:32029622). Translocates to mitochondria following cleavage and disrupts the outer mitochondrial membrane leading to the release of cytochrome c (PubMed:10480954, PubMed:32029622, PubMed:9727491) Induces ICE-like proteases and apoptosis Induces ICE-like proteases and apoptosis Does not induce apoptosis Induces ICE-like proteases and apoptosis","subcellular_location":"Mitochondrion membrane","url":"https://www.uniprot.org/uniprotkb/P55957/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/BID","classification":"Not Classified","n_dependent_lines":16,"n_total_lines":1208,"dependency_fraction":0.013245033112582781},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/BID","total_profiled":1310},"omim":[{"mim_id":"619660","title":"BCL2-LIKE 15; BCL2L15","url":"https://www.omim.org/entry/619660"},{"mim_id":"616466","title":"UNC5 NETRIN RECEPTOR D; UNC5D","url":"https://www.omim.org/entry/616466"},{"mim_id":"613558","title":"DEAFNESS, AUTOSOMAL DOMINANT 51; DFNA51","url":"https://www.omim.org/entry/613558"},{"mim_id":"612787","title":"PSEUDOURIDYLATE SYNTHASE 10; PUS10","url":"https://www.omim.org/entry/612787"},{"mim_id":"610423","title":"PHOSPHOFURIN ACIDIC CLUSTER SORTING PROTEIN 2; PACS2","url":"https://www.omim.org/entry/610423"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"},{"location":"Mitochondria","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"bone 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America","url":"https://pubmed.ncbi.nlm.nih.gov/14769944","citation_count":31,"is_preprint":false},{"pmid":"33462413","id":"PMC_33462413","title":"Stepwise activation of the pro-apoptotic protein Bid at mitochondrial membranes.","date":"2021","source":"Cell death and differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/33462413","citation_count":30,"is_preprint":false},{"pmid":"11997142","id":"PMC_11997142","title":"Sequence and functional similarities between pro-apoptotic Bid and plant lipid transfer proteins.","date":"2002","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/11997142","citation_count":30,"is_preprint":false},{"pmid":"16908521","id":"PMC_16908521","title":"Acyl coenzyme A-binding protein augments bid-induced mitochondrial damage and cell death by activating mu-calpain.","date":"2006","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/16908521","citation_count":28,"is_preprint":false},{"pmid":"37968414","id":"PMC_37968414","title":"BID-seq for transcriptome-wide quantitative sequencing of mRNA pseudouridine at base resolution.","date":"2023","source":"Nature protocols","url":"https://pubmed.ncbi.nlm.nih.gov/37968414","citation_count":27,"is_preprint":false},{"pmid":"19839062","id":"PMC_19839062","title":"BH3-only protein bid participates in the Bcl-2 network in healthy liver cells.","date":"2009","source":"Hepatology (Baltimore, Md.)","url":"https://pubmed.ncbi.nlm.nih.gov/19839062","citation_count":27,"is_preprint":false},{"pmid":"25398540","id":"PMC_25398540","title":"RIP1-dependent Bid cleavage mediates TNFα-induced but Caspase-3-independent cell death in L929 fibroblastoma cells.","date":"2015","source":"Apoptosis : an international journal on programmed cell death","url":"https://pubmed.ncbi.nlm.nih.gov/25398540","citation_count":27,"is_preprint":false},{"pmid":"12172380","id":"PMC_12172380","title":"Temporal and spatial profile of Bid cleavage after experimental traumatic brain injury.","date":"2002","source":"Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/12172380","citation_count":27,"is_preprint":false},{"pmid":"20356928","id":"PMC_20356928","title":"Activity of protein kinase CK2 uncouples Bid cleavage from caspase-8 activation.","date":"2010","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/20356928","citation_count":26,"is_preprint":false},{"pmid":"21113148","id":"PMC_21113148","title":"Proapoptotic Bid mediates the Atr-directed DNA damage response to replicative stress.","date":"2010","source":"Cell death and differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/21113148","citation_count":26,"is_preprint":false},{"pmid":"21304825","id":"PMC_21304825","title":"Stage-specific expression of TNFα regulates bad/bid-mediated apoptosis and RIP1/ROS-mediated secondary necrosis in Birnavirus-infected fish cells.","date":"2011","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/21304825","citation_count":26,"is_preprint":false},{"pmid":"20646170","id":"PMC_20646170","title":"BH3-only protein Bid is dispensable for seizure-induced neuronal death and the associated nuclear accumulation of apoptosis-inducing factor.","date":"2010","source":"Journal of neurochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/20646170","citation_count":26,"is_preprint":false},{"pmid":"18173728","id":"PMC_18173728","title":"Bid is required in NPe6-PDT-induced apoptosis.","date":"2008","source":"Photochemistry and photobiology","url":"https://pubmed.ncbi.nlm.nih.gov/18173728","citation_count":25,"is_preprint":false},{"pmid":"25368155","id":"PMC_25368155","title":"BID mediates selective killing of APC-deficient cells in intestinal tumor suppression by nonsteroidal antiinflammatory drugs.","date":"2014","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/25368155","citation_count":25,"is_preprint":false},{"pmid":"20023601","id":"PMC_20023601","title":"Deficiency of Bid protein reduces sepsis-induced apoptosis and inflammation, while improving septic survival.","date":"2010","source":"Shock (Augusta, Ga.)","url":"https://pubmed.ncbi.nlm.nih.gov/20023601","citation_count":25,"is_preprint":false},{"pmid":"16582605","id":"PMC_16582605","title":"BID as a double agent in cell life and death.","date":"2006","source":"Cell cycle (Georgetown, Tex.)","url":"https://pubmed.ncbi.nlm.nih.gov/16582605","citation_count":24,"is_preprint":false},{"pmid":"21859891","id":"PMC_21859891","title":"BID binds to replication protein A and stimulates ATR function following replicative stress.","date":"2011","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/21859891","citation_count":24,"is_preprint":false},{"pmid":"19661444","id":"PMC_19661444","title":"The BH3-only protein bid does not mediate death-receptor-induced liver injury in obstructive cholestasis.","date":"2009","source":"The American journal of pathology","url":"https://pubmed.ncbi.nlm.nih.gov/19661444","citation_count":24,"is_preprint":false},{"pmid":"21339359","id":"PMC_21339359","title":"Bid and Bim collaborate during induction of T cell death in persistent infection.","date":"2011","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/21339359","citation_count":24,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":47700,"output_tokens":8685,"usd":0.136687,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":18473,"output_tokens":5240,"usd":0.111683,"stage2_stop_reason":"end_turn"},"total_usd":0.24837,"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\": 1998,\n      \"finding\": \"Caspase-8 cleaves BID, and the C-terminal fragment (tBid) translocates to mitochondria where it triggers cytochrome c release; immunodepletion of BID eliminated cytochrome c releasing activity; a BH3 domain mutation diminished activity; Bcl2 antagonized tBid activity\",\n      \"method\": \"Protein purification, peptide mass fingerprinting, in vitro caspase cleavage assay, immunodepletion, site-directed mutagenesis, cell-free cytochrome c release assay\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstitution in vitro with mutagenesis, immunodepletion controls, replicated across multiple subsequent labs\",\n      \"pmids\": [\"9727491\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"BID is a BH3-only protein that heterodimerizes with both BAX and BCL-2 via its BH3 domain; an intact BH3 domain is required to bind the BH1 domain of BCL-2 or BAX; BH3 mutagenesis dissociated binding from death-promoting activity; BID expression induces ICE-like proteases and apoptosis\",\n      \"method\": \"Interactive (yeast two-hybrid) cloning, site-directed mutagenesis, co-immunoprecipitation, cell death assays\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — mutagenesis dissecting binding vs. function, replicated extensively\",\n      \"pmids\": [\"8918887\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"BID is phosphorylated by casein kinase I (CKI) and casein kinase II (CKII); phosphorylated BID is insensitive to caspase-8 cleavage in vitro; inhibition of CKI/CKII accelerated Fas-mediated apoptosis and BID cleavage; a non-phosphorylatable BID mutant was more toxic than wild-type\",\n      \"method\": \"In vitro kinase assay, site-directed mutagenesis, pharmacological kinase inhibition, Fas-mediated apoptosis assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay plus mutagenesis plus cellular pharmacology, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"11583622\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"BID is phosphorylated by ATM kinase following DNA double-strand breaks on two ATM consensus sites; BID-deficient cells fail to accumulate in S phase after etoposide treatment; a non-phosphorylatable BID mutant did not restore S phase arrest and increased sensitivity to etoposide-induced apoptosis; BID partially localizes to the nucleus in healthy cells\",\n      \"method\": \"Phosphorylation mapping, ATM kinase assay, Bid-/- cell reconstitution with wild-type vs. phospho-mutant BID, cell cycle analysis, nuclear fractionation\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — two independent Cell papers (PMIDs 16122425 and 16122426) using phospho-mapping, mutagenesis, and KO reconstitution\",\n      \"pmids\": [\"16122425\", \"16122426\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"PACS-2 translocates BID to mitochondria in response to apoptotic inducers, initiating tBid formation, cytochrome c release, and caspase-3 activation; depletion of PACS-2 prevents BID-mediated apoptosis\",\n      \"method\": \"siRNA knockdown, subcellular fractionation, Western blot for tBid/cytochrome c/caspase-3, live-cell imaging of mitochondria-ER contacts\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal functional assays with siRNA and reconstitution, single lab with multiple readouts\",\n      \"pmids\": [\"15692567\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"BID, BIM, and PUMA are essential activators of BAX and BAK; triple knockout of Bid/Bim/Puma phenocopies Bax/Bak double knockout (persistent interdigital webs, imperforate vaginas) and prevents BAX/BAK homo-oligomerization and cytochrome c release in neurons and T lymphocytes\",\n      \"method\": \"Triple-knockout mouse genetics, epistasis analysis, cytochrome c release assay, BAX/BAK oligomerization assay\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic epistasis with multiple cell types and multiple orthogonal readouts\",\n      \"pmids\": [\"21127253\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"NMR solution structure of the human BID BH3–BAK complex identified the activation site at the canonical BH3-binding groove of BAK; BAK BH1 groove mutations prevented MOMP but not BID binding; BAK BH3 mutations allowed BID binding but blocked oligomerization; BID follows a 'hit-and-run' mechanism dissociating from the trigger site to allow BAK oligomerization; NOXA and BAD are predicted to clash with the trigger site and are not activators of BAK\",\n      \"method\": \"NMR structure determination, site-directed mutagenesis of BAK BH1 and BH3 domains, MOMP assay, binding assay\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — NMR structure plus mutagenesis plus functional MOMP validation in single study\",\n      \"pmids\": [\"23604079\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"BID preferentially activates BAK while BIM preferentially activates BAX; cells lacking BAK are relatively resistant to agents requiring BID activation (topoisomerase inhibitors, TRAIL)\",\n      \"method\": \"BH3 profiling, BAK/BAX knockout cell lines, apoptosis assays, clinical correlation\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple KO cell lines with functional apoptosis assays and biochemical BH3 profiling\",\n      \"pmids\": [\"24074954\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Calpain cleaves BID between Gly70 and Arg71 in cisplatin-treated cells, yielding a 14-kDa fragment; calpain-cleaved BID induces cytochrome c release from isolated mitochondria; calpain inhibitors but not caspase or cathepsin L inhibitors prevented BID cleavage\",\n      \"method\": \"In vitro calpain cleavage of recombinant BID, cleavage site mapping, isolated mitochondria cytochrome c release assay, pharmacological inhibitor panel\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with recombinant proteins plus cleavage site mapping, single lab\",\n      \"pmids\": [\"11940658\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Granzyme B cleaves cytosolic BID to produce truncated BID, which is the sole cytosolic protein responsible for GrB-induced cytochrome c release in a cell-free system; GrB also acts directly on mitochondria to open the permeability transition pore independently of cytosolic proteins\",\n      \"method\": \"Cell-free in vitro system, immunodepletion, recombinant protein assays, mitochondrial membrane potential measurement\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with immunodepletion establishing BID as sole cytosolic mediator\",\n      \"pmids\": [\"11114298\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Bid-deficient cells from multiple tissues are resistant to granzyme B-induced cell death; cytochrome c remains in mitochondria of Bid-deficient cells treated with granzyme B; surviving Bid-deficient cells proliferate normally\",\n      \"method\": \"Bid-/- mouse-derived primary cells, granzyme B treatment, cytochrome c localization by fractionation, cell viability and proliferation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO cells from multiple tissues with defined molecular readout (cytochrome c retention)\",\n      \"pmids\": [\"15574417\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Lysosomal cysteine cathepsins (B, L, S, K, H) cleave BID in vitro; cathepsin-mediated BID cleavage triggers the mitochondrial apoptosis pathway; E-64d (cysteine protease inhibitor) prevented BID cleavage and apoptosis\",\n      \"method\": \"In vitro protease cleavage assays with purified cathepsins and recombinant BID, pharmacological inhibitor studies, cell death assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with multiple purified enzymes plus cellular inhibitor validation\",\n      \"pmids\": [\"18469004\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"BID is a p53 transcriptional target; the human and mouse BID genomic loci contain p53-binding DNA response elements that bind p53 and mediate p53-dependent transactivation; BID-null MEFs are more resistant to adriamycin and 5-fluorouracil\",\n      \"method\": \"Reporter gene assay with p53 response elements, chromatin immunoprecipitation-like binding assay, BID-null MEF apoptosis assays\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — p53 response element binding and reporter assay plus KO cellular phenotype, single lab\",\n      \"pmids\": [\"12402042\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"BID interacts with NOD1, NOD2, and the IKK complex, impacting NF-κB and ERK signaling; BID-deficient colonocytes and macrophages are markedly defective in cytokine production in response to NOD activation; Bid-/- mice are unresponsive to NOD agonists in experimental colitis\",\n      \"method\": \"Genome-wide RNAi screen, co-immunoprecipitation of BID with NOD1/NOD2/IKK, Bid-/- macrophage cytokine assays, in vivo colitis model\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP plus KO in vivo and in vitro with multiple readouts, single lab but genome-wide screen validation\",\n      \"pmids\": [\"21552281\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Recombinant BID and Bax cause complete cytochrome c loss from isolated mitochondria in vitro while preserving inner membrane ultrastructure and protein import function; BID and BAX act only on the outer mitochondrial membrane; inner membrane lesions during apoptosis are secondary caspase-dependent events\",\n      \"method\": \"Isolated mitochondria in vitro assay, electron microscopy of ultrastructure, mitochondrial protein import assay, caspase inhibition experiments\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with recombinant proteins plus structural (EM) and functional validation, replicated\",\n      \"pmids\": [\"10973993\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"BID-induced mitochondrial membrane permeabilization can be inhibited by PTPC inhibitors (cyclosporin A, bongkrekic acid); full-length BID preferentially permeabilizes membranes containing both ANT and BAX; tBid acts on membranes containing ANT alone, suggesting functional interaction with ANT (adenine nucleotide translocase)\",\n      \"method\": \"Intact cell microinjection, isolated mitochondria assay, proteoliposome reconstitution, planar bilayer electrophysiology\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with proteoliposomes and electrophysiology, single lab\",\n      \"pmids\": [\"11175349\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"tBid requires interaction with BAX and the BAX C-terminal transmembrane domain to induce ionic channel formation in liposomes; cut BID alone cannot induce BAX oligomerization in liposomes; an additional mitochondrial factor is required for BAX oligomerization\",\n      \"method\": \"Liposome reconstitution, planar bilayer electrophysiology, gel filtration, recombinant protein interaction assays\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution in defined lipid system with electrophysiology, single lab\",\n      \"pmids\": [\"11964155\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Cleavage by caspase-8 (not effector caspases) is the primary event activating BID during TRAIL-induced apoptosis; additionally, association of tBid with the outer mitochondrial membrane via helices α6 and α7 is required for full apoptotic activity; a tBid mutant lacking these helices has diminished activity despite an intact BH3 domain\",\n      \"method\": \"CRISPR/Cas9 BID-KO and BID/BAX/BAK TKO cells, reconstitution with wild-type and caspase-resistant/BH3-defective/helix-deletion mutants, TRAIL apoptosis assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO with systematic reconstitution using multiple point mutants, clean causal assignment\",\n      \"pmids\": [\"27053107\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"BID controls BAX activation in AIF-mediated caspase-independent necroptosis; BID is processed by calpains into tBid (non-cleavable BID-G70A or BID-Δ68-71 abolishes BAX activation); tBid localizes to mitochondria to facilitate BAX activation and programmed necrotic death\",\n      \"method\": \"Bid-/- and calpain-resistant BID mutant MEFs, BAX activation assay, necroptosis (MNNG) model, reintroduction of wild-type vs. mutant BID\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO cells with rescue by wild-type but not calpain-cleavage-resistant mutants, multiple readouts\",\n      \"pmids\": [\"21738214\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ATM-mediated BID phosphorylation maintains quiescence of haematopoietic stem cells (HSCs); loss of BID phosphorylation leads to escape from quiescence, HSC pool exhaustion, and reduced repopulating potential; BID phosphorylation protects HSCs from irradiation by regulating oxidative stress; loss of phosphorylation or ATM knockout increases mitochondrial BID correlating with increased mitochondrial ROS\",\n      \"method\": \"Knock-in phospho-mutant BID mice, HSC quiescence assays (BrdU), competitive reconstitution assays, ROS measurement, irradiation survival\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo knock-in phospho-mutant mice with multiple orthogonal functional readouts\",\n      \"pmids\": [\"22446738\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"BID associates with RPA70 N-terminal basic cleft; BID stimulates association of RPA with ATR-ATRIP complex; disruption of BID-RPA interaction impairs ATR-ATRIP chromatin association, CHK1 activation, and recovery of DNA replication following hydroxyurea\",\n      \"method\": \"Co-immunoprecipitation, NMR mapping of BID-RPA70 interaction, BID mutants disrupting RPA binding, chromatin fractionation, CHK1 phosphorylation assay, DNA replication recovery assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — NMR-guided interaction mapping plus functional mutagenesis with multiple ATR pathway readouts\",\n      \"pmids\": [\"21859891\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"BID functions at the level of the ATR damage sensor complex following replicative stress; BID is found in nuclear foci with RPA and associates with ATR/ATRIP/RPA complex; BID-deficient cells show reduced ATR/ATRIP chromatin loading, reduced CHK1 activation, and impaired recovery of DNA synthesis\",\n      \"method\": \"Nuclear foci colocalization, co-immunoprecipitation of BID with ATR/ATRIP/RPA, Bid-/- cell chromatin fractionation, CHK1 phosphorylation assay\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP plus KO phenotype with multiple ATR-pathway readouts\",\n      \"pmids\": [\"21113148\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Humanin (HN) peptide binds purified BID and tBid in vitro; HN blocks tBid-induced cytochrome c and SMAC release from isolated mitochondria; HN inhibits tBid-induced BAX and BAK oligomerization in mitochondrial membranes; inactive HN mutants fail to bind BID or block these activities\",\n      \"method\": \"In vitro binding assay with purified proteins, isolated mitochondria cytochrome c/SMAC release assay, chemical cross-linking/gel filtration for oligomerization, gene transfection\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with mutagenesis of HN, single lab\",\n      \"pmids\": [\"15661737\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Membrane-insertion analysis shows that the α6 helix of tBid inserts into membranes only as part of the α6-α7 hairpin (not individually), indicating synergistic hairpin insertion into mitochondrial membrane is required for function\",\n      \"method\": \"Glycosylation mapping of chimeric single-helix segments in a model membrane insertion system\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct membrane insertion assay with glycosylation reporter, single lab\",\n      \"pmids\": [\"15323553\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Stepwise activation of cBid at real mitochondrial membranes: upon mitochondrial binding, cBid reorganizes to expose the BH3 domain while maintaining overall structural integrity; membrane-bound cBid associates with MTCH2 and remains primed until interacting with BAX, which triggers cBid fragmentation, large conformational changes, and BAX-mediated MOMP\",\n      \"method\": \"Spin-label ESR spectroscopy, site-directed PEGylation at real mitochondrial membranes, co-immunoprecipitation with MTCH2\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — ESR and site-directed PEGylation at authentic mitochondrial membranes, mechanistically detailed, single lab\",\n      \"pmids\": [\"33462413\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In live cells, BID adopts an extended conformation at the mitochondrial outer membrane critical for its association with the membrane and for intermolecular contacts within BID oligomers; direct intermolecular FRET contacts between BID and BAX were observed in live cells, confirming BID as a component of the BID-BAX permeabilization network\",\n      \"method\": \"Confocal FRET microscopy in live cells, FRET efficiency measurement, conformational analysis\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live-cell FRET with conformational analysis, single lab\",\n      \"pmids\": [\"27763642\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Three novel BID isoforms are generated by alternative splicing: BidS (lacks BH3 domain, inhibits tBid pro-apoptotic effects and Fas-mediated apoptosis), BidEL (induces apoptosis), and BidES (induces apoptosis but partially inhibits tBid); expression of these isoforms is regulated during granulocyte maturation and they differ in subcellular localization\",\n      \"method\": \"RT-PCR/gene structure analysis, endogenous protein detection, cell death assays, subcellular localization\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — endogenous isoform identification plus functional apoptosis assays, single lab\",\n      \"pmids\": [\"14583606\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"BID translocates to mitochondria during erastin-induced ferroptosis; CRISPR/Cas9 Bid knockout preserves mitochondrial integrity and function, and mediates neuroprotection against ferroptosis and oxytosis; BID inhibitor BI-6c9 inhibited ferroptosis; ferroptosis inhibitors (ferrostatin-1, liproxstatin-1) prevented BID-dependent mitochondrial dysfunction\",\n      \"method\": \"CRISPR/Cas9 BID knockout, mitochondrial membrane potential measurement, ATP assay, mitochondrial fragmentation imaging, pharmacological inhibitors\",\n      \"journal\": \"Redox biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with multiple mitochondrial readouts, single lab\",\n      \"pmids\": [\"28384611\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Caspase-1 directly cleaves BID to generate tBid; in GSDMD-deficient cells, tBid-induced MOMP drives SMAC release to relieve IAP inhibition of caspase-3, enabling rapid secondary necrosis; caspase-1-driven activation of caspase-8/-9 and Bid cleavage act synergistically\",\n      \"method\": \"GSDMD-/- and GSDMD/BID double-KO cells, caspase activation assays, SMAC release measurement, caspase-1 cleavage assay\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — double-KO genetic dissection with multiple caspase readouts, single lab\",\n      \"pmids\": [\"32345661\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"BID translocation to mitochondria precedes AIF release; BID-mediated mitochondrial AIF release causes rapid nuclear translocation and caspase-independent neuronal death; BID siRNA and BID inhibitor prevented AIF nuclear translocation and cell death; tBid-induced death was inhibited by AIF siRNA, establishing AIF as the main downstream effector of BID in glutamate-induced neuronal death\",\n      \"method\": \"Fluorescence video microscopy, BID siRNA, small molecule BID inhibitor, AIF siRNA, caspase-3 inhibitor comparison\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live-cell imaging plus siRNA epistasis, single lab\",\n      \"pmids\": [\"18535584\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"BID sequence has significant similarity to plant lipid transfer proteins; isolated BID shows lipid transfer activity higher than plant LTPs; lysolipids alter BID association with mitochondria and stimulate BID-induced cytochrome c release; BID enhances incorporation of fluorescent lysolipids into mitochondria\",\n      \"method\": \"Sequence analysis, in vitro lipid transfer assay, isolated mitochondria cytochrome c release assay with exogenous lipids, fluorescent lipid incorporation assay\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro biochemical assays, single lab, no mutagenesis validation of lipid-transfer relevance\",\n      \"pmids\": [\"11997142\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Activity of protein kinase CK2 temporally uncouples caspase-8 activation from BID cleavage during TRAIL-induced apoptosis; CK2 inhibition (DRB) or dominant-negative CK2α largely eliminated the lag time between caspase-8 activation and BID cleavage in individual cells\",\n      \"method\": \"Single-cell FRET probes for caspase-8 (IETD) and full-length BID, CK2 inhibitor and dominant-negative expression, time-lapse microscopy\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — FRET-based single-cell kinetics with pharmacological and genetic CK2 manipulation, single lab\",\n      \"pmids\": [\"20356928\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"PLZF transcription factor binds a high-affinity site upstream of the BID transcriptional start site (EMSA) and represses BID expression; PLZF-expressing Jurkat cells show retarded mitochondrial membrane potential loss and resistance to apoptosis\",\n      \"method\": \"Differential expression screen, EMSA for PLZF binding to BID promoter, inducible PLZF expression, annexin V/TUNEL apoptosis assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — EMSA plus functional transcriptional repression assay with inducible system, single lab\",\n      \"pmids\": [\"14769944\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Caspase-cleaved arrestin-2-(1-380) directly binds tBID and doubles tBid-induced cytochrome c release from isolated mitochondria; arrestin-2 pro-apoptotic action requires BID: it does not facilitate apoptosis in BID KO cells, and rescue of BID expression restores activity\",\n      \"method\": \"Co-immunoprecipitation of arrestin-2 fragment with tBID, isolated mitochondria cytochrome c release assay, BID-/- cells with reconstitution, caspase-3 activity assay\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding plus KO epistasis plus reconstitution, single lab\",\n      \"pmids\": [\"24141717\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"In some cell types, tBID induces BAX insertion into mitochondria in vivo and in vitro; tBID relieves inhibition of the BAX transmembrane signal-anchor by the N-terminal domain; however, Bid-null MEFs support BAX insertion into mitochondria in response to TNFα or E1A without BID, demonstrating a parallel BID-independent pathway for BAX membrane insertion in which cytochrome c release is uncoupled from BAX insertion\",\n      \"method\": \"tBid reconstitution in vitro, Bid-null MEF apoptosis and BAX insertion assays, confocal BAX localization\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro reconstitution plus KO genetics showing both BID-dependent and independent routes\",\n      \"pmids\": [\"11139284\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Human granzyme B efficiently cleaves human or mouse BID, but mouse granzyme B is highly resistant to cleaving BID, indicating the BID pathway is not a major primary mediator of mouse granzyme B cytotoxicity; substrate specificity differences mapped to tetrapeptide recognition\",\n      \"method\": \"Substrate specificity profiling, in vitro cleavage assay with human vs. mouse granzyme B and BID substrates\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro biochemical specificity assay comparing species variants, single lab\",\n      \"pmids\": [\"17179148\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"BID cleavage at Asp60 and/or Asp75 (caspase-8/granzyme B target residues) is required for mitochondrial permeabilization and apoptosis induced by etoposide and gamma-radiation in p53-mutant T leukemic cells; this activation is independent of death receptor or mitochondrial inducer caspase activation; caspase-2 knockdown did not prevent BID activation by DNA damage\",\n      \"method\": \"Dominant-negative caspase constructs, CrmA expression, siRNA against caspase-2, BID cleavage site mapping, mitochondrial permeabilization assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple genetic/pharmacological tools excluding known upstream activators, single lab\",\n      \"pmids\": [\"15117953\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"BID is a BH3-only BCL-2 family protein that functions as a molecular bridge between the death receptor extrinsic pathway and the mitochondrial intrinsic pathway: caspase-8 (and alternatively granzyme B, calpains, or cathepsins) cleaves full-length BID, and the resulting tBid translocates to the outer mitochondrial membrane via its α6-α7 hydrophobic hairpin helices where—in a stepwise process involving MTCH2 association followed by a 'hit-and-run' interaction at the canonical BH3-binding groove of BAK (or BAX)—it promotes BAK/BAX homo-oligomerization and MOMP, releasing cytochrome c, SMAC, and AIF to activate downstream caspase-dependent and caspase-independent death; phosphorylation of BID by casein kinases I/II protects it from caspase-8 cleavage, while ATM-mediated phosphorylation recruits nuclear BID to the ATR/ATRIP/RPA sensor complex to enforce S-phase checkpoint arrest and maintain haematopoietic stem cell quiescence, establishing BID as a dual-function integrator of apoptotic and DNA-damage-response signaling.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"BID is a BH3-only BCL-2 family protein that functions as the principal molecular bridge coupling proteolytic death signals to mitochondrial outer-membrane permeabilization (MOMP) and apoptosis [#0, #1]. Cytosolic full-length BID is cleaved by caspase-8 to yield a C-terminal fragment (tBid) that translocates to mitochondria and triggers cytochrome c release; an intact BH3 domain and Bcl-2 antagonism define this as a canonical apoptotic switch [#0, #1]. Diverse proteases converge on BID to generate active tBid—granzyme B during cytotoxic-lymphocyte killing [#9, #10], calpains during cisplatin treatment and AIF-mediated necroptosis [#8, #18], lysosomal cathepsins [#11], and caspase-1 during inflammatory secondary necrosis [#28]—establishing BID as a shared effector node for extrinsic, lysosomal, and inflammatory death routes. Together with BIM and PUMA, BID is an essential direct activator of BAX and BAK, and BID-deficient or triple-knockout cells fail to oligomerize BAX/BAK or release cytochrome c [#5, #7]. At the membrane, tBid inserts via its synergistic α6–α7 hydrophobic hairpin [#17, #23], engages MTCH2 in a primed state, and then activates BAK at its canonical BH3-binding groove through a 'hit-and-run' mechanism that permits BAK homo-oligomerization and MOMP [#6, #24], with BID preferentially activating BAK over BAX [#7]. BID activity acts strictly on the outer membrane to release cytochrome c, SMAC, and AIF, driving both caspase-dependent and caspase-independent death [#14, #29]. BID expression is induced as a p53 transcriptional target [#12] and repressed by PLZF [#32], while phosphorylation by casein kinases I/II protects it from caspase-8 cleavage and tempers death-receptor signaling [#2, #31]. Beyond apoptosis, ATM-dependent phosphorylation directs nuclear BID into the ATR/ATRIP/RPA replication-stress sensor complex—where it binds the RPA70 N-terminal cleft to support CHK1 activation and S-phase checkpoint recovery [#3, #20, #21]—and this DNA-damage-response function maintains haematopoietic stem cell quiescence and limits mitochondrial oxidative stress [#19]. BID also participates in NOD1/NOD2–IKK innate-immune signaling [#13] and is required for mitochondrial dysfunction in ferroptosis [#27], extending its role beyond classical cell death.\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Established BID as a BH3-only protein whose death-promoting activity is separable from its physical interactions, defining the structural basis of its function.\",\n      \"evidence\": \"Yeast two-hybrid cloning, BH3 mutagenesis, and co-immunoprecipitation showing BH3-dependent binding to BAX and BCL-2\",\n      \"pmids\": [\"8918887\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the upstream activator that processes BID\", \"Mechanism of BAX/BCL-2 binding versus death promotion left unresolved\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Answered how the extrinsic and intrinsic pathways connect by showing caspase-8 cleaves BID into tBid that triggers mitochondrial cytochrome c release.\",\n      \"evidence\": \"In vitro caspase cleavage, immunodepletion, BH3 mutagenesis, and cell-free cytochrome c release assay\",\n      \"pmids\": [\"9727491\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How tBid permeabilizes the membrane was not resolved\", \"Whether other proteases activate BID was unknown\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Defined the membrane target of BID by showing recombinant BID/BAX act only on the outer mitochondrial membrane, sparing inner-membrane function.\",\n      \"evidence\": \"Isolated mitochondria assays with EM ultrastructure and protein-import readouts; cell-free granzyme B reconstitution with immunodepletion\",\n      \"pmids\": [\"10973993\", \"11114298\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Required mitochondrial co-factors for BAX oligomerization not identified\", \"Channel-forming versus pore-inducing mechanism unresolved\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Showed that casein kinase phosphorylation gates BID's susceptibility to caspase-8 cleavage, establishing post-translational control of the death switch.\",\n      \"evidence\": \"In vitro kinase assay, phospho-mutant mutagenesis, and pharmacological CKI/CKII inhibition in Fas apoptosis\",\n      \"pmids\": [\"11583622\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinetic coupling to caspase-8 in living cells not yet measured\", \"Physiological signals controlling CKI/CKII activity on BID unknown\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Broadened BID activation beyond caspases by identifying calpain cleavage and p53-dependent transcriptional induction as alternative routes to BID activity.\",\n      \"evidence\": \"In vitro calpain cleavage with site mapping and inhibitor panels; p53 response-element reporter and BID-null MEF chemoresistance\",\n      \"pmids\": [\"11940658\", \"12402042\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"p53 transactivation evidence is Medium-confidence and single-lab\", \"Relative in vivo contribution of calpain versus caspase cleavage unclear\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Demonstrated that DNA-damage-induced BID cleavage can occur independently of death-receptor or known mitochondrial caspases, hinting at a distinct stress-response role.\",\n      \"evidence\": \"Dominant-negative caspases, CrmA, caspase-2 siRNA, and cleavage-site mapping in p53-mutant leukemic cells; granzyme B species-specificity profiling\",\n      \"pmids\": [\"15117953\", \"17179148\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Identity of the protease cleaving BID after DNA damage not defined\", \"Mouse granzyme B resistance limits mouse-model interpretation\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Revealed a non-apoptotic nuclear function: ATM phosphorylates BID after double-strand breaks to enforce S-phase arrest, separating BID's DNA-damage role from its death role.\",\n      \"evidence\": \"Phospho-mapping, ATM kinase assay, Bid-/- reconstitution with phospho-mutants, cell-cycle analysis, and nuclear fractionation; PACS-2-dependent mitochondrial translocation\",\n      \"pmids\": [\"16122425\", \"16122426\", \"15692567\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular target of nuclear BID at the checkpoint not yet identified\", \"PACS-2 translocation evidence is Medium-confidence single-lab\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Extended BID's protease repertoire to lysosomal cathepsins and established BID as the link between mitochondria and caspase-independent AIF-driven neuronal death.\",\n      \"evidence\": \"In vitro cathepsin cleavage with E-64d; live imaging and siRNA epistasis placing AIF downstream of BID\",\n      \"pmids\": [\"18469004\", \"18535584\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cathepsin cleavage sites in cells not mapped\", \"AIF-pathway evidence rests on siRNA in a single neuronal system\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Defined BID as a genetically essential direct activator of BAX/BAK and placed it at the ATR damage-sensor complex during replicative stress.\",\n      \"evidence\": \"Triple-knockout mouse epistasis with oligomerization assays; nuclear foci colocalization and Co-IP of BID with ATR/ATRIP/RPA in Bid-/- cells; single-cell CK2 kinetics\",\n      \"pmids\": [\"21127253\", \"21113148\", \"20356928\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct activator role does not exclude redundancy with BIM/PUMA in specific tissues\", \"Precise step of ATR-complex assembly stimulated by BID not yet defined\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Mapped BID's nuclear interaction surface to RPA70 and uncovered roles in NOD innate immunity and calpain-driven necroptosis, expanding its functional range.\",\n      \"evidence\": \"NMR-guided BID-RPA70 mapping with functional mutants; genome-wide RNAi and reciprocal Co-IP with NOD1/NOD2/IKK plus in vivo colitis; calpain-resistant BID mutant rescue in necroptosis\",\n      \"pmids\": [\"21859891\", \"21552281\", \"21738214\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How a single protein partitions between mitochondrial, nuclear, and IKK pools is unresolved\", \"Whether NOD/IKK function requires BID cleavage is unclear\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Showed that ATM-BID phosphorylation maintains haematopoietic stem cell quiescence and limits mitochondrial ROS, giving the DNA-damage function an in vivo physiological output.\",\n      \"evidence\": \"Knock-in phospho-mutant mice with quiescence, competitive reconstitution, ROS, and irradiation-survival assays\",\n      \"pmids\": [\"22446738\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking BID phosphorylation to mitochondrial ROS control not defined\", \"Connection between nuclear checkpoint role and stem-cell phenotype incompletely mapped\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Provided the structural and selectivity logic of BID-mediated activation: a 'hit-and-run' engagement of the BAK BH3 groove and a preference for BAK over BAX.\",\n      \"evidence\": \"NMR structure of BID BH3–BAK with BH1/BH3 mutagenesis and MOMP assays; BH3 profiling across BAK/BAX-knockout lines\",\n      \"pmids\": [\"23604079\", \"24074954\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of BAK-over-BAX preference not fully explained\", \"Transient nature of the trigger interaction limits direct visualization\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Confirmed caspase-8 as the primary physiological activator during TRAIL apoptosis and showed the α6–α7 helices anchor tBid to mitochondria for full activity.\",\n      \"evidence\": \"CRISPR BID-KO and BID/BAX/BAK TKO reconstitution with caspase-resistant, BH3-defective, and helix-deletion mutants; live-cell FRET of BID-BAX contacts\",\n      \"pmids\": [\"27053107\", \"27763642\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Role of effector caspases in BID activation excluded only in the TRAIL context\", \"Live-cell FRET (Medium) needs corroboration of oligomer stoichiometry\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Linked BID to inflammatory cell death by showing caspase-1 cleaves BID to drive SMAC release and secondary necrosis when GSDMD is absent.\",\n      \"evidence\": \"GSDMD-/- and GSDMD/BID double-knockout cells with caspase and SMAC-release assays\",\n      \"pmids\": [\"32345661\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab study without in vivo validation\", \"Physiological settings where caspase-1–BID axis dominates not defined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Resolved the stepwise membrane activation pathway, showing membrane-bound cBid exposes its BH3 domain, associates with MTCH2, and remains primed until BAX-triggered fragmentation.\",\n      \"evidence\": \"Spin-label ESR, site-directed PEGylation at authentic mitochondrial membranes, and Co-IP with MTCH2\",\n      \"pmids\": [\"33462413\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of MTCH2 association in vivo not established\", \"How the primed state is released specifically by BAX versus BAK unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How a single BID protein is partitioned and regulated across its mitochondrial death, nuclear checkpoint, innate-immune, and ferroptosis functions—and what determines which output dominates in a given cell—remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying model coordinating cytosolic, mitochondrial, and nuclear BID pools\", \"Determinants of protease and partner selection in different stress contexts not defined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [30]},\n      {\"term_id\": \"GO:0140313\", \"supporting_discovery_ids\": [1, 5, 6]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 5, 6, 7]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [20, 21]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [0, 14, 24]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [9, 0]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [3, 20, 21]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [0, 5, 17, 18, 28]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [3, 20, 21]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [9, 10, 13]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [19, 27]}\n    ],\n    \"complexes\": [\"ATR/ATRIP/RPA replication-stress sensor complex\"],\n    \"partners\": [\"BAK\", \"BAX\", \"BCL-2\", \"MTCH2\", \"RPA70\", \"PACS-2\", \"NOD2\", \"AIF\"],\n    \"other_free_text\": []\n  }\n}\n```","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":9,"faith_total":9,"faith_pct":100.0}}