{"gene":"MAD2L1","run_date":"2026-04-28T18:30:27","timeline":{"discoveries":[{"year":1996,"finding":"hsMAD2 (MAD2L1) is a necessary component of the mitotic checkpoint in HeLa cells; antibody electroporation caused premature mitotic exit. MAD2 localizes to kinetochores after chromosome condensation but is absent from kinetochores at metaphase, suggesting it monitors spindle-kinetochore attachment completion.","method":"Antibody electroporation (loss-of-function), immunofluorescence localization","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 — direct functional antibody interference plus subcellular localization, foundational paper replicated widely","pmids":["8824189"],"is_preprint":false},{"year":1997,"finding":"MAD2 associates with the cyclosome/anaphase-promoting complex (APC/C) upon mitotic checkpoint activation, and purified MAD2 arrests cycling Xenopus egg extracts in metaphase by blocking cyclin B ubiquitination, establishing MAD2 as a direct inhibitor of APC/C.","method":"Co-immunoprecipitation, Xenopus egg extract cell-free assay, in vitro ubiquitination assay","journal":"Proceedings of the National Academy of Sciences","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution in egg extracts plus biochemical co-IP, replicated by multiple subsequent studies","pmids":["9356466"],"is_preprint":false},{"year":1998,"finding":"MAD2 prevents APC/C activation by forming a ternary hMAD2-CDC20-APC complex; MAD2 injection into Xenopus embryos arrests cells in mitosis with inactive APC. MAD2 exists in two folded states (tetramer and monomer), both binding CDC20, but only the tetramer inhibits APC/C, indicating structural state is critical for checkpoint signaling.","method":"Xenopus embryo microinjection, in vitro APC activity assay, gel filtration/biochemical fractionation, co-immunoprecipitation","journal":"Genes & Development","confidence":"High","confidence_rationale":"Tier 1 — multiple orthogonal biochemical and cell-based assays in a single study; foundational for understanding MAD2-CDC20-APC/C inhibition","pmids":["9637688"],"is_preprint":false},{"year":2000,"finding":"Solution NMR structure of human MAD2 determined; MAD2 has a novel three-layered α/β fold. The minimal MAD2-binding region of CDC20 is a 40-residue segment N-terminal to its WD40 repeats, and the C-terminal flexible region of MAD2 is required for CDC20 binding, becoming structured upon complex formation.","method":"NMR spectroscopy, deletion mutagenesis, NMR titration","journal":"Nature Structural Biology","confidence":"High","confidence_rationale":"Tier 1 — atomic structure plus mutagenesis defining binding interface","pmids":["10700282"],"is_preprint":false},{"year":2001,"finding":"RNAi-mediated suppression of Mad1 in mammalian cells causes loss of Mad2 kinetochore localization and impairment of the spindle checkpoint. Binding of Mad2 to Mad1 or Cdc20 peptides triggers extensive rearrangement of Mad2 tertiary structure, suggesting a common conformational change mechanism upon ligand binding.","method":"RNAi knockdown, NMR spectroscopy, peptide-binding structural analysis","journal":"Molecular Cell","confidence":"High","confidence_rationale":"Tier 1/2 — NMR structural data plus cell-based RNAi with defined phenotype; replicated by subsequent conformational studies","pmids":["11804586"],"is_preprint":false},{"year":2001,"finding":"MAD2 haplo-insufficiency (deletion of one MAD2 allele) results in a defective mitotic checkpoint, premature sister-chromatid separation in the presence of spindle inhibitors, and elevated chromosome mis-segregation rates, demonstrating partial loss of MAD2 is sufficient to cause chromosomal instability.","method":"Gene deletion (heterozygous knockout in mice and human cancer cells), flow cytometry, chromosome analysis","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 — clean genetic loss-of-function with specific quantitative phenotypic readouts, replicated in two systems","pmids":["11201745"],"is_preprint":false},{"year":2001,"finding":"Mad2 interaction with Mad1 is crucial for localization of Mad2 to kinetochores; at kinetochores, Mad2 interacts with Cdc20. Mad2 forms mutually exclusive, oligomerization-independent complexes with Mad1 and Cdc20. A monomeric Mad2 point mutant still causes cell cycle arrest of comparable strength, showing oligomerization is not required for checkpoint function.","method":"Co-immunoprecipitation, kinetochore localization by immunofluorescence, Xenopus embryo injection, mutational analysis","journal":"The EMBO Journal","confidence":"High","confidence_rationale":"Tier 2 — reciprocal co-IPs, mutagenesis, and cell-based functional assays; replicated across multiple labs","pmids":["11707408"],"is_preprint":false},{"year":2001,"finding":"HsMad1 and HsMAD2 associate with nuclear pore complexes throughout interphase, as demonstrated by co-localization with nucleoporin antibodies and co-purification with enriched nuclear envelope fractions, suggesting a non-mitotic role for the Mad1/Mad2 complex.","method":"Immunofluorescence co-localization, subcellular fractionation/co-purification","journal":"Journal of Cell Science","confidence":"Medium","confidence_rationale":"Tier 2/3 — localization established by two orthogonal methods (IF + fractionation) but functional consequence not fully defined","pmids":["11181178"],"is_preprint":false},{"year":2002,"finding":"BubR1 and Mad2 each independently inhibit Cdc20-APC/C activation. At physiological concentrations, BubR1 and Mad2 mutually promote each other's binding to Cdc20 and act synergistically to quantitatively inhibit APC/C; BubR1 is ~12-fold more potent than Mad2 as an APC/C inhibitor. BubR1-Cdc20 inhibition does not require BubR1 kinase activity.","method":"In vitro APC/C activity assay with purified recombinant proteins, quantitative biochemistry","journal":"Molecular Biology of the Cell","confidence":"High","confidence_rationale":"Tier 1 — reconstituted in vitro with purified proteins, quantitative analysis of synergy","pmids":["11907259"],"is_preprint":false},{"year":2002,"finding":"A novel MAD2-binding protein, CMT2 (later identified as p31comet), interacts with MAD2. Formation of the CMT2-MAD2 complex coincides with dissociation of the p55CDC-MAD2 complex upon spindle attachment completion. CMT2 overexpression causes premature securin destruction and mitotic exit, while CMT2 depletion delays anaphase onset.","method":"Co-immunoprecipitation, overexpression and depletion functional assays","journal":"The EMBO Journal","confidence":"Medium","confidence_rationale":"Tier 2/3 — co-IP plus gain/loss-of-function with defined phenotype; single study","pmids":["12456649"],"is_preprint":false},{"year":2003,"finding":"Aurora B kinase activity is required for kinetochore localization of spindle checkpoint components BubR1, Mad2, and Cenp-E. Aurora B inhibition with ZM447439 or Aurora B RNAi prevents mitotic arrest after spindle damage and abolishes kinetochore recruitment of Mad2, linking chromosome alignment sensing to checkpoint protein localization.","method":"Small molecule Aurora kinase inhibition (ZM447439), RNAi, immunofluorescence localization","journal":"Journal of Cell Biology","confidence":"High","confidence_rationale":"Tier 2 — pharmacological inhibition plus genetic RNAi with specific kinetochore localization readout; highly cited and replicated","pmids":["12719470"],"is_preprint":false},{"year":2003,"finding":"Nuf2 and Hec1 (Ndc80 complex components) are required for retention of Mad1 and Mad2 at kinetochores; RNAi depletion of either protein causes >5-fold reduction of Mad1 and Mad2 at kinetochores, which is microtubule-dependent and reversible upon spindle depolymerization.","method":"RNAi knockdown, immunofluorescence quantification of kinetochore localization","journal":"Current Biology","confidence":"Medium","confidence_rationale":"Tier 2 — RNAi with quantitative kinetochore localization readout, single lab","pmids":["14654001"],"is_preprint":false},{"year":2003,"finding":"MAD2 phosphorylation on multiple serine residues occurs in a cell cycle-dependent manner in vivo; only unphosphorylated MAD2 interacts with Mad1 or the APC/C. A phospho-mimicking MAD2 mutant (S→D) fails to interact with Mad1 or APC/C and acts as a dominant-negative antagonist of wild-type MAD2.","method":"In vivo phosphorylation analysis, co-immunoprecipitation, dominant-negative overexpression","journal":"The EMBO Journal","confidence":"Medium","confidence_rationale":"Tier 2 — phospho-mimetic mutagenesis plus co-IP demonstrating regulatory mechanism; single lab","pmids":["12574116"],"is_preprint":false},{"year":2004,"finding":"Mad2 adopts two distinct natively folded conformations at equilibrium without cofactor binding (termed N1-Mad2/O-Mad2 and N2-Mad2/C-Mad2). NMR structure of N2-Mad2 determined. N2/C-Mad2 is more potent in APC/C inhibition. Interconversion is slow in vitro but accelerated by a Mad1 fragment. Overexpression of a Mad2 mutant that sequesters N2-Mad2 partially blocks checkpoint signaling in cells.","method":"NMR spectroscopy, in vitro APC/C inhibition assay, equilibrium conformational analysis, cell overexpression","journal":"Nature Structural & Molecular Biology","confidence":"High","confidence_rationale":"Tier 1 — NMR structure plus in vitro functional assay plus cell-based validation; foundational two-state model paper","pmids":["15024386"],"is_preprint":false},{"year":2005,"finding":"The Mad1-bound closed conformer of Mad2 (C-Mad2) serves as a template/receptor for cytosolic open Mad2 (O-Mad2) at kinetochores; O-Mad2 and C-Mad2 interaction is essential for the spindle checkpoint. This interaction enables conversion of O-Mad2 into C-Mad2 bound to Cdc20, constituting the 'Mad2 template model' for amplification of the checkpoint signal away from kinetochores.","method":"Mutational analysis, fluorescence microscopy (live imaging, FRAP), co-immunoprecipitation, epistasis","journal":"Current Biology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (mutagenesis + live imaging + co-IP), replicated independently across labs","pmids":["15694304"],"is_preprint":false},{"year":2006,"finding":"The molecular determinants of the O-Mad2:C-Mad2 conformational dimer were characterized. Mutation of individual interface residues abrogates the SAC in S. cerevisiae. NMR chemical shift perturbations show O-Mad2 undergoes major conformational rearrangement upon binding C-Mad2. p31comet competes with O-Mad2 for C-Mad2 binding, explaining its negative regulatory role on the SAC.","method":"NMR spectroscopy, yeast genetic assay (SAC abolition), mutational analysis, co-immunoprecipitation","journal":"The EMBO Journal","confidence":"High","confidence_rationale":"Tier 1/2 — NMR structural data plus genetic assay plus mutagenesis, multiple orthogonal methods","pmids":["16525508"],"is_preprint":false},{"year":1999,"finding":"Mad2 binds to phosphorylated kinetochores but not to unphosphorylated ones in lysed PtK1 cells, providing a molecular link between attachment-sensitive kinetochore phosphorylation and Mad2 recruitment to unattached kinetochores.","method":"Lysed cell in vitro kinetochore binding assay, phosphatase treatment, immunofluorescence","journal":"Current Biology","confidence":"Medium","confidence_rationale":"Tier 2 — cell-free biochemical assay with direct mechanistic readout; single lab","pmids":["10375530"],"is_preprint":false},{"year":1999,"finding":"FAT10, an MHC-encoded ubiquitin-like protein, noncovalently associates with MAD2 as identified by yeast two-hybrid screening and co-immunoprecipitation, suggesting FAT10 may modulate MAD2 activity during cell growth.","method":"Yeast two-hybrid, co-immunoprecipitation","journal":"Proceedings of the National Academy of Sciences","confidence":"Low","confidence_rationale":"Tier 3 — yeast two-hybrid plus co-IP without deep mechanistic follow-up in the initial study","pmids":["10200259"],"is_preprint":false},{"year":2007,"finding":"Crystal structure of the O-Mad2:C-Mad2 conformational dimer reveals an asymmetric interface explaining selective dimerization. Buried hydrophobic residues undergo rearrangement correlated with the topological change. The structure supports a catalytic model where C-Mad2 template facilitates O-Mad2 binding to Cdc20.","method":"X-ray crystallography, mutational functional validation","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with functional mutagenesis validation; landmark structural paper","pmids":["18022367"],"is_preprint":false},{"year":2008,"finding":"Crystal structure of the symmetric C-Mad2:C-Mad2 (C-C) dimer determined, revealing the structural basis for unliganded C-Mad2 (but not O-Mad2 or ligand-bound C-Mad2) forming symmetric dimers. The Mad1-Mad2 core complex facilitates conversion of O-Mad2 to C-Mad2 in vitro.","method":"X-ray crystallography, in vitro conformational conversion assay, cell-based functional assays","journal":"PLoS Biology","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus in vitro reconstitution plus cell biology validation","pmids":["18318601"],"is_preprint":false},{"year":2008,"finding":"Mps1 catalytic activity is required for recruitment of Mad2 (but not Mad1) to kinetochores; catalytically inactive Mps1 restores Mad1 kinetochore localization but not Mad2. Mps1 kinase activity restrains anaphase during unperturbed mitosis.","method":"RNAi complementation with catalytically inactive mutant and analogue-sensitive allele, immunofluorescence","journal":"Journal of Cell Biology","confidence":"High","confidence_rationale":"Tier 2 — RNAi rescue with kinase-dead and analogue-sensitive alleles, dissects Mad1 vs Mad2 localization requirements","pmids":["18541701"],"is_preprint":false},{"year":2008,"finding":"SCF(β-TrCP) degrades REST during G2, allowing transcriptional derepression of Mad2 (a REST target gene). Expression of stable REST (unable to bind β-TrCP) or oncogenic REST-FS inhibits Mad2 expression, producing checkpoint defects analogous to Mad2+/- cells, demonstrating transcriptional regulation of Mad2 by the β-TrCP-REST axis.","method":"Unbiased protein interaction screen, co-immunoprecipitation, stable mutant expression, flow cytometry, fluorescence microscopy","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 — identification of transcriptional regulatory mechanism with multiple orthogonal methods and defined phenotypic readouts","pmids":["18354482"],"is_preprint":false},{"year":2010,"finding":"Sustained Mps1 activity during mitosis is required for recruitment of open Mad2 (O-Mad2) to the Mad1-C-Mad2 core complex at kinetochores. Mps1 inhibition after mitotic entry leaves the Mad1-C-Mad2 core complex kinetochore-bound but abolishes O-Mad2 recruitment. Mps1 can dimerize and transphosphorylate in cells, promoting its own release from kinetochores to facilitate O-Mad2 recruitment.","method":"Novel Mps1 inhibitor (AZ3146), immunofluorescence, co-immunoprecipitation","journal":"Journal of Cell Biology","confidence":"High","confidence_rationale":"Tier 2 — pharmacological inhibition with temporal control, dissects sequential steps of Mad2 recruitment, replicated mechanism","pmids":["20624899"],"is_preprint":false},{"year":2010,"finding":"Phosphorylation of MAD2 on S195 inhibits its conformational transition from O-Mad2 to C-Mad2. Phospho-mimicking Mad2(S195D) fails to bind Cdc20 but retains binding to high-affinity ligands Mad1 and MBP1. Overexpression of Mad2(S195D) causes checkpoint defects in human cells.","method":"Phospho-mimetic mutagenesis, intein-mediated semisynthesis of phosphorylated protein, NMR, co-immunoprecipitation, cell-based checkpoint assay","journal":"Proceedings of the National Academy of Sciences","confidence":"High","confidence_rationale":"Tier 1/2 — phospho-semisynthesis plus NMR plus mutagenesis plus cell assay; mechanistically rigorous","pmids":["21041666"],"is_preprint":false},{"year":2011,"finding":"Endogenous human mitotic checkpoint complex (MCC) is assembled by first forming a BUBR1:BUB3:CDC20 complex in G2, followed by selective incorporation of closed MAD2 (C-MAD2) during mitosis. A recombinant MCC containing C-MAD2 effectively inhibits APC/C, whereas BUBR1:BUB3:CDC20 alone is ineffective at comparable concentrations.","method":"Biochemical fractionation, co-immunoprecipitation, in vitro APC/C inhibition assay, expression of conformation-locked MAD2 mutant","journal":"Cell Cycle","confidence":"High","confidence_rationale":"Tier 1/2 — reconstitution in vitro with defined components plus native complex isolation; mechanistically defines MCC assembly requirements","pmids":["22037211"],"is_preprint":false},{"year":2011,"finding":"BUBR1 directly interacts with closed MAD2 (C-MAD2) via Arg133 and Gln134 of C-MAD2; this interaction is essential for MCC-mediated inhibition of APC/C. The same C-MAD2 residues are required for MAD2 dimerization and p31comet binding.","method":"Co-immunoprecipitation with mutant proteins, in vitro APC/C inhibition assay","journal":"Journal of Biological Chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — mutational mapping of interaction interface with functional consequence; single lab","pmids":["21525009"],"is_preprint":false},{"year":2012,"finding":"Mad2 inhibits Cdc20 by binding directly to a site on Cdc20 required for APC/C binding; Mad2 and APC/C compete for Cdc20 in vitro. A Cdc20 mutant that does not stably bind Mad2 abrogates the SAC in vivo, uncovering a second mechanism by which the SAC inhibits APC/C.","method":"In vitro competition assay, co-immunoprecipitation, cell-based SAC functional assay with Cdc20 mutant","journal":"Journal of Cell Biology","confidence":"High","confidence_rationale":"Tier 1/2 — in vitro reconstitution competition assay plus in vivo mutagenesis validation; identifies second inhibitory mechanism","pmids":["23007648"],"is_preprint":false},{"year":2012,"finding":"Mad2 overexpression hyperstabilizes kinetochore-microtubule (k-MT) attachments independent of the mitotic checkpoint by altering centromeric localization and activity of Aurora B kinase. This checkpoint-independent function of Mad2 requires Cdc20 and explains why Mad2 overexpression increases chromosome missegregation.","method":"Mad2 overexpression, Mad1 depletion (to uncouple checkpoint), k-MT attachment stability assay, Aurora B localization/activity measurement","journal":"Current Biology","confidence":"High","confidence_rationale":"Tier 2 — genetic dissection using Mad1 depletion to separate checkpoint from non-checkpoint function, multiple readouts","pmids":["22405866"],"is_preprint":false},{"year":2013,"finding":"Tpr (nuclear pore complex protein) stabilizes Mad1 and Mad2 protein levels before mitosis by forming a complex (TM2 complex) with them during interphase and mitosis. Tpr is required for Mad1-C-Mad2 recruitment to NPCs and for normal Mad2 levels at kinetochores; overexpression of GFP-Mad2 restores SAC response in Tpr-depleted cells. Tpr may regulate SAC proteostasis through SUMO-isopeptidases SENP1 and SENP2 at NPCs.","method":"Co-immunoprecipitation, protein half-life measurement, RNAi depletion, rescue by GFP-Mad2 overexpression, immunofluorescence","journal":"Journal of Cell Biology","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal methods (co-IP, protein stability, RNAi rescue), single lab","pmids":["24344181"],"is_preprint":false},{"year":2015,"finding":"TRIP13 AAA+ ATPase, aided by adapter protein p31comet, converts MAD2 from the signaling-active closed conformer (C-MAD2) to the inactive open conformer (O-MAD2), thereby inactivating the spindle assembly checkpoint and promoting disassembly of mitotic checkpoint complexes. The PCH-2 (C. elegans TRIP13 ortholog) structure reveals it as a new AAA+ protein remodeler with substrate-recognition domain related to NSF and p97.","method":"Cryo-EM/structural analysis of C. elegans TRIP13, in vitro MAD2 conformational conversion assay, functional genetics","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 — structure plus in vitro reconstitution of MAD2 conformational conversion; replicated by subsequent structural studies","pmids":["25918846"],"is_preprint":false},{"year":2015,"finding":"TRIP13, together with p31comet, prevents APC/C inhibition by free MCC components but cannot reactivate APC/C already bound to MCC. Crystal structure of human TRIP13 determined. TRIP13 and p31comet catalyze conversion of C-Mad2 to O-Mad2 through local unfolding of the Mad2 C-terminal region without disrupting the stable folded core, as shown by NMR.","method":"NMR spectroscopy, X-ray crystallography of human TRIP13, in vitro APC/C inhibition assay, mutagenesis","journal":"Nature Communications","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus NMR plus in vitro functional assay plus mutagenesis in one study","pmids":["29208896"],"is_preprint":false},{"year":2015,"finding":"Structure of an intermediate Mad2 conformer (I-Mad2) bound to C-Mad2 determined by X-ray crystallography and NMR; I-Mad2 retains O-Mad2 fold but core elements move toward C-Mad2 configuration. An allosteric network connects the C-Mad2-binding site to the conformationally malleable C-terminal region. Mutations at the I-Mad2:C-Mad2 interface hinder I-Mad2 formation and impede the structural transition.","method":"X-ray crystallography, NMR spectroscopy, mutagenesis","journal":"Proceedings of the National Academy of Sciences","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus NMR plus mutagenesis defining allosteric mechanism","pmids":["26305957"],"is_preprint":false},{"year":2018,"finding":"Cryo-EM structures of the TRIP13-p31comet-C-MAD2-CDC20 complex reveal that p31comet recruits C-MAD2 to TRIP13 hexameric ring, positioning MAD2's N-terminus (MAD2NT) into TRIP13's axial pore. ATP-driven translocation pushes on and rotates the p31comet-C-MAD2 complex, unwinding a region of the αA helix of C-MAD2 required to stabilize its β-sheet, destabilizing C-MAD2 in favor of O-MAD2.","method":"Cryo-electron microscopy, molecular modeling","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 — high-resolution cryo-EM structure defining complete mechanistic model for TRIP13-mediated MAD2 remodeling","pmids":["29973720"],"is_preprint":false},{"year":2021,"finding":"Kinetochore-catalyzed Mad2-Cdc20 assembly occurs through a tripartite mechanism: localized delivery of Mad2 and Cdc20 substrates, plus two phosphorylation-dependent interactions that geometrically constrain their positions and prime Cdc20 for interaction with Mad2. This was established with a probe specifically monitoring the assembly reaction at kinetochores in living cells.","method":"Live-cell imaging with specific biosensor probe for Mad2-Cdc20 assembly at kinetochores, phosphorylation-dependent interaction analysis","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 — real-time in vivo monitoring of reaction plus phosphorylation mechanism dissection; mechanistically detailed single study","pmids":["33384372"],"is_preprint":false},{"year":2020,"finding":"MAD2 associates with shugoshin 2 (SGO2) in a SAC-activated manner to create a separase inhibitor (SGO2-MAD2 complex) that can functionally replace securin. SGO2-MAD2 sequesters most separase in securin-knockout cells and uses a pseudo-substrate sequence to block the active site of separase. TRIP13-p31comet liberates separase from SGO2-MAD2 in vitro.","method":"Co-immunoprecipitation, in vitro reconstitution (separase inhibition assay), genetic knockouts, TRIP13-p31comet in vitro disassembly assay","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1/2 — in vitro reconstitution plus multiple genetic knockouts with specific functional readout; identifies a new securin-independent MAD2 function","pmids":["32322060"],"is_preprint":false},{"year":1997,"finding":"Human MAD2 (hMAD2) interacts with the C-terminal 30 amino acids of the insulin receptor (IR) cytoplasmic domain but not with IGF-IR; this interaction does not require IR kinase activity and is reduced upon IR autophosphorylation/activation, suggesting MAD2 is released from activated IR.","method":"Yeast two-hybrid, GST pulldown in vitro, co-immunoprecipitation from mammalian cells","journal":"Journal of Biological Chemistry","confidence":"Low","confidence_rationale":"Tier 3 — single co-IP/pulldown with no functional mechanistic follow-up; unclear if relevant to canonical MAD2 checkpoint function","pmids":["9092546"],"is_preprint":false},{"year":2004,"finding":"TRRAP (HAT cofactor) controls mitotic checkpoint integrity by regulating transcription of Mad1 and Mad2 genes through histone H4 and H3 acetylation at their promoters. Trrap associates with HATs Tip60 and PCAF at Mad1/Mad2 promoters in a cell cycle-dependent manner; ectopic expression of Mad1 and Mad2 fully restores the mitotic checkpoint in Trrap-deficient cells.","method":"Chromatin immunoprecipitation, RNAi/conditional knockout, ectopic expression rescue","journal":"The EMBO Journal","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP plus functional rescue establishes transcriptional regulatory mechanism; single lab","pmids":["15549134"],"is_preprint":false},{"year":2009,"finding":"Nek2 kinase physically associates with Mad2 and Cdc20 and can phosphorylate both proteins in vitro; overexpression of Nek2 enhances Mad2-induced mitotic delay, suggesting Nek2 regulates the Mad2-Cdc20 mitotic checkpoint complex.","method":"Co-immunoprecipitation, in vitro kinase assay, overexpression functional assay","journal":"Experimental and Molecular Pathology","confidence":"Low","confidence_rationale":"Tier 3 — co-IP plus in vitro kinase assay without deep mechanistic validation of phospho-site function","pmids":["20034488"],"is_preprint":false},{"year":2013,"finding":"Chk1 co-localizes and physically associates with Mad2 in cells under unstressed and DNA-damaged conditions. Chk1 phosphorylates Mad2 in vitro on multiple sites; a Mad2 mutant lacking all six Chk1 phosphorylatable sites cannot be phosphorylated by Chk1, suggesting a crosslink between DNA damage and mitotic spindle checkpoints.","method":"Co-immunoprecipitation, in vitro kinase assay, mutagenesis, immunofluorescence co-localization","journal":"Cell Cycle","confidence":"Low","confidence_rationale":"Tier 3 — co-IP and in vitro kinase assay, but in vivo functional consequence of specific phospho-sites not established","pmids":["23454898"],"is_preprint":false},{"year":2014,"finding":"The FAT10-MAD2 interaction interface was mapped to FAT10's first ubiquitin-like domain (NMR structure determined). Disruption of FAT10-MAD2 interaction through mutation of specific MAD2-binding residues dramatically limited FAT10's pro-malignant capacity (tumor growth in vivo, aneuploidy, proliferation, migration, invasion) without affecting FAT10's other interactions.","method":"NMR structure of FAT10 domain, mutagenesis of binding interface, in vivo tumor xenograft assay, in vitro cellular assays","journal":"Proceedings of the National Academy of Sciences","confidence":"Medium","confidence_rationale":"Tier 2 — structural mapping plus mutagenesis plus in vivo functional validation; single lab","pmids":["25422469"],"is_preprint":false}],"current_model":"MAD2L1 (MAD2) is a conformationally metamorphic spindle assembly checkpoint protein that exists in open (O-Mad2) and closed (C-Mad2) states; at unattached kinetochores, the Mad1-bound C-Mad2 template recruits cytosolic O-Mad2 and catalyzes its conversion to C-Mad2 bound to CDC20, forming the mitotic checkpoint complex (MCC: MAD2-BUBR1-BUB3-CDC20) that inhibits APC/C-dependent ubiquitination of securin and cyclin B to prevent premature anaphase; checkpoint silencing is driven by TRIP13 AAA+ ATPase and p31comet, which remodel C-Mad2 back to O-Mad2, disassembling the MCC, while MAD2 phosphorylation (e.g., S195) and transcriptional regulation (via REST/β-TrCP and TRRAP/HAT complexes) provide additional control of its activity and abundance."},"narrative":{"teleology":[{"year":1996,"claim":"Establishing MAD2 as an essential vertebrate SAC component resolved whether the yeast checkpoint had a functional human counterpart and showed MAD2 monitors kinetochore–spindle attachment.","evidence":"Antibody electroporation in HeLa cells caused premature mitotic exit; immunofluorescence showed kinetochore localization only on unattached chromosomes","pmids":["8824189"],"confidence":"High","gaps":["Downstream target of MAD2 inhibition not identified","Mechanism of MAD2 removal from attached kinetochores unknown"]},{"year":1997,"claim":"Demonstrating that MAD2 directly associates with and inhibits APC/C via CDC20 identified the molecular target of checkpoint signaling.","evidence":"Co-IP of MAD2 with APC/C in mitotic cells; purified MAD2 arrested Xenopus extracts in metaphase by blocking cyclin B ubiquitination","pmids":["9356466"],"confidence":"High","gaps":["Structural basis of MAD2–CDC20 interaction unknown","Whether MAD2 alone is sufficient for full APC/C inhibition unclear"]},{"year":1998,"claim":"Discovery that MAD2 forms a ternary MAD2–CDC20–APC complex and that its oligomeric state determines inhibitory potency revealed that MAD2's structural conformation is functionally critical.","evidence":"Xenopus embryo injection, in vitro APC assay, and gel filtration showed tetrameric but not monomeric MAD2 inhibited APC/C","pmids":["9637688"],"confidence":"High","gaps":["Atomic structure not yet determined","Nature of the two folded states unresolved"]},{"year":2000,"claim":"The NMR structure of MAD2 revealed a novel α/β fold and showed that the flexible C-terminal region becomes ordered upon CDC20 binding, providing the first atomic framework for the checkpoint interaction.","evidence":"Solution NMR structure determination plus deletion mutagenesis defining the minimal CDC20-binding region","pmids":["10700282"],"confidence":"High","gaps":["Whether MAD2 adopts multiple stable folds not yet recognized","Structure of the MAD2–CDC20 complex not determined"]},{"year":2001,"claim":"Multiple studies established that Mad1 recruits Mad2 to kinetochores via direct binding, that Mad2 forms mutually exclusive complexes with Mad1 and Cdc20, and that Mad2 haploinsufficiency causes chromosomal instability — linking checkpoint protein dosage to genome integrity.","evidence":"RNAi of Mad1 ablated Mad2 kinetochore localization; NMR showed ligand binding triggers extensive conformational change; heterozygous Mad2 knockout in mice and human cells produced premature chromatid separation; monomeric Mad2 mutant retained checkpoint activity","pmids":["11804586","11201745","11707408"],"confidence":"High","gaps":["Identity of the two Mad2 conformational states not yet defined at atomic level","Mechanism of signal amplification from kinetochores unclear"]},{"year":2002,"claim":"Identification of BubR1 as a synergistic co-inhibitor with Mad2 of APC/C-Cdc20, and discovery of p31comet as a Mad2-binding negative regulator, defined both the composition of the inhibitory signal and its silencing mechanism.","evidence":"In vitro APC/C inhibition with purified proteins showed BubR1–Mad2 synergy; co-IP identified CMT2/p31comet whose overexpression caused premature securin destruction","pmids":["11907259","12456649"],"confidence":"High","gaps":["Stoichiometry and order of MCC assembly unknown","Molecular mechanism of p31comet-mediated checkpoint silencing unresolved"]},{"year":2003,"claim":"Aurora B and the Ndc80 complex were shown to act upstream of Mad2 kinetochore recruitment, while cell-cycle-dependent phosphorylation of Mad2 was found to negatively regulate its binding to Mad1 and APC/C.","evidence":"Aurora B inhibition/RNAi abolished Mad2 kinetochore localization; Nuf2/Hec1 RNAi reduced Mad1/Mad2 at kinetochores; phospho-mimetic Mad2 mutants lost Mad1/APC interactions","pmids":["12719470","14654001","12574116"],"confidence":"High","gaps":["Specific kinase(s) phosphorylating Mad2 in vivo not identified","Hierarchy between Aurora B and Ndc80 in Mad2 recruitment unclear"]},{"year":2004,"claim":"NMR determination of two natively folded Mad2 states (O-Mad2 and C-Mad2) and demonstration that C-Mad2 is the active APC/C inhibitor established the two-state model, while TRRAP-dependent histone acetylation was shown to control Mad2 transcription.","evidence":"NMR structures of both conformers; C-Mad2 was more potent in APC/C inhibition; ChIP showed TRRAP/HAT at Mad2 promoter; Mad2 ectopic expression rescued Trrap-deficient cells","pmids":["15024386","15549134"],"confidence":"High","gaps":["How Mad1 catalyzes O-to-C conversion not structurally resolved","Whether transcriptional regulation is tissue-specific unknown"]},{"year":2005,"claim":"The 'Mad2 template model' was established: kinetochore-bound Mad1–C-Mad2 serves as a template/receptor for cytosolic O-Mad2, catalytically converting it to C-Mad2–Cdc20 and amplifying checkpoint signaling away from kinetochores.","evidence":"Mutagenesis, FRAP live imaging, and co-IP showed O-Mad2:C-Mad2 interaction is essential for checkpoint function","pmids":["15694304"],"confidence":"High","gaps":["Structural basis of O-Mad2:C-Mad2 asymmetric dimer unknown","Rate-limiting step of catalytic conversion not defined"]},{"year":2006,"claim":"Characterization of the O-Mad2:C-Mad2 conformational dimer interface by NMR and yeast genetics, plus demonstration that p31comet competes with O-Mad2 for C-Mad2 binding, unified the template and silencing models.","evidence":"NMR chemical shift perturbation, interface mutations abolished SAC in S. cerevisiae, competition assays showed p31comet displaces O-Mad2","pmids":["16525508"],"confidence":"High","gaps":["Crystal structure of O-Mad2:C-Mad2 dimer not yet obtained","Whether p31comet binding is regulated by post-translational modification unknown"]},{"year":2007,"claim":"The crystal structure of the asymmetric O-Mad2:C-Mad2 dimer provided atomic-level validation of the template model and revealed the hydrophobic rearrangements underlying the topological conformational switch.","evidence":"X-ray crystallography with functional mutagenesis validation","pmids":["18022367"],"confidence":"High","gaps":["Structure of complete Mad1–C-Mad2 core complex at kinetochore not determined","Energetic landscape of O-to-C transition not quantified"]},{"year":2008,"claim":"Mps1 kinase was established as the key catalyst for O-Mad2 recruitment to kinetochore-bound Mad1–C-Mad2, the REST–β-TrCP degradation axis was shown to control MAD2 transcription, and the C-Mad2 symmetric dimer structure was determined.","evidence":"Catalytically inactive Mps1 restored Mad1 but not Mad2 kinetochore localization; stable REST mutant suppressed Mad2 expression producing checkpoint defects; crystal structure of C-C Mad2 dimer","pmids":["18541701","18354482","18318601"],"confidence":"High","gaps":["Direct Mps1 substrates enabling O-Mad2 recruitment not identified","Whether REST regulation of Mad2 is relevant in non-transformed cells unclear"]},{"year":2010,"claim":"Sustained Mps1 activity was shown to be continuously required for O-Mad2 recruitment even after the Mad1–C-Mad2 core is kinetochore-bound, and phosphorylation at S195 was demonstrated to block the O-to-C conformational transition by inhibiting Cdc20 binding.","evidence":"Timed Mps1 inhibitor (AZ3146) treatment dissected sequential steps; intein-mediated semisynthesis of pS195-Mad2 plus NMR showed blocked conformational change","pmids":["20624899","21041666"],"confidence":"High","gaps":["Kinase responsible for S195 phosphorylation in vivo not identified","Whether Mps1 directly phosphorylates Mad2 or acts indirectly unknown"]},{"year":2011,"claim":"The order of MCC assembly was defined — BUB3–BUBR1–CDC20 forms in G2, then C-Mad2 joins during mitosis — and the C-Mad2 residues (R133/Q134) mediating BUBR1 interaction were mapped, establishing that C-Mad2 incorporation is the rate-limiting, mitosis-specific step.","evidence":"Biochemical fractionation of endogenous MCC across cell cycle; mutagenesis showed R133A/Q134A abolished BUBR1 binding and APC/C inhibition","pmids":["22037211","21525009"],"confidence":"High","gaps":["Whether MCC assembly occurs only at kinetochores or also in the cytoplasm not resolved","Structural model of complete MCC not yet available"]},{"year":2012,"claim":"A second mechanism of APC/C inhibition was uncovered — Mad2 directly competes with APC/C for a binding site on Cdc20 — and Mad2 overexpression was found to hyperstabilize kinetochore–microtubule attachments independently of the SAC via Aurora B relocalization.","evidence":"In vitro competition assay showed Mad2 and APC/C compete for Cdc20; Cdc20 mutant unable to bind Mad2 abrogated SAC; Mad2 overexpression with Mad1 depletion still altered k-MT stability and Aurora B localization","pmids":["23007648","22405866"],"confidence":"High","gaps":["Relative contribution of two inhibitory mechanisms in vivo not quantified","Mechanism by which Mad2–Cdc20 alters Aurora B localization unknown"]},{"year":2015,"claim":"TRIP13 was structurally and biochemically established as the AAA+ ATPase that, with p31comet adaptor, catalyzes C-Mad2→O-Mad2 remodeling to silence the SAC, while the I-Mad2 intermediate structure revealed the allosteric pathway of the conformational transition.","evidence":"Cryo-EM/crystal structures of TRIP13; NMR showed local unfolding of Mad2 C-terminal region; crystal structure of I-Mad2:C-Mad2 defined intermediate state and allosteric network","pmids":["25918846","29208896","26305957"],"confidence":"High","gaps":["Kinetics of TRIP13-mediated remodeling in vivo not measured","Whether TRIP13 acts on MCC-bound or free C-Mad2 in cells debated"]},{"year":2018,"claim":"Cryo-EM of the TRIP13–p31comet–C-MAD2–CDC20 complex revealed the complete mechanical mechanism: p31comet positions MAD2's N-terminus into TRIP13's axial pore, and ATP-driven translocation unwinds the αA helix, destabilizing the β-sheet that locks the closed conformation.","evidence":"Cryo-electron microscopy of the quaternary complex with molecular modeling","pmids":["29973720"],"confidence":"High","gaps":["Whether TRIP13 can remodel MCC already bound to APC/C in vivo remains unclear","Regulation of TRIP13 activity during the cell cycle not defined"]},{"year":2020,"claim":"Discovery that C-Mad2 forms a complex with SGO2 that directly inhibits separase via a pseudo-substrate mechanism — functionally replacing securin — revealed a checkpoint-effector role for MAD2 beyond APC/C inhibition.","evidence":"Co-IP, in vitro separase inhibition reconstitution, genetic knockouts; TRIP13–p31comet disassembled SGO2–MAD2 in vitro","pmids":["32322060"],"confidence":"High","gaps":["Whether SGO2–MAD2 complex has physiological importance outside securin-null contexts unknown","Structural basis of SGO2–MAD2 interaction not determined"]},{"year":2021,"claim":"Real-time biosensor imaging at kinetochores revealed that Mad2–Cdc20 assembly is driven by a tripartite mechanism requiring localized substrate delivery plus two phosphorylation-dependent interactions that geometrically constrain Cdc20 for Mad2 capture.","evidence":"Live-cell imaging with kinetochore-specific biosensor probe; phosphorylation-dependent interaction dissection","pmids":["33384372"],"confidence":"High","gaps":["Identities of all kinases providing the two phosphorylation inputs at kinetochores not fully resolved","Whether geometric constraint model applies in all cell types unknown"]},{"year":null,"claim":"Key unresolved questions include: the in vivo kinase(s) responsible for MAD2 S195 phosphorylation and its cell-cycle timing; the structural basis of the complete kinetochore-bound Mad1–C-Mad2 catalytic platform; whether TRIP13 acts preferentially on free C-Mad2, MCC, or APC/C-bound MCC in living cells; and the physiological significance of the SGO2–MAD2 separase inhibitory complex in wild-type cells.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No in vivo identification of the S195 kinase","No structure of kinetochore-assembled Mad1–Mad2 catalytic platform","Relative TRIP13 substrates (free C-Mad2 vs MCC vs APC/C-bound MCC) not resolved in cells"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[1,2,8,24,26,34]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[14,15,18]}],"localization":[{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[0,4,10,11,16,22,33]},{"term_id":"GO:0005635","term_label":"nuclear envelope","supporting_discovery_ids":[7,28]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[14,24]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0,1,5,8,14,24,26,33]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[1,2,8,24,29,32]}],"complexes":["MCC (MAD2-BUBR1-BUB3-CDC20)","Mad1-C-Mad2 core complex","SGO2-MAD2 separase inhibitory complex"],"partners":["CDC20","MAD1L1","BUBR1","BUB3","MAD2L1BP","TRIP13","SGO2","TPR"],"other_free_text":[]},"mechanistic_narrative":"MAD2L1 (MAD2) is a conformationally metamorphic spindle assembly checkpoint (SAC) protein that prevents premature anaphase by inhibiting the anaphase-promoting complex/cyclosome (APC/C). MAD2 exists in interconvertible open (O-Mad2) and closed (C-Mad2) conformations; at unattached kinetochores, Mad1-bound C-Mad2 acts as a template to catalyze conversion of cytosolic O-Mad2 into C-Mad2 complexed with CDC20, generating the mitotic checkpoint complex (MCC: MAD2–BUBR1–BUB3–CDC20) that directly inhibits APC/C-mediated ubiquitination of securin and cyclin B [PMID:15694304, PMID:22037211, PMID:9637688]. Checkpoint silencing is driven by the AAA+ ATPase TRIP13, which, together with the adaptor p31comet, threads the MAD2 N-terminus through its axial pore and mechanically unfolds C-Mad2 back to inactive O-Mad2, disassembling the MCC [PMID:29973720, PMID:25918846]. Beyond canonical SAC signaling, C-Mad2 forms a separase-inhibitory complex with shugoshin 2 (SGO2) that functionally substitutes for securin, and MAD2 overexpression hyperstabilizes kinetochore–microtubule attachments independently of the checkpoint through effects on Aurora B localization [PMID:32322060, PMID:22405866]. MAD2 abundance is transcriptionally controlled by the β-TrCP–REST degradation axis and TRRAP-associated histone acetyltransferases, while phosphorylation at S195 inhibits the O-to-C conformational transition and Cdc20 binding, providing post-translational tuning of checkpoint strength [PMID:18354482, PMID:21041666, PMID:15549134]."},"prefetch_data":{"uniprot":{"accession":"Q13257","full_name":"Mitotic spindle assembly checkpoint protein MAD2A","aliases":["Mitotic arrest deficient 2-like protein 1","MAD2-like protein 1"],"length_aa":205,"mass_kda":23.5,"function":"Component of the spindle-assembly checkpoint that prevents the onset of anaphase until all chromosomes are properly aligned at the metaphase plate (PubMed:15024386, PubMed:29162720). In the closed conformation (C-MAD2) forms a heterotetrameric complex with MAD1L1 at unattached kinetochores during prometaphase, the complex recruits open conformation molecules of MAD2L1 (O-MAD2) and then promotes the conversion of O-MAD2 to C-MAD2 (PubMed:29162720). Required for the execution of the mitotic checkpoint which monitors the process of kinetochore-spindle attachment and inhibits the activity of the anaphase promoting complex by sequestering CDC20 until all chromosomes are aligned at the metaphase plate (PubMed:10700282, PubMed:11804586, PubMed:15024386)","subcellular_location":"Nucleus; Chromosome, centromere, kinetochore; Cytoplasm; Cytoplasm, cytoskeleton, spindle pole","url":"https://www.uniprot.org/uniprotkb/Q13257/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/MAD2L1","classification":"Common Essential","n_dependent_lines":1185,"n_total_lines":1208,"dependency_fraction":0.9809602649006622},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"ANAPC4","stoichiometry":0.2},{"gene":"ARFGAP2","stoichiometry":0.2},{"gene":"CAPZB","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/MAD2L1","total_profiled":1310},"omim":[{"mim_id":"621142","title":"CHROMOSOME 15 OPEN READING FRAME 39; C15ORF39","url":"https://www.omim.org/entry/621142"},{"mim_id":"618796","title":"SAC3 DOMAIN-CONTAINING PROTEIN 1; SAC3D1","url":"https://www.omim.org/entry/618796"},{"mim_id":"618136","title":"MAD2L1-BINDING PROTEIN; MAD2L1BP","url":"https://www.omim.org/entry/618136"},{"mim_id":"616401","title":"SPINDLE APPARATUS COILED-COIL PROTEIN 1; SPDL1","url":"https://www.omim.org/entry/616401"},{"mim_id":"615890","title":"DYNEIN, CYTOPLASMIC 1, LIGHT INTERMEDIATE CHAIN 1; DYNC1LI1","url":"https://www.omim.org/entry/615890"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"bone marrow","ntpm":5.5},{"tissue":"lymphoid tissue","ntpm":5.3}],"url":"https://www.proteinatlas.org/search/MAD2L1"},"hgnc":{"alias_symbol":["MAD2","HSMAD2"],"prev_symbol":[]},"alphafold":{"accession":"Q13257","domains":[{"cath_id":"3.30.900.10","chopping":"12-198","consensus_level":"high","plddt":95.8148,"start":12,"end":198}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13257","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q13257-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q13257-F1-predicted_aligned_error_v6.png","plddt_mean":93.81},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MAD2L1","jax_strain_url":"https://www.jax.org/strain/search?query=MAD2L1"},"sequence":{"accession":"Q13257","fasta_url":"https://rest.uniprot.org/uniprotkb/Q13257.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q13257/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13257"}},"corpus_meta":[{"pmid":"12719470","id":"PMC_12719470","title":"Aurora B couples chromosome 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of MAD2 expression to mitotic checkpoint control in ovarian cancer cells.","date":"2002","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/11912137","citation_count":128,"is_preprint":false},{"pmid":"19000814","id":"PMC_19000814","title":"Protein metamorphosis: the two-state behavior of Mad2.","date":"2008","source":"Structure (London, England : 1993)","url":"https://pubmed.ncbi.nlm.nih.gov/19000814","citation_count":126,"is_preprint":false},{"pmid":"16525508","id":"PMC_16525508","title":"Determinants of conformational dimerization of Mad2 and its inhibition by p31comet.","date":"2006","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/16525508","citation_count":117,"is_preprint":false},{"pmid":"29973720","id":"PMC_29973720","title":"Mechanism for remodelling of the cell cycle checkpoint protein MAD2 by the ATPase 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MAD2 localizes to kinetochores after chromosome condensation but is absent from kinetochores at metaphase, suggesting it monitors spindle-kinetochore attachment completion.\",\n      \"method\": \"Antibody electroporation (loss-of-function), immunofluorescence localization\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct functional antibody interference plus subcellular localization, foundational paper replicated widely\",\n      \"pmids\": [\"8824189\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"MAD2 associates with the cyclosome/anaphase-promoting complex (APC/C) upon mitotic checkpoint activation, and purified MAD2 arrests cycling Xenopus egg extracts in metaphase by blocking cyclin B ubiquitination, establishing MAD2 as a direct inhibitor of APC/C.\",\n      \"method\": \"Co-immunoprecipitation, Xenopus egg extract cell-free assay, in vitro ubiquitination assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution in egg extracts plus biochemical co-IP, replicated by multiple subsequent studies\",\n      \"pmids\": [\"9356466\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"MAD2 prevents APC/C activation by forming a ternary hMAD2-CDC20-APC complex; MAD2 injection into Xenopus embryos arrests cells in mitosis with inactive APC. MAD2 exists in two folded states (tetramer and monomer), both binding CDC20, but only the tetramer inhibits APC/C, indicating structural state is critical for checkpoint signaling.\",\n      \"method\": \"Xenopus embryo microinjection, in vitro APC activity assay, gel filtration/biochemical fractionation, co-immunoprecipitation\",\n      \"journal\": \"Genes & Development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — multiple orthogonal biochemical and cell-based assays in a single study; foundational for understanding MAD2-CDC20-APC/C inhibition\",\n      \"pmids\": [\"9637688\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Solution NMR structure of human MAD2 determined; MAD2 has a novel three-layered α/β fold. The minimal MAD2-binding region of CDC20 is a 40-residue segment N-terminal to its WD40 repeats, and the C-terminal flexible region of MAD2 is required for CDC20 binding, becoming structured upon complex formation.\",\n      \"method\": \"NMR spectroscopy, deletion mutagenesis, NMR titration\",\n      \"journal\": \"Nature Structural Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — atomic structure plus mutagenesis defining binding interface\",\n      \"pmids\": [\"10700282\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"RNAi-mediated suppression of Mad1 in mammalian cells causes loss of Mad2 kinetochore localization and impairment of the spindle checkpoint. Binding of Mad2 to Mad1 or Cdc20 peptides triggers extensive rearrangement of Mad2 tertiary structure, suggesting a common conformational change mechanism upon ligand binding.\",\n      \"method\": \"RNAi knockdown, NMR spectroscopy, peptide-binding structural analysis\",\n      \"journal\": \"Molecular Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — NMR structural data plus cell-based RNAi with defined phenotype; replicated by subsequent conformational studies\",\n      \"pmids\": [\"11804586\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"MAD2 haplo-insufficiency (deletion of one MAD2 allele) results in a defective mitotic checkpoint, premature sister-chromatid separation in the presence of spindle inhibitors, and elevated chromosome mis-segregation rates, demonstrating partial loss of MAD2 is sufficient to cause chromosomal instability.\",\n      \"method\": \"Gene deletion (heterozygous knockout in mice and human cancer cells), flow cytometry, chromosome analysis\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean genetic loss-of-function with specific quantitative phenotypic readouts, replicated in two systems\",\n      \"pmids\": [\"11201745\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Mad2 interaction with Mad1 is crucial for localization of Mad2 to kinetochores; at kinetochores, Mad2 interacts with Cdc20. Mad2 forms mutually exclusive, oligomerization-independent complexes with Mad1 and Cdc20. A monomeric Mad2 point mutant still causes cell cycle arrest of comparable strength, showing oligomerization is not required for checkpoint function.\",\n      \"method\": \"Co-immunoprecipitation, kinetochore localization by immunofluorescence, Xenopus embryo injection, mutational analysis\",\n      \"journal\": \"The EMBO Journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IPs, mutagenesis, and cell-based functional assays; replicated across multiple labs\",\n      \"pmids\": [\"11707408\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"HsMad1 and HsMAD2 associate with nuclear pore complexes throughout interphase, as demonstrated by co-localization with nucleoporin antibodies and co-purification with enriched nuclear envelope fractions, suggesting a non-mitotic role for the Mad1/Mad2 complex.\",\n      \"method\": \"Immunofluorescence co-localization, subcellular fractionation/co-purification\",\n      \"journal\": \"Journal of Cell Science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — localization established by two orthogonal methods (IF + fractionation) but functional consequence not fully defined\",\n      \"pmids\": [\"11181178\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"BubR1 and Mad2 each independently inhibit Cdc20-APC/C activation. At physiological concentrations, BubR1 and Mad2 mutually promote each other's binding to Cdc20 and act synergistically to quantitatively inhibit APC/C; BubR1 is ~12-fold more potent than Mad2 as an APC/C inhibitor. BubR1-Cdc20 inhibition does not require BubR1 kinase activity.\",\n      \"method\": \"In vitro APC/C activity assay with purified recombinant proteins, quantitative biochemistry\",\n      \"journal\": \"Molecular Biology of the Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted in vitro with purified proteins, quantitative analysis of synergy\",\n      \"pmids\": [\"11907259\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"A novel MAD2-binding protein, CMT2 (later identified as p31comet), interacts with MAD2. Formation of the CMT2-MAD2 complex coincides with dissociation of the p55CDC-MAD2 complex upon spindle attachment completion. CMT2 overexpression causes premature securin destruction and mitotic exit, while CMT2 depletion delays anaphase onset.\",\n      \"method\": \"Co-immunoprecipitation, overexpression and depletion functional assays\",\n      \"journal\": \"The EMBO Journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — co-IP plus gain/loss-of-function with defined phenotype; single study\",\n      \"pmids\": [\"12456649\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Aurora B kinase activity is required for kinetochore localization of spindle checkpoint components BubR1, Mad2, and Cenp-E. Aurora B inhibition with ZM447439 or Aurora B RNAi prevents mitotic arrest after spindle damage and abolishes kinetochore recruitment of Mad2, linking chromosome alignment sensing to checkpoint protein localization.\",\n      \"method\": \"Small molecule Aurora kinase inhibition (ZM447439), RNAi, immunofluorescence localization\",\n      \"journal\": \"Journal of Cell Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — pharmacological inhibition plus genetic RNAi with specific kinetochore localization readout; highly cited and replicated\",\n      \"pmids\": [\"12719470\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Nuf2 and Hec1 (Ndc80 complex components) are required for retention of Mad1 and Mad2 at kinetochores; RNAi depletion of either protein causes >5-fold reduction of Mad1 and Mad2 at kinetochores, which is microtubule-dependent and reversible upon spindle depolymerization.\",\n      \"method\": \"RNAi knockdown, immunofluorescence quantification of kinetochore localization\",\n      \"journal\": \"Current Biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — RNAi with quantitative kinetochore localization readout, single lab\",\n      \"pmids\": [\"14654001\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"MAD2 phosphorylation on multiple serine residues occurs in a cell cycle-dependent manner in vivo; only unphosphorylated MAD2 interacts with Mad1 or the APC/C. A phospho-mimicking MAD2 mutant (S→D) fails to interact with Mad1 or APC/C and acts as a dominant-negative antagonist of wild-type MAD2.\",\n      \"method\": \"In vivo phosphorylation analysis, co-immunoprecipitation, dominant-negative overexpression\",\n      \"journal\": \"The EMBO Journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — phospho-mimetic mutagenesis plus co-IP demonstrating regulatory mechanism; single lab\",\n      \"pmids\": [\"12574116\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Mad2 adopts two distinct natively folded conformations at equilibrium without cofactor binding (termed N1-Mad2/O-Mad2 and N2-Mad2/C-Mad2). NMR structure of N2-Mad2 determined. N2/C-Mad2 is more potent in APC/C inhibition. Interconversion is slow in vitro but accelerated by a Mad1 fragment. Overexpression of a Mad2 mutant that sequesters N2-Mad2 partially blocks checkpoint signaling in cells.\",\n      \"method\": \"NMR spectroscopy, in vitro APC/C inhibition assay, equilibrium conformational analysis, cell overexpression\",\n      \"journal\": \"Nature Structural & Molecular Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — NMR structure plus in vitro functional assay plus cell-based validation; foundational two-state model paper\",\n      \"pmids\": [\"15024386\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The Mad1-bound closed conformer of Mad2 (C-Mad2) serves as a template/receptor for cytosolic open Mad2 (O-Mad2) at kinetochores; O-Mad2 and C-Mad2 interaction is essential for the spindle checkpoint. This interaction enables conversion of O-Mad2 into C-Mad2 bound to Cdc20, constituting the 'Mad2 template model' for amplification of the checkpoint signal away from kinetochores.\",\n      \"method\": \"Mutational analysis, fluorescence microscopy (live imaging, FRAP), co-immunoprecipitation, epistasis\",\n      \"journal\": \"Current Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (mutagenesis + live imaging + co-IP), replicated independently across labs\",\n      \"pmids\": [\"15694304\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The molecular determinants of the O-Mad2:C-Mad2 conformational dimer were characterized. Mutation of individual interface residues abrogates the SAC in S. cerevisiae. NMR chemical shift perturbations show O-Mad2 undergoes major conformational rearrangement upon binding C-Mad2. p31comet competes with O-Mad2 for C-Mad2 binding, explaining its negative regulatory role on the SAC.\",\n      \"method\": \"NMR spectroscopy, yeast genetic assay (SAC abolition), mutational analysis, co-immunoprecipitation\",\n      \"journal\": \"The EMBO Journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — NMR structural data plus genetic assay plus mutagenesis, multiple orthogonal methods\",\n      \"pmids\": [\"16525508\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Mad2 binds to phosphorylated kinetochores but not to unphosphorylated ones in lysed PtK1 cells, providing a molecular link between attachment-sensitive kinetochore phosphorylation and Mad2 recruitment to unattached kinetochores.\",\n      \"method\": \"Lysed cell in vitro kinetochore binding assay, phosphatase treatment, immunofluorescence\",\n      \"journal\": \"Current Biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — cell-free biochemical assay with direct mechanistic readout; single lab\",\n      \"pmids\": [\"10375530\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"FAT10, an MHC-encoded ubiquitin-like protein, noncovalently associates with MAD2 as identified by yeast two-hybrid screening and co-immunoprecipitation, suggesting FAT10 may modulate MAD2 activity during cell growth.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — yeast two-hybrid plus co-IP without deep mechanistic follow-up in the initial study\",\n      \"pmids\": [\"10200259\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Crystal structure of the O-Mad2:C-Mad2 conformational dimer reveals an asymmetric interface explaining selective dimerization. Buried hydrophobic residues undergo rearrangement correlated with the topological change. The structure supports a catalytic model where C-Mad2 template facilitates O-Mad2 binding to Cdc20.\",\n      \"method\": \"X-ray crystallography, mutational functional validation\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with functional mutagenesis validation; landmark structural paper\",\n      \"pmids\": [\"18022367\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Crystal structure of the symmetric C-Mad2:C-Mad2 (C-C) dimer determined, revealing the structural basis for unliganded C-Mad2 (but not O-Mad2 or ligand-bound C-Mad2) forming symmetric dimers. The Mad1-Mad2 core complex facilitates conversion of O-Mad2 to C-Mad2 in vitro.\",\n      \"method\": \"X-ray crystallography, in vitro conformational conversion assay, cell-based functional assays\",\n      \"journal\": \"PLoS Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus in vitro reconstitution plus cell biology validation\",\n      \"pmids\": [\"18318601\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Mps1 catalytic activity is required for recruitment of Mad2 (but not Mad1) to kinetochores; catalytically inactive Mps1 restores Mad1 kinetochore localization but not Mad2. Mps1 kinase activity restrains anaphase during unperturbed mitosis.\",\n      \"method\": \"RNAi complementation with catalytically inactive mutant and analogue-sensitive allele, immunofluorescence\",\n      \"journal\": \"Journal of Cell Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — RNAi rescue with kinase-dead and analogue-sensitive alleles, dissects Mad1 vs Mad2 localization requirements\",\n      \"pmids\": [\"18541701\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"SCF(β-TrCP) degrades REST during G2, allowing transcriptional derepression of Mad2 (a REST target gene). Expression of stable REST (unable to bind β-TrCP) or oncogenic REST-FS inhibits Mad2 expression, producing checkpoint defects analogous to Mad2+/- cells, demonstrating transcriptional regulation of Mad2 by the β-TrCP-REST axis.\",\n      \"method\": \"Unbiased protein interaction screen, co-immunoprecipitation, stable mutant expression, flow cytometry, fluorescence microscopy\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — identification of transcriptional regulatory mechanism with multiple orthogonal methods and defined phenotypic readouts\",\n      \"pmids\": [\"18354482\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Sustained Mps1 activity during mitosis is required for recruitment of open Mad2 (O-Mad2) to the Mad1-C-Mad2 core complex at kinetochores. Mps1 inhibition after mitotic entry leaves the Mad1-C-Mad2 core complex kinetochore-bound but abolishes O-Mad2 recruitment. Mps1 can dimerize and transphosphorylate in cells, promoting its own release from kinetochores to facilitate O-Mad2 recruitment.\",\n      \"method\": \"Novel Mps1 inhibitor (AZ3146), immunofluorescence, co-immunoprecipitation\",\n      \"journal\": \"Journal of Cell Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — pharmacological inhibition with temporal control, dissects sequential steps of Mad2 recruitment, replicated mechanism\",\n      \"pmids\": [\"20624899\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Phosphorylation of MAD2 on S195 inhibits its conformational transition from O-Mad2 to C-Mad2. Phospho-mimicking Mad2(S195D) fails to bind Cdc20 but retains binding to high-affinity ligands Mad1 and MBP1. Overexpression of Mad2(S195D) causes checkpoint defects in human cells.\",\n      \"method\": \"Phospho-mimetic mutagenesis, intein-mediated semisynthesis of phosphorylated protein, NMR, co-immunoprecipitation, cell-based checkpoint assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — phospho-semisynthesis plus NMR plus mutagenesis plus cell assay; mechanistically rigorous\",\n      \"pmids\": [\"21041666\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Endogenous human mitotic checkpoint complex (MCC) is assembled by first forming a BUBR1:BUB3:CDC20 complex in G2, followed by selective incorporation of closed MAD2 (C-MAD2) during mitosis. A recombinant MCC containing C-MAD2 effectively inhibits APC/C, whereas BUBR1:BUB3:CDC20 alone is ineffective at comparable concentrations.\",\n      \"method\": \"Biochemical fractionation, co-immunoprecipitation, in vitro APC/C inhibition assay, expression of conformation-locked MAD2 mutant\",\n      \"journal\": \"Cell Cycle\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — reconstitution in vitro with defined components plus native complex isolation; mechanistically defines MCC assembly requirements\",\n      \"pmids\": [\"22037211\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"BUBR1 directly interacts with closed MAD2 (C-MAD2) via Arg133 and Gln134 of C-MAD2; this interaction is essential for MCC-mediated inhibition of APC/C. The same C-MAD2 residues are required for MAD2 dimerization and p31comet binding.\",\n      \"method\": \"Co-immunoprecipitation with mutant proteins, in vitro APC/C inhibition assay\",\n      \"journal\": \"Journal of Biological Chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mutational mapping of interaction interface with functional consequence; single lab\",\n      \"pmids\": [\"21525009\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Mad2 inhibits Cdc20 by binding directly to a site on Cdc20 required for APC/C binding; Mad2 and APC/C compete for Cdc20 in vitro. A Cdc20 mutant that does not stably bind Mad2 abrogates the SAC in vivo, uncovering a second mechanism by which the SAC inhibits APC/C.\",\n      \"method\": \"In vitro competition assay, co-immunoprecipitation, cell-based SAC functional assay with Cdc20 mutant\",\n      \"journal\": \"Journal of Cell Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — in vitro reconstitution competition assay plus in vivo mutagenesis validation; identifies second inhibitory mechanism\",\n      \"pmids\": [\"23007648\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Mad2 overexpression hyperstabilizes kinetochore-microtubule (k-MT) attachments independent of the mitotic checkpoint by altering centromeric localization and activity of Aurora B kinase. This checkpoint-independent function of Mad2 requires Cdc20 and explains why Mad2 overexpression increases chromosome missegregation.\",\n      \"method\": \"Mad2 overexpression, Mad1 depletion (to uncouple checkpoint), k-MT attachment stability assay, Aurora B localization/activity measurement\",\n      \"journal\": \"Current Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic dissection using Mad1 depletion to separate checkpoint from non-checkpoint function, multiple readouts\",\n      \"pmids\": [\"22405866\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Tpr (nuclear pore complex protein) stabilizes Mad1 and Mad2 protein levels before mitosis by forming a complex (TM2 complex) with them during interphase and mitosis. Tpr is required for Mad1-C-Mad2 recruitment to NPCs and for normal Mad2 levels at kinetochores; overexpression of GFP-Mad2 restores SAC response in Tpr-depleted cells. Tpr may regulate SAC proteostasis through SUMO-isopeptidases SENP1 and SENP2 at NPCs.\",\n      \"method\": \"Co-immunoprecipitation, protein half-life measurement, RNAi depletion, rescue by GFP-Mad2 overexpression, immunofluorescence\",\n      \"journal\": \"Journal of Cell Biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (co-IP, protein stability, RNAi rescue), single lab\",\n      \"pmids\": [\"24344181\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TRIP13 AAA+ ATPase, aided by adapter protein p31comet, converts MAD2 from the signaling-active closed conformer (C-MAD2) to the inactive open conformer (O-MAD2), thereby inactivating the spindle assembly checkpoint and promoting disassembly of mitotic checkpoint complexes. The PCH-2 (C. elegans TRIP13 ortholog) structure reveals it as a new AAA+ protein remodeler with substrate-recognition domain related to NSF and p97.\",\n      \"method\": \"Cryo-EM/structural analysis of C. elegans TRIP13, in vitro MAD2 conformational conversion assay, functional genetics\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — structure plus in vitro reconstitution of MAD2 conformational conversion; replicated by subsequent structural studies\",\n      \"pmids\": [\"25918846\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TRIP13, together with p31comet, prevents APC/C inhibition by free MCC components but cannot reactivate APC/C already bound to MCC. Crystal structure of human TRIP13 determined. TRIP13 and p31comet catalyze conversion of C-Mad2 to O-Mad2 through local unfolding of the Mad2 C-terminal region without disrupting the stable folded core, as shown by NMR.\",\n      \"method\": \"NMR spectroscopy, X-ray crystallography of human TRIP13, in vitro APC/C inhibition assay, mutagenesis\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus NMR plus in vitro functional assay plus mutagenesis in one study\",\n      \"pmids\": [\"29208896\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Structure of an intermediate Mad2 conformer (I-Mad2) bound to C-Mad2 determined by X-ray crystallography and NMR; I-Mad2 retains O-Mad2 fold but core elements move toward C-Mad2 configuration. An allosteric network connects the C-Mad2-binding site to the conformationally malleable C-terminal region. Mutations at the I-Mad2:C-Mad2 interface hinder I-Mad2 formation and impede the structural transition.\",\n      \"method\": \"X-ray crystallography, NMR spectroscopy, mutagenesis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus NMR plus mutagenesis defining allosteric mechanism\",\n      \"pmids\": [\"26305957\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Cryo-EM structures of the TRIP13-p31comet-C-MAD2-CDC20 complex reveal that p31comet recruits C-MAD2 to TRIP13 hexameric ring, positioning MAD2's N-terminus (MAD2NT) into TRIP13's axial pore. ATP-driven translocation pushes on and rotates the p31comet-C-MAD2 complex, unwinding a region of the αA helix of C-MAD2 required to stabilize its β-sheet, destabilizing C-MAD2 in favor of O-MAD2.\",\n      \"method\": \"Cryo-electron microscopy, molecular modeling\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — high-resolution cryo-EM structure defining complete mechanistic model for TRIP13-mediated MAD2 remodeling\",\n      \"pmids\": [\"29973720\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Kinetochore-catalyzed Mad2-Cdc20 assembly occurs through a tripartite mechanism: localized delivery of Mad2 and Cdc20 substrates, plus two phosphorylation-dependent interactions that geometrically constrain their positions and prime Cdc20 for interaction with Mad2. This was established with a probe specifically monitoring the assembly reaction at kinetochores in living cells.\",\n      \"method\": \"Live-cell imaging with specific biosensor probe for Mad2-Cdc20 assembly at kinetochores, phosphorylation-dependent interaction analysis\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — real-time in vivo monitoring of reaction plus phosphorylation mechanism dissection; mechanistically detailed single study\",\n      \"pmids\": [\"33384372\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MAD2 associates with shugoshin 2 (SGO2) in a SAC-activated manner to create a separase inhibitor (SGO2-MAD2 complex) that can functionally replace securin. SGO2-MAD2 sequesters most separase in securin-knockout cells and uses a pseudo-substrate sequence to block the active site of separase. TRIP13-p31comet liberates separase from SGO2-MAD2 in vitro.\",\n      \"method\": \"Co-immunoprecipitation, in vitro reconstitution (separase inhibition assay), genetic knockouts, TRIP13-p31comet in vitro disassembly assay\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — in vitro reconstitution plus multiple genetic knockouts with specific functional readout; identifies a new securin-independent MAD2 function\",\n      \"pmids\": [\"32322060\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"Human MAD2 (hMAD2) interacts with the C-terminal 30 amino acids of the insulin receptor (IR) cytoplasmic domain but not with IGF-IR; this interaction does not require IR kinase activity and is reduced upon IR autophosphorylation/activation, suggesting MAD2 is released from activated IR.\",\n      \"method\": \"Yeast two-hybrid, GST pulldown in vitro, co-immunoprecipitation from mammalian cells\",\n      \"journal\": \"Journal of Biological Chemistry\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single co-IP/pulldown with no functional mechanistic follow-up; unclear if relevant to canonical MAD2 checkpoint function\",\n      \"pmids\": [\"9092546\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"TRRAP (HAT cofactor) controls mitotic checkpoint integrity by regulating transcription of Mad1 and Mad2 genes through histone H4 and H3 acetylation at their promoters. Trrap associates with HATs Tip60 and PCAF at Mad1/Mad2 promoters in a cell cycle-dependent manner; ectopic expression of Mad1 and Mad2 fully restores the mitotic checkpoint in Trrap-deficient cells.\",\n      \"method\": \"Chromatin immunoprecipitation, RNAi/conditional knockout, ectopic expression rescue\",\n      \"journal\": \"The EMBO Journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP plus functional rescue establishes transcriptional regulatory mechanism; single lab\",\n      \"pmids\": [\"15549134\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Nek2 kinase physically associates with Mad2 and Cdc20 and can phosphorylate both proteins in vitro; overexpression of Nek2 enhances Mad2-induced mitotic delay, suggesting Nek2 regulates the Mad2-Cdc20 mitotic checkpoint complex.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, overexpression functional assay\",\n      \"journal\": \"Experimental and Molecular Pathology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — co-IP plus in vitro kinase assay without deep mechanistic validation of phospho-site function\",\n      \"pmids\": [\"20034488\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Chk1 co-localizes and physically associates with Mad2 in cells under unstressed and DNA-damaged conditions. Chk1 phosphorylates Mad2 in vitro on multiple sites; a Mad2 mutant lacking all six Chk1 phosphorylatable sites cannot be phosphorylated by Chk1, suggesting a crosslink between DNA damage and mitotic spindle checkpoints.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, mutagenesis, immunofluorescence co-localization\",\n      \"journal\": \"Cell Cycle\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — co-IP and in vitro kinase assay, but in vivo functional consequence of specific phospho-sites not established\",\n      \"pmids\": [\"23454898\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The FAT10-MAD2 interaction interface was mapped to FAT10's first ubiquitin-like domain (NMR structure determined). Disruption of FAT10-MAD2 interaction through mutation of specific MAD2-binding residues dramatically limited FAT10's pro-malignant capacity (tumor growth in vivo, aneuploidy, proliferation, migration, invasion) without affecting FAT10's other interactions.\",\n      \"method\": \"NMR structure of FAT10 domain, mutagenesis of binding interface, in vivo tumor xenograft assay, in vitro cellular assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — structural mapping plus mutagenesis plus in vivo functional validation; single lab\",\n      \"pmids\": [\"25422469\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MAD2L1 (MAD2) is a conformationally metamorphic spindle assembly checkpoint protein that exists in open (O-Mad2) and closed (C-Mad2) states; at unattached kinetochores, the Mad1-bound C-Mad2 template recruits cytosolic O-Mad2 and catalyzes its conversion to C-Mad2 bound to CDC20, forming the mitotic checkpoint complex (MCC: MAD2-BUBR1-BUB3-CDC20) that inhibits APC/C-dependent ubiquitination of securin and cyclin B to prevent premature anaphase; checkpoint silencing is driven by TRIP13 AAA+ ATPase and p31comet, which remodel C-Mad2 back to O-Mad2, disassembling the MCC, while MAD2 phosphorylation (e.g., S195) and transcriptional regulation (via REST/β-TrCP and TRRAP/HAT complexes) provide additional control of its activity and abundance.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"MAD2L1 (MAD2) is a conformationally metamorphic spindle assembly checkpoint (SAC) protein that prevents premature anaphase by inhibiting the anaphase-promoting complex/cyclosome (APC/C). MAD2 exists in interconvertible open (O-Mad2) and closed (C-Mad2) conformations; at unattached kinetochores, Mad1-bound C-Mad2 acts as a template to catalyze conversion of cytosolic O-Mad2 into C-Mad2 complexed with CDC20, generating the mitotic checkpoint complex (MCC: MAD2–BUBR1–BUB3–CDC20) that directly inhibits APC/C-mediated ubiquitination of securin and cyclin B [PMID:15694304, PMID:22037211, PMID:9637688]. Checkpoint silencing is driven by the AAA+ ATPase TRIP13, which, together with the adaptor p31comet, threads the MAD2 N-terminus through its axial pore and mechanically unfolds C-Mad2 back to inactive O-Mad2, disassembling the MCC [PMID:29973720, PMID:25918846]. Beyond canonical SAC signaling, C-Mad2 forms a separase-inhibitory complex with shugoshin 2 (SGO2) that functionally substitutes for securin, and MAD2 overexpression hyperstabilizes kinetochore–microtubule attachments independently of the checkpoint through effects on Aurora B localization [PMID:32322060, PMID:22405866]. MAD2 abundance is transcriptionally controlled by the β-TrCP–REST degradation axis and TRRAP-associated histone acetyltransferases, while phosphorylation at S195 inhibits the O-to-C conformational transition and Cdc20 binding, providing post-translational tuning of checkpoint strength [PMID:18354482, PMID:21041666, PMID:15549134].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Establishing MAD2 as an essential vertebrate SAC component resolved whether the yeast checkpoint had a functional human counterpart and showed MAD2 monitors kinetochore–spindle attachment.\",\n      \"evidence\": \"Antibody electroporation in HeLa cells caused premature mitotic exit; immunofluorescence showed kinetochore localization only on unattached chromosomes\",\n      \"pmids\": [\"8824189\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream target of MAD2 inhibition not identified\", \"Mechanism of MAD2 removal from attached kinetochores unknown\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Demonstrating that MAD2 directly associates with and inhibits APC/C via CDC20 identified the molecular target of checkpoint signaling.\",\n      \"evidence\": \"Co-IP of MAD2 with APC/C in mitotic cells; purified MAD2 arrested Xenopus extracts in metaphase by blocking cyclin B ubiquitination\",\n      \"pmids\": [\"9356466\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of MAD2–CDC20 interaction unknown\", \"Whether MAD2 alone is sufficient for full APC/C inhibition unclear\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Discovery that MAD2 forms a ternary MAD2–CDC20–APC complex and that its oligomeric state determines inhibitory potency revealed that MAD2's structural conformation is functionally critical.\",\n      \"evidence\": \"Xenopus embryo injection, in vitro APC assay, and gel filtration showed tetrameric but not monomeric MAD2 inhibited APC/C\",\n      \"pmids\": [\"9637688\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic structure not yet determined\", \"Nature of the two folded states unresolved\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"The NMR structure of MAD2 revealed a novel α/β fold and showed that the flexible C-terminal region becomes ordered upon CDC20 binding, providing the first atomic framework for the checkpoint interaction.\",\n      \"evidence\": \"Solution NMR structure determination plus deletion mutagenesis defining the minimal CDC20-binding region\",\n      \"pmids\": [\"10700282\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether MAD2 adopts multiple stable folds not yet recognized\", \"Structure of the MAD2–CDC20 complex not determined\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Multiple studies established that Mad1 recruits Mad2 to kinetochores via direct binding, that Mad2 forms mutually exclusive complexes with Mad1 and Cdc20, and that Mad2 haploinsufficiency causes chromosomal instability — linking checkpoint protein dosage to genome integrity.\",\n      \"evidence\": \"RNAi of Mad1 ablated Mad2 kinetochore localization; NMR showed ligand binding triggers extensive conformational change; heterozygous Mad2 knockout in mice and human cells produced premature chromatid separation; monomeric Mad2 mutant retained checkpoint activity\",\n      \"pmids\": [\"11804586\", \"11201745\", \"11707408\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the two Mad2 conformational states not yet defined at atomic level\", \"Mechanism of signal amplification from kinetochores unclear\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Identification of BubR1 as a synergistic co-inhibitor with Mad2 of APC/C-Cdc20, and discovery of p31comet as a Mad2-binding negative regulator, defined both the composition of the inhibitory signal and its silencing mechanism.\",\n      \"evidence\": \"In vitro APC/C inhibition with purified proteins showed BubR1–Mad2 synergy; co-IP identified CMT2/p31comet whose overexpression caused premature securin destruction\",\n      \"pmids\": [\"11907259\", \"12456649\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and order of MCC assembly unknown\", \"Molecular mechanism of p31comet-mediated checkpoint silencing unresolved\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Aurora B and the Ndc80 complex were shown to act upstream of Mad2 kinetochore recruitment, while cell-cycle-dependent phosphorylation of Mad2 was found to negatively regulate its binding to Mad1 and APC/C.\",\n      \"evidence\": \"Aurora B inhibition/RNAi abolished Mad2 kinetochore localization; Nuf2/Hec1 RNAi reduced Mad1/Mad2 at kinetochores; phospho-mimetic Mad2 mutants lost Mad1/APC interactions\",\n      \"pmids\": [\"12719470\", \"14654001\", \"12574116\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific kinase(s) phosphorylating Mad2 in vivo not identified\", \"Hierarchy between Aurora B and Ndc80 in Mad2 recruitment unclear\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"NMR determination of two natively folded Mad2 states (O-Mad2 and C-Mad2) and demonstration that C-Mad2 is the active APC/C inhibitor established the two-state model, while TRRAP-dependent histone acetylation was shown to control Mad2 transcription.\",\n      \"evidence\": \"NMR structures of both conformers; C-Mad2 was more potent in APC/C inhibition; ChIP showed TRRAP/HAT at Mad2 promoter; Mad2 ectopic expression rescued Trrap-deficient cells\",\n      \"pmids\": [\"15024386\", \"15549134\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How Mad1 catalyzes O-to-C conversion not structurally resolved\", \"Whether transcriptional regulation is tissue-specific unknown\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"The 'Mad2 template model' was established: kinetochore-bound Mad1–C-Mad2 serves as a template/receptor for cytosolic O-Mad2, catalytically converting it to C-Mad2–Cdc20 and amplifying checkpoint signaling away from kinetochores.\",\n      \"evidence\": \"Mutagenesis, FRAP live imaging, and co-IP showed O-Mad2:C-Mad2 interaction is essential for checkpoint function\",\n      \"pmids\": [\"15694304\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of O-Mad2:C-Mad2 asymmetric dimer unknown\", \"Rate-limiting step of catalytic conversion not defined\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Characterization of the O-Mad2:C-Mad2 conformational dimer interface by NMR and yeast genetics, plus demonstration that p31comet competes with O-Mad2 for C-Mad2 binding, unified the template and silencing models.\",\n      \"evidence\": \"NMR chemical shift perturbation, interface mutations abolished SAC in S. cerevisiae, competition assays showed p31comet displaces O-Mad2\",\n      \"pmids\": [\"16525508\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Crystal structure of O-Mad2:C-Mad2 dimer not yet obtained\", \"Whether p31comet binding is regulated by post-translational modification unknown\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"The crystal structure of the asymmetric O-Mad2:C-Mad2 dimer provided atomic-level validation of the template model and revealed the hydrophobic rearrangements underlying the topological conformational switch.\",\n      \"evidence\": \"X-ray crystallography with functional mutagenesis validation\",\n      \"pmids\": [\"18022367\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of complete Mad1–C-Mad2 core complex at kinetochore not determined\", \"Energetic landscape of O-to-C transition not quantified\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Mps1 kinase was established as the key catalyst for O-Mad2 recruitment to kinetochore-bound Mad1–C-Mad2, the REST–β-TrCP degradation axis was shown to control MAD2 transcription, and the C-Mad2 symmetric dimer structure was determined.\",\n      \"evidence\": \"Catalytically inactive Mps1 restored Mad1 but not Mad2 kinetochore localization; stable REST mutant suppressed Mad2 expression producing checkpoint defects; crystal structure of C-C Mad2 dimer\",\n      \"pmids\": [\"18541701\", \"18354482\", \"18318601\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct Mps1 substrates enabling O-Mad2 recruitment not identified\", \"Whether REST regulation of Mad2 is relevant in non-transformed cells unclear\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Sustained Mps1 activity was shown to be continuously required for O-Mad2 recruitment even after the Mad1–C-Mad2 core is kinetochore-bound, and phosphorylation at S195 was demonstrated to block the O-to-C conformational transition by inhibiting Cdc20 binding.\",\n      \"evidence\": \"Timed Mps1 inhibitor (AZ3146) treatment dissected sequential steps; intein-mediated semisynthesis of pS195-Mad2 plus NMR showed blocked conformational change\",\n      \"pmids\": [\"20624899\", \"21041666\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase responsible for S195 phosphorylation in vivo not identified\", \"Whether Mps1 directly phosphorylates Mad2 or acts indirectly unknown\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"The order of MCC assembly was defined — BUB3–BUBR1–CDC20 forms in G2, then C-Mad2 joins during mitosis — and the C-Mad2 residues (R133/Q134) mediating BUBR1 interaction were mapped, establishing that C-Mad2 incorporation is the rate-limiting, mitosis-specific step.\",\n      \"evidence\": \"Biochemical fractionation of endogenous MCC across cell cycle; mutagenesis showed R133A/Q134A abolished BUBR1 binding and APC/C inhibition\",\n      \"pmids\": [\"22037211\", \"21525009\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether MCC assembly occurs only at kinetochores or also in the cytoplasm not resolved\", \"Structural model of complete MCC not yet available\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"A second mechanism of APC/C inhibition was uncovered — Mad2 directly competes with APC/C for a binding site on Cdc20 — and Mad2 overexpression was found to hyperstabilize kinetochore–microtubule attachments independently of the SAC via Aurora B relocalization.\",\n      \"evidence\": \"In vitro competition assay showed Mad2 and APC/C compete for Cdc20; Cdc20 mutant unable to bind Mad2 abrogated SAC; Mad2 overexpression with Mad1 depletion still altered k-MT stability and Aurora B localization\",\n      \"pmids\": [\"23007648\", \"22405866\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contribution of two inhibitory mechanisms in vivo not quantified\", \"Mechanism by which Mad2–Cdc20 alters Aurora B localization unknown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"TRIP13 was structurally and biochemically established as the AAA+ ATPase that, with p31comet adaptor, catalyzes C-Mad2→O-Mad2 remodeling to silence the SAC, while the I-Mad2 intermediate structure revealed the allosteric pathway of the conformational transition.\",\n      \"evidence\": \"Cryo-EM/crystal structures of TRIP13; NMR showed local unfolding of Mad2 C-terminal region; crystal structure of I-Mad2:C-Mad2 defined intermediate state and allosteric network\",\n      \"pmids\": [\"25918846\", \"29208896\", \"26305957\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinetics of TRIP13-mediated remodeling in vivo not measured\", \"Whether TRIP13 acts on MCC-bound or free C-Mad2 in cells debated\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Cryo-EM of the TRIP13–p31comet–C-MAD2–CDC20 complex revealed the complete mechanical mechanism: p31comet positions MAD2's N-terminus into TRIP13's axial pore, and ATP-driven translocation unwinds the αA helix, destabilizing the β-sheet that locks the closed conformation.\",\n      \"evidence\": \"Cryo-electron microscopy of the quaternary complex with molecular modeling\",\n      \"pmids\": [\"29973720\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether TRIP13 can remodel MCC already bound to APC/C in vivo remains unclear\", \"Regulation of TRIP13 activity during the cell cycle not defined\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Discovery that C-Mad2 forms a complex with SGO2 that directly inhibits separase via a pseudo-substrate mechanism — functionally replacing securin — revealed a checkpoint-effector role for MAD2 beyond APC/C inhibition.\",\n      \"evidence\": \"Co-IP, in vitro separase inhibition reconstitution, genetic knockouts; TRIP13–p31comet disassembled SGO2–MAD2 in vitro\",\n      \"pmids\": [\"32322060\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether SGO2–MAD2 complex has physiological importance outside securin-null contexts unknown\", \"Structural basis of SGO2–MAD2 interaction not determined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Real-time biosensor imaging at kinetochores revealed that Mad2–Cdc20 assembly is driven by a tripartite mechanism requiring localized substrate delivery plus two phosphorylation-dependent interactions that geometrically constrain Cdc20 for Mad2 capture.\",\n      \"evidence\": \"Live-cell imaging with kinetochore-specific biosensor probe; phosphorylation-dependent interaction dissection\",\n      \"pmids\": [\"33384372\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identities of all kinases providing the two phosphorylation inputs at kinetochores not fully resolved\", \"Whether geometric constraint model applies in all cell types unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include: the in vivo kinase(s) responsible for MAD2 S195 phosphorylation and its cell-cycle timing; the structural basis of the complete kinetochore-bound Mad1–C-Mad2 catalytic platform; whether TRIP13 acts preferentially on free C-Mad2, MCC, or APC/C-bound MCC in living cells; and the physiological significance of the SGO2–MAD2 separase inhibitory complex in wild-type cells.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in vivo identification of the S195 kinase\", \"No structure of kinetochore-assembled Mad1–Mad2 catalytic platform\", \"Relative TRIP13 substrates (free C-Mad2 vs MCC vs APC/C-bound MCC) not resolved in cells\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 2, 8, 24, 26, 34]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [14, 15, 18]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [0, 4, 10, 11, 16, 22, 33]},\n      {\"term_id\": \"GO:0005635\", \"supporting_discovery_ids\": [7, 28]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [14, 24]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0, 1, 5, 8, 14, 24, 26, 33]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 2, 8, 24, 29, 32]}\n    ],\n    \"complexes\": [\n      \"MCC (MAD2-BUBR1-BUB3-CDC20)\",\n      \"Mad1-C-Mad2 core complex\",\n      \"SGO2-MAD2 separase inhibitory complex\"\n    ],\n    \"partners\": [\n      \"CDC20\",\n      \"MAD1L1\",\n      \"BUBR1\",\n      \"BUB3\",\n      \"MAD2L1BP\",\n      \"TRIP13\",\n      \"SGO2\",\n      \"TPR\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}