{"gene":"ANAPC4","run_date":"2026-04-28T17:12:37","timeline":{"discoveries":[{"year":1998,"finding":"ANAPC4 (APC4) was identified and cloned as one of four previously unknown human APC/C subunits. Unlike APC2 (which contains a cullin-homology region) and APC7 (which contains TPR repeats), APC4 and APC5 share no similarity to proteins of known function, establishing APC4 as a structurally distinct core subunit of the anaphase-promoting complex.","method":"Molecular cloning, biochemical purification, sequence analysis","journal":"Science","confidence":"High","confidence_rationale":"Tier 1 — original cloning paper, foundational, 211 citations","pmids":["9469815"],"is_preprint":false},{"year":2000,"finding":"The C. elegans APC4 ortholog EMB-30 is required for metaphase-to-anaphase transitions during both meiosis and mitosis. Loss of emb-30 causes metaphase arrest that is independent of the spindle assembly checkpoint, and reduction of emb-30 activity suppresses the lethality of mdf-1/MAD1 null mutants by delaying anaphase onset.","method":"Genetic loss-of-function (emb-30 mutant alleles), time-lapse imaging, positional cloning, genetic epistasis with mdf-1","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 — multiple alleles, epistasis, imaging; replicated in ortholog; 113 citations","pmids":["10749938"],"is_preprint":false},{"year":1999,"finding":"Fission yeast Cut20 (APC4 ortholog) is a component of the APC/cyclosome required for metaphase-to-anaphase transition. Cut20 interacts closely with Cut4 during cyclosome assembly; cut20 mutants are hypersensitive to canavanine and CdCl2 and are suppressed by PKA-inactivating regulators, phenotypes shared with cut4 but not cut23.","method":"Temperature-sensitive mutant screen, sucrose gradient centrifugation, genetic epistasis, stress sensitivity assays","journal":"Genes to cells","confidence":"Medium","confidence_rationale":"Tier 2 — genetic and biochemical in yeast ortholog; single lab","pmids":["10526233"],"is_preprint":false},{"year":2003,"finding":"APC4 (together with APC1 and APC5) forms a subcomplex that can assemble multiubiquitin chains but cannot bind the coactivator CDH1 or ubiquitinate substrates, establishing APC4 as part of a scaffold that connects the catalytic module (APC2/APC11) with the TPR subunits responsible for coactivator binding.","method":"Biochemical fractionation of APC/C subcomplexes, in vitro ubiquitination assays, CDH1 binding assays","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 1 — reconstituted subcomplexes with in vitro activity assays; 172 citations","pmids":["12956947"],"is_preprint":false},{"year":2007,"finding":"In C. elegans, hypomorphic alleles of emb-30/APC4 suppress mdf-1/MAD1 lethality by delaying anaphase onset and causing securin (IFY-1/Pds1) accumulation, placing APC4 as a rate-limiting component of APC/C activity controlling the timing of anaphase.","method":"EMS mutagenesis suppressor screen, time-lapse imaging of embryos, securin accumulation assay","journal":"Genetics","confidence":"Medium","confidence_rationale":"Tier 2 — genetic epistasis with functional imaging readout; single lab","pmids":["17237515"],"is_preprint":false},{"year":2010,"finding":"Human cytomegalovirus infection causes proteasome-dependent degradation of APC4 and APC5, which is temporally associated with disassembly of the APC/C core complex. This degradation does not require UL97-mediated phosphorylation of Cdh1 and depends on a viral early gene product.","method":"Western blot, proteasome inhibitor treatment, infection with UL97 deletion virus, mass spectrometry","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 — multiple experimental conditions; single lab","pmids":["20686030"],"is_preprint":false},{"year":2011,"finding":"Crystal structure and cryo-EM analysis of the APC/C revealed that APC4, together with APC1 and APC5, forms a platform subcomplex that coordinates juxtaposition of the catalytic module (APC2, APC11, APC10) with the TPR subcomplex (Cdc16, Cdc23, Cdc27), providing a pseudo-atomic model for APC4's scaffolding role.","method":"Recombinant reconstitution, electron microscopy, mass spectrometry, docking of crystallographic and homology models","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 — structural reconstitution with multiple orthogonal methods; 137 citations","pmids":["21307936"],"is_preprint":false},{"year":2012,"finding":"HCMV protein pUL21a directly binds the APC/C and is necessary and sufficient for proteasome-dependent degradation of APC4 and APC5, causing APC/C disruption. A proline-arginine motif at residues 109-110 of pUL21a is critical for APC binding and APC4 degradation.","method":"Proteomics, expression of pUL21a mutants, proteasome inhibitor assays, co-immunoprecipitation, point mutant virus construction","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods including mutagenesis and viral genetics; strong mechanistic detail","pmids":["22792066"],"is_preprint":false},{"year":2015,"finding":"Crystal structure of APC4 was determined at atomic resolution, revealing that APC4 comprises a WD40 domain split by a long α-helical domain. In the context of the APC/C (fitted to a 3.6-Å cryo-EM map), regions of APC4 disordered in the crystal become ordered through contacts with APC5.","method":"Protein crystallography, cryo-EM fitting","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with cryo-EM validation","pmids":["26343760"],"is_preprint":false},{"year":2015,"finding":"HCMV protein UL21a induces degradation of APC1 in addition to APC4 and APC5. In uninfected cells, depletion of any one of APC1, APC4, APC5, or APC8 triggers a cellular mechanism that specifically reduces levels of all three platform subunits (APC1, APC4, APC5), revealing an interdependency in platform subcomplex stability.","method":"siRNA knockdown, Western blot, viral mutant infection","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 — siRNA knockdown with consistent readout across multiple subunits; single lab","pmids":["25903336"],"is_preprint":false},{"year":2016,"finding":"APC4 and APC5 are part of the APC/C platform subcomplex (with APC1 and APC15) that connects the catalytic module to the TPR lobe. The WD40 domain of APC1 mediates coactivator-induced conformational changes that expose the UbcH10-binding site, with APC4/APC5 providing structural scaffolding for this allosteric transition.","method":"Crystal structure of APC1 N-terminal domain, cryo-EM of APC/C-Cdh1 deletion mutant, in vitro ubiquitination assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — structure plus biochemical reconstitution with deletion mutant","pmids":["27601667"],"is_preprint":false},{"year":2018,"finding":"APC4 is the major SUMO target within the APC/C during mitosis in human cells, with SUMO acceptor lysines at positions 772 and 798. SUMOylation of APC4 peaks during mitosis and is critical for timely APC/C activation and anaphase onset, and increases APC/C ubiquitylation activity toward a subset of substrates including KIF18B.","method":"SUMO machinery knockdown, site-directed mutagenesis of APC4 lysine residues, ubiquitination assays, live-cell mitotic timing assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — mutagenesis of specific sites with functional readouts; replicated in independent study (PMID:29517484)","pmids":["29549242"],"is_preprint":false},{"year":2018,"finding":"SUMOylation of the APC4 subunit peaks during mitosis and is critical for timely APC/C activation and anaphase onset. A functionally important SUMO-interacting motif (SIM) was identified in the cullin-homology domain of APC2, located near the APC4 SUMOylation sites, suggesting a SUMO-SIM interaction mechanism that regulates APC/C activity.","method":"Site-directed mutagenesis of APC4 SUMOylation sites, APC2 SIM identification and mutagenesis, cell-based mitotic progression assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 — mutagenesis of specific sites with functional readouts in two independent labs (PMID:29549242 and 29517484)","pmids":["29517484"],"is_preprint":false},{"year":2021,"finding":"Cryo-EM structural analysis of SUMOylated APC/C revealed that SUMOylation of APC4 causes a substantial rearrangement of the WHB domain of APC2 (APC2WHB). This repositioning reduces APC/CCdc20 affinity for the mitotic checkpoint complex (MCC), attenuating MCC-mediated suppression and allowing more efficient substrate ubiquitination when the SAC is silenced.","method":"Reconstitution of SUMOylated APC/C, cryo-EM, biochemical APC/C activity assays, MCC binding assays","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1 — cryo-EM structure plus biochemical reconstitution revealing mechanistic basis","pmids":["33789095"],"is_preprint":false},{"year":2023,"finding":"Accumulated lactate inhibits the SUMO protease SENP1 by forming a complex with zinc in the SENP1 active site. SENP1 inhibition stabilizes SUMOylation of APC4 at two specific residues, which drives UBE2C binding to the APC/C, stimulating timed degradation of cell cycle proteins and efficient mitotic exit in proliferating human cells.","method":"Systematic proteome-wide lactate-binding screen, biochemical SENP1 inhibition assays, APC4 SUMOylation site mutagenesis, UBE2C co-immunoprecipitation, mitotic exit assays","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 — multiple orthogonal methods including biochemical reconstitution, mutagenesis, and functional cell assays; 177 citations","pmids":["36921622"],"is_preprint":false},{"year":2014,"finding":"In Pqbp1-conditional knockout mice with microcephaly, Apc4 was identified as a hub gene in the network of APC/C-related genes whose expression is disrupted. Exogenous Apc4 expression rescued cell cycle length, proliferation, and cell phenotypes of NSPCs caused by Pqbp1-cKO, demonstrating that Apc4 is a functionally limiting node in APC/C-mediated M-phase progression in neural stem progenitor cells.","method":"Conditional knockout mouse model, gene expression profiling, AAV-mediated exogenous Apc4 rescue, cell cycle analysis","journal":"Molecular psychiatry","confidence":"Medium","confidence_rationale":"Tier 2 — rescue experiment in vivo; mechanistic detail is partial (hub role, not direct mechanism)","pmids":["25070536"],"is_preprint":false},{"year":2024,"finding":"Deletion of Anapc4 specifically in neurons (conditional KO) revealed that APC/C controls USP1 protein levels in neurons and regulates the number of primary neurites exiting somata. APC4 SUMOylation was found not to impact primary neurite number or USP1 levels, distinguishing the structural APC4 scaffolding function from its SUMOylation-dependent mitotic role.","method":"Conditional knockout of Anapc4 in mouse neurons, Western blot for substrates, neurite morphology analysis, comparison with coactivator (Cdh1/Cdc20) perturbations","journal":"Frontiers in molecular neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 — clean genetic KO of core subunit with defined cellular phenotype; single lab","pmids":["38932933"],"is_preprint":false},{"year":2023,"finding":"Homozygous missense variants in CDC23 (APC8) that cause female infertility in humans lead to decreased CDC23 and APC4 protein levels and accumulation of securin and cyclin B1 in mouse oocytes, demonstrating that APC4 protein stability depends on the integrity of the APC/C platform and is required for oocyte maturation.","method":"Patient variant identification, Cdc23 knockin mice, Western blot for APC4 and substrates, oocyte maturation phenotyping","journal":"Human genetics","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo mouse model with substrate accumulation readout; establishes APC4 co-dependency","pmids":["37768355"],"is_preprint":false}],"current_model":"ANAPC4 (APC4) is a core scaffolding subunit of the APC/C ubiquitin ligase whose WD40 and α-helical domains form part of the platform subcomplex (with APC1 and APC5) that bridges the catalytic module (APC2/APC11) to the TPR coactivator-binding lobe; its protein stability is interdependent with other platform subunits, and its SUMOylation at K772/K798—regulated by the SENP1 SUMO protease (itself inhibited by lactate) and promoted by CDK1—drives UBE2C recruitment and APC/C activation, with the resulting structural rearrangement of APC2's WHB domain attenuating MCC inhibition to ensure timely anaphase onset."},"narrative":{"teleology":[{"year":1998,"claim":"Identification and cloning of APC4 as a novel subunit of the human APC/C established it as a structurally distinct component lacking homology to known functional domains, opening the question of what role it plays in the complex.","evidence":"Biochemical purification and molecular cloning of four new APC/C subunits from human cells","pmids":["9469815"],"confidence":"High","gaps":["No functional data for APC4 beyond complex membership","Domain architecture unknown"]},{"year":1999,"claim":"Genetic studies in fission yeast (Cut20) and C. elegans (EMB-30) demonstrated that APC4 orthologs are essential for the metaphase-to-anaphase transition, establishing a conserved requirement for this subunit in APC/C-dependent cell division across eukaryotes.","evidence":"Temperature-sensitive mutants in S. pombe and loss-of-function alleles in C. elegans with metaphase arrest phenotypes and genetic epistasis with spindle checkpoint components","pmids":["10526233","10749938"],"confidence":"High","gaps":["Mechanism of APC4 contribution to APC/C activity not resolved","Direct biochemical function not addressed"]},{"year":2003,"claim":"Biochemical reconstitution revealed that APC4 forms a subcomplex with APC1 and APC5 that supports multiubiquitin chain formation but cannot bind coactivators, defining APC4's role as a scaffold bridging the catalytic and coactivator-binding modules.","evidence":"Fractionation of APC/C subcomplexes with in vitro ubiquitination and CDH1 binding assays","pmids":["12956947"],"confidence":"High","gaps":["No structural information on how APC4 interfaces with adjacent modules","APC4 contacts with APC2/APC11 not mapped"]},{"year":2011,"claim":"Cryo-EM and crystal structures provided the first pseudo-atomic model of APC4 within the assembled APC/C, showing it coordinates the platform that juxtaposes the catalytic and TPR modules, and later revealing a split WD40 domain stabilized by contacts with APC5.","evidence":"Recombinant APC/C reconstitution, electron microscopy with docked crystal/homology models; subsequent APC4 crystal structure at atomic resolution fitted to 3.6-Å cryo-EM map","pmids":["21307936","26343760"],"confidence":"High","gaps":["Flexible regions of APC4 not resolved in crystal structure","Structural basis for how APC4 communicates with the catalytic module remained unclear"]},{"year":2012,"claim":"The discovery that HCMV protein pUL21a directly targets APC4 and APC5 for proteasomal degradation, and that siRNA knockdown of any single platform subunit destabilizes all three (APC1/APC4/APC5), revealed an interdependent stability mechanism within the platform subcomplex.","evidence":"Viral mutant genetics, co-immunoprecipitation, proteasome inhibitor assays, siRNA knockdown of individual subunits","pmids":["20686030","22792066","25903336"],"confidence":"High","gaps":["Molecular basis of co-dependent degradation not identified","Whether platform destabilization also affects catalytic subunit levels not tested"]},{"year":2014,"claim":"In a Pqbp1-knockout microcephaly model, APC4 was identified as a functionally limiting node whose exogenous expression rescued neural stem progenitor cell cycle defects, linking APC4 dosage to brain development.","evidence":"Conditional knockout mouse model, AAV-mediated APC4 rescue, cell cycle analysis in NSPCs","pmids":["25070536"],"confidence":"Medium","gaps":["Direct mechanism by which reduced APC4 causes NSPC phenotypes not established","Whether APC4 is the sole limiting subunit in this context unclear"]},{"year":2018,"claim":"Two independent studies established that APC4 is the major SUMO target on the APC/C during mitosis, with K772 and K798 as the acceptor sites, and that this modification is required for timely APC/C activation and anaphase onset; a SUMO-interacting motif on APC2 was identified as the likely intramolecular receptor.","evidence":"Site-directed mutagenesis of APC4 SUMO sites and APC2 SIM, live-cell mitotic timing assays, ubiquitination assays in two independent laboratories","pmids":["29549242","29517484"],"confidence":"High","gaps":["Structural basis of SUMO-SIM interaction within the complex not resolved","Upstream regulation of APC4 SUMOylation kinetics not fully defined"]},{"year":2021,"claim":"Cryo-EM of SUMOylated APC/C revealed that APC4 SUMOylation repositions the APC2 WHB domain, reducing MCC affinity and thereby attenuating spindle assembly checkpoint inhibition—providing the structural mechanism for how SUMOylation activates APC/C at anaphase onset.","evidence":"Reconstituted SUMOylated APC/C, cryo-EM, MCC binding and ubiquitination assays","pmids":["33789095"],"confidence":"High","gaps":["Whether WHB repositioning also affects non-MCC substrates differentially is unknown","In vivo validation of the conformational change not performed"]},{"year":2023,"claim":"Lactate was identified as a physiological regulator of APC4 SUMOylation through inhibition of the SUMO protease SENP1 via zinc complexation, connecting cellular metabolic state to APC/C activation timing and mitotic exit.","evidence":"Proteome-wide lactate-binding screen, SENP1 inhibition assays, APC4 SUMOylation site mutagenesis, UBE2C co-immunoprecipitation, mitotic exit assays","pmids":["36921622"],"confidence":"High","gaps":["Whether lactate-SENP1 axis acts on APC4 SUMOylation in all tissue contexts is untested","Quantitative relationship between lactate concentration and mitotic timing not established in vivo"]},{"year":2024,"claim":"Conditional deletion of Anapc4 in postmitotic neurons demonstrated a SUMOylation-independent scaffolding function controlling USP1 levels and neurite number, distinguishing APC4's structural role from its mitosis-specific SUMOylation-dependent regulatory function.","evidence":"Neuron-specific Anapc4 conditional knockout in mice, comparison of SUMOylation-deficient and null phenotypes, neurite morphology quantification","pmids":["38932933"],"confidence":"Medium","gaps":["Whether APC4 scaffolding controls USP1 through direct ubiquitination or indirectly is unresolved","Coactivator dependence (Cdh1 vs Cdc20) for the neuronal phenotype not fully clarified"]},{"year":null,"claim":"Key open questions include the structural basis of the APC4-SUMO/APC2-SIM interaction at atomic resolution, identification of the full substrate repertoire whose ubiquitination specifically depends on APC4 SUMOylation, and the molecular mechanism by which APC4 loss destabilizes APC1/APC5 and whether this involves a dedicated quality-control pathway.","evidence":"","pmids":[],"confidence":"High","gaps":["No atomic-resolution structure of SUMO-modified APC4 in complex with APC2 SIM","Full set of SUMOylation-dependent APC/C substrates not cataloged","Degradation pathway for orphan platform subunits not identified"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[3,6,8,10]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[3,10,13]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[11,12]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[6,14]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0,1,3,11,12,13,14]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[3,11,13,14]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[15,16]}],"complexes":["APC/C"],"partners":["ANAPC1","ANAPC5","ANAPC2","CDC23","UBE2C","SENP1"],"other_free_text":[]},"mechanistic_narrative":"ANAPC4 (APC4) is a core scaffolding subunit of the anaphase-promoting complex/cyclosome (APC/C), a multi-subunit E3 ubiquitin ligase essential for metaphase-to-anaphase transition and mitotic exit. APC4 contains a split WD40 domain interrupted by a long α-helical domain and, together with APC1 and APC5, forms the platform subcomplex that bridges the catalytic module (APC2/APC11) to the TPR coactivator-binding lobe; these platform subunits exhibit co-dependent protein stability [PMID:12956947, PMID:26343760, PMID:25903336]. APC4 is the principal SUMO target within the APC/C during mitosis: SUMOylation at K772 and K798—promoted by CDK1 activity and stabilized by lactate-mediated inhibition of the SUMO protease SENP1—drives UBE2C recruitment, repositions the APC2 WHB domain to attenuate mitotic checkpoint complex (MCC) inhibition, and is required for timely anaphase onset [PMID:29549242, PMID:29517484, PMID:33789095, PMID:36921622]. Beyond cell division, APC4 functions in postmitotic neurons where its scaffolding role—independent of SUMOylation—controls USP1 levels and primary neurite number [PMID:38932933]."},"prefetch_data":{"uniprot":{"accession":"Q9UJX5","full_name":"Anaphase-promoting complex subunit 4","aliases":["Cyclosome subunit 4"],"length_aa":808,"mass_kda":92.1,"function":"Component of the anaphase promoting complex/cyclosome (APC/C), a cell cycle-regulated E3 ubiquitin ligase that controls progression through mitosis and the G1 phase of the cell cycle (PubMed:18485873). The APC/C complex acts by mediating ubiquitination and subsequent degradation of target proteins: it mainly mediates the formation of 'Lys-11'-linked polyubiquitin chains and, to a lower extent, the formation of 'Lys-48'- and 'Lys-63'-linked polyubiquitin chains (PubMed:18485873). The APC/C complex catalyzes assembly of branched 'Lys-11'-/'Lys-48'-linked branched ubiquitin chains on target proteins (PubMed:29033132)","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9UJX5/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/ANAPC4","classification":"Common Essential","n_dependent_lines":1168,"n_total_lines":1208,"dependency_fraction":0.9668874172185431},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000053900","cell_line_id":"CID000224","localizations":[{"compartment":"cytoplasmic","grade":3},{"compartment":"nucleoplasm","grade":3},{"compartment":"cytoskeleton","grade":2},{"compartment":"big_aggregates","grade":1}],"interactors":[{"gene":"ANAPC16","stoichiometry":10.0},{"gene":"ANAPC2","stoichiometry":10.0},{"gene":"CDC16","stoichiometry":10.0},{"gene":"CDC26","stoichiometry":10.0},{"gene":"CDC23","stoichiometry":10.0},{"gene":"ANAPC10","stoichiometry":10.0},{"gene":"ANAPC7","stoichiometry":10.0},{"gene":"ANAPC1","stoichiometry":10.0},{"gene":"ANAPC5","stoichiometry":10.0},{"gene":"CDC27","stoichiometry":10.0}],"url":"https://opencell.sf.czbiohub.org/target/CID000224","total_profiled":1310},"omim":[{"mim_id":"612157","title":"SENTRIN-SPECIFIC PROTEASE FAMILY, MEMBER 1; SENP1","url":"https://www.omim.org/entry/612157"},{"mim_id":"606947","title":"ANAPHASE-PROMOTING COMPLEX, SUBUNIT 4; ANAPC4","url":"https://www.omim.org/entry/606947"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ANAPC4"},"hgnc":{"alias_symbol":["APC4"],"prev_symbol":[]},"alphafold":{"accession":"Q9UJX5","domains":[{"cath_id":"-","chopping":"571-717","consensus_level":"medium","plddt":83.4144,"start":571,"end":717},{"cath_id":"1.20.1270","chopping":"249-541","consensus_level":"medium","plddt":83.3189,"start":249,"end":541}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UJX5","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UJX5-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UJX5-F1-predicted_aligned_error_v6.png","plddt_mean":80.94},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ANAPC4","jax_strain_url":"https://www.jax.org/strain/search?query=ANAPC4"},"sequence":{"accession":"Q9UJX5","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9UJX5.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9UJX5/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UJX5"}},"corpus_meta":[{"pmid":"9469815","id":"PMC_9469815","title":"Identification 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downstream of the checkpoint.\",\n      \"method\": \"Genetic loss-of-function analysis, time-lapse imaging, positional cloning, epistasis with spindle checkpoint mutants in C. elegans\",\n      \"journal\": \"Molecular Biology of the Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with defined phenotype plus genetic epistasis, replicated in other organisms\",\n      \"pmids\": [\"10749938\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Fission yeast APC4 ortholog Cut20 is a component of the APC/cyclosome required for metaphase-anaphase transition; Cut20 interacts closely with Cut4 (APC1) in the assembly process of the cyclosome, and cut20 mutants show hypersensitivity to cellular stressors and are suppressed by PKA-inactivating regulators.\",\n      \"method\": \"Temperature-sensitive mutant screen, sucrose gradient sedimentation, genetic epistasis, and stress sensitivity assays in S. pombe\",\n      \"journal\": \"Genes to Cells\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods in a well-defined genetic system\",\n      \"pmids\": [\"10526233\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Cryo-EM, mass spectrometry, and docking of crystal structures into a pseudo-atomic model showed that APC4 (together with APC1 and APC5) functions as a scaffolding subunit that coordinates the juxtaposition of the catalytic module (APC2/APC11/APC10) and TPR phosphorylation sites relative to co-activator and substrate recognition.\",\n      \"method\": \"Cryo-electron microscopy, mass spectrometry, crystallography, and recombinant APC/C reconstitution\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — structural determination combined with reconstitution, replicated across subsequent structural studies\",\n      \"pmids\": [\"21307936\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Crystal structure of Apc4 revealed a WD40 domain split by a long α-helical domain; within the APC/C context, regions of Apc4 disordered in the crystal assume order through contacts to Apc5, defining the molecular interface between these two platform subunits.\",\n      \"method\": \"X-ray crystallography of Apc4 and fitting into 3.6-Å cryo-EM map of APC/C\",\n      \"journal\": \"Journal of Molecular Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — atomic-resolution crystal structure validated in EM context\",\n      \"pmids\": [\"26343760\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"APC4 is the major SUMO target within the human APC/C complex; SUMOylation occurs at lysines 772 and 798 of APC4, peaks during mitosis, and is critical for timely APC/C activation and anaphase onset, increasing APC/C ubiquitylation activity toward substrates including KIF18B.\",\n      \"method\": \"SUMO site mapping by mass spectrometry, site-directed mutagenesis (K772R/K798R), siRNA knockdown, mitotic timing by live imaging in human cell lines, in vitro ubiquitination assays\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — site identification, mutagenesis, functional readout, multiple orthogonal methods\",\n      \"pmids\": [\"29549242\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Sumoylation of APC4 peaks during mitosis and is critical for timely APC/C activation and anaphase onset; a SUMO-interacting motif (SIM) in the cullin-homology domain of APC2, located near the APC4 sumoylation sites, also contributes to APC/C activation.\",\n      \"method\": \"APC4 SUMO site mutagenesis, co-immunoprecipitation, mitotic timing assays in Xenopus egg extracts and human cells\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — independent replication of APC4 SUMOylation finding with additional SIM discovery, multiple orthogonal methods\",\n      \"pmids\": [\"29517484\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Cryo-EM structure of SUMOylated APC/C showed that SUMOylation on APC4 causes substantial rearrangement of the WHB domain of APC2 (APC2WHB), which reduces the affinity of APC/CCdc20 for the mitotic checkpoint complex (MCC), thereby attenuating MCC-mediated suppression and allowing more efficient ubiquitination of substrates when the SAC is silenced.\",\n      \"method\": \"Reconstitution of SUMOylated APC/C, cryo-EM structure determination, biochemical MCC-binding assays, ubiquitination assays\",\n      \"journal\": \"Cell Reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structure combined with reconstitution and biochemical assays\",\n      \"pmids\": [\"33789095\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Accumulated lactate inhibits the SUMO protease SENP1 by forming a complex with zinc in its active site; SENP1 inhibition stabilizes SUMOylation of APC4 at two residues, which drives UBE2C binding to APC/C, stimulating timed mitotic exit. This mechanism operates at mitotic entry when lactate reaches its apex, and persistent lactate accumulation drives aberrant APC/C remodelling causing mitotic slippage.\",\n      \"method\": \"Systematic proteome-wide lactate-protein interaction screen, biochemical SENP1 inhibition assays with lactate, APC4 SUMOylation site mutagenesis, UBE2C co-immunoprecipitation, mitotic timing assays, pharmacological rescue experiments in human cells\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — multiple orthogonal biochemical and cell biological methods, mechanistic chain from lactate to SENP1 to APC4 SUMOylation to APC/C activity established rigorously\",\n      \"pmids\": [\"36921622\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"During HCMV infection, APC4 and APC5 subunits undergo proteasome-dependent degradation that is temporally associated with APC/C disassembly; this degradation requires viral early gene products but not UL97-mediated phosphorylation of Cdh1.\",\n      \"method\": \"Western blot, proteasome inhibitor treatment, UL97 deletion virus analysis, mass spectrometry of Cdh1 phosphorylation sites in human fibroblasts\",\n      \"journal\": \"Journal of Virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean viral genetics with defined molecular phenotype, single lab\",\n      \"pmids\": [\"20686030\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"HCMV protein pUL21a binds the APC/C and is necessary and sufficient to cause proteasome-dependent degradation of APC4 and APC5, disrupting APC/C assembly; residues Pro109-Arg110 in pUL21a are critical for APC binding and APC4/5 degradation.\",\n      \"method\": \"Proteomics pulldown, expression of pUL21a alone (sufficiency), PR-AA point mutant virus, proteasome inhibitor rescue, Western blot in human cells\",\n      \"journal\": \"PLoS Pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — necessity and sufficiency established with mutagenesis and genetic rescue, multiple orthogonal methods\",\n      \"pmids\": [\"22792066\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"UL21a induces proteasome-dependent degradation of APC1 in addition to APC4 and APC5; furthermore, depletion of any single platform subunit (APC1, APC4, or APC5) or APC8 in uninfected cells triggers a cellular mechanism that co-degrades all three platform subunits, revealing a cellular interdependence for platform subunit stability.\",\n      \"method\": \"siRNA knockdown of individual APC subunits, Western blot, proteasome inhibitor treatment in human cells\",\n      \"journal\": \"Journal of Virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — systematic knockdown across multiple subunits with consistent readout, single lab\",\n      \"pmids\": [\"25903336\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"APC4 (together with APC1, APC5, APC15) forms the APC/C platform subcomplex; the WD40 domain of APC1 within this platform is required to mediate a coactivator-induced conformational change that promotes UbcH10 binding to the catalytic module, activating APC/C ubiquitination initiation activity.\",\n      \"method\": \"Cryo-EM structure of APC/C-Cdh1 with Apc1(WD40) deletion, in vitro ubiquitination assays with UbcH10 and Ube2S\",\n      \"journal\": \"PNAS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structure combined with biochemical deletion mutagenesis and functional assays\",\n      \"pmids\": [\"27601667\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Deletion of APC4 (a core APC/C subunit) in neurons revealed that APC/C ubiquitylates USP1 and controls neurite number; APC4 knockout altered USP1 protein levels and increased the number of primary neurites exiting somata, while APC4 SUMOylation was not required for these neuronal functions.\",\n      \"method\": \"Conditional knockout of APC4 in cultured mouse neurons, Western blot for APC/C substrates, morphometric analysis of neurite number, APC4 SUMOylation site mutant\",\n      \"journal\": \"Frontiers in Molecular Neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean genetic KO with defined cellular phenotype, single lab, moderate mechanistic follow-up\",\n      \"pmids\": [\"38932933\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In Cdc23Y329C/Y329C mice modeling CDC23 variants causing female infertility, APC4 protein levels were reduced alongside CDC23, and oocytes accumulated securin and cyclin B1, demonstrating that APC4 stability is dependent on CDC23 and that APC/C containing normal APC4 is required for oocyte maturation.\",\n      \"method\": \"Mouse knockin model, Western blot for APC4 and APC/C substrates in oocytes, pharmacological rescue with AZ3146\",\n      \"journal\": \"Human Genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo mouse model with biochemical readout, single lab\",\n      \"pmids\": [\"37768355\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"APC4 (ANAPC4) is a core structural scaffolding subunit of the APC/C E3 ubiquitin ligase that, together with APC1 and APC5, forms the platform subcomplex connecting the catalytic module (APC2/APC11) to the TPR lobe; its WD40 domain undergoes mitotically-timed SUMOylation at K772/K798 — stabilized by lactate-mediated inhibition of the SUMO protease SENP1 — which drives UBE2C recruitment, repositions the APC2 WHB domain to reduce MCC affinity, and thereby stimulates APC/C ubiquitin ligase activity to promote timely metaphase-to-anaphase transition.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1998,\n      \"finding\": \"ANAPC4 (APC4) was identified and cloned as one of four previously unknown human APC/C subunits. Unlike APC2 (which contains a cullin-homology region) and APC7 (which contains TPR repeats), APC4 and APC5 share no similarity to proteins of known function, establishing APC4 as a structurally distinct core subunit of the anaphase-promoting complex.\",\n      \"method\": \"Molecular cloning, biochemical purification, sequence analysis\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — original cloning paper, foundational, 211 citations\",\n      \"pmids\": [\"9469815\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"The C. elegans APC4 ortholog EMB-30 is required for metaphase-to-anaphase transitions during both meiosis and mitosis. Loss of emb-30 causes metaphase arrest that is independent of the spindle assembly checkpoint, and reduction of emb-30 activity suppresses the lethality of mdf-1/MAD1 null mutants by delaying anaphase onset.\",\n      \"method\": \"Genetic loss-of-function (emb-30 mutant alleles), time-lapse imaging, positional cloning, genetic epistasis with mdf-1\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple alleles, epistasis, imaging; replicated in ortholog; 113 citations\",\n      \"pmids\": [\"10749938\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Fission yeast Cut20 (APC4 ortholog) is a component of the APC/cyclosome required for metaphase-to-anaphase transition. Cut20 interacts closely with Cut4 during cyclosome assembly; cut20 mutants are hypersensitive to canavanine and CdCl2 and are suppressed by PKA-inactivating regulators, phenotypes shared with cut4 but not cut23.\",\n      \"method\": \"Temperature-sensitive mutant screen, sucrose gradient centrifugation, genetic epistasis, stress sensitivity assays\",\n      \"journal\": \"Genes to cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic and biochemical in yeast ortholog; single lab\",\n      \"pmids\": [\"10526233\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"APC4 (together with APC1 and APC5) forms a subcomplex that can assemble multiubiquitin chains but cannot bind the coactivator CDH1 or ubiquitinate substrates, establishing APC4 as part of a scaffold that connects the catalytic module (APC2/APC11) with the TPR subunits responsible for coactivator binding.\",\n      \"method\": \"Biochemical fractionation of APC/C subcomplexes, in vitro ubiquitination assays, CDH1 binding assays\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted subcomplexes with in vitro activity assays; 172 citations\",\n      \"pmids\": [\"12956947\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"In C. elegans, hypomorphic alleles of emb-30/APC4 suppress mdf-1/MAD1 lethality by delaying anaphase onset and causing securin (IFY-1/Pds1) accumulation, placing APC4 as a rate-limiting component of APC/C activity controlling the timing of anaphase.\",\n      \"method\": \"EMS mutagenesis suppressor screen, time-lapse imaging of embryos, securin accumulation assay\",\n      \"journal\": \"Genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with functional imaging readout; single lab\",\n      \"pmids\": [\"17237515\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Human cytomegalovirus infection causes proteasome-dependent degradation of APC4 and APC5, which is temporally associated with disassembly of the APC/C core complex. This degradation does not require UL97-mediated phosphorylation of Cdh1 and depends on a viral early gene product.\",\n      \"method\": \"Western blot, proteasome inhibitor treatment, infection with UL97 deletion virus, mass spectrometry\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple experimental conditions; single lab\",\n      \"pmids\": [\"20686030\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Crystal structure and cryo-EM analysis of the APC/C revealed that APC4, together with APC1 and APC5, forms a platform subcomplex that coordinates juxtaposition of the catalytic module (APC2, APC11, APC10) with the TPR subcomplex (Cdc16, Cdc23, Cdc27), providing a pseudo-atomic model for APC4's scaffolding role.\",\n      \"method\": \"Recombinant reconstitution, electron microscopy, mass spectrometry, docking of crystallographic and homology models\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — structural reconstitution with multiple orthogonal methods; 137 citations\",\n      \"pmids\": [\"21307936\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"HCMV protein pUL21a directly binds the APC/C and is necessary and sufficient for proteasome-dependent degradation of APC4 and APC5, causing APC/C disruption. A proline-arginine motif at residues 109-110 of pUL21a is critical for APC binding and APC4 degradation.\",\n      \"method\": \"Proteomics, expression of pUL21a mutants, proteasome inhibitor assays, co-immunoprecipitation, point mutant virus construction\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including mutagenesis and viral genetics; strong mechanistic detail\",\n      \"pmids\": [\"22792066\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Crystal structure of APC4 was determined at atomic resolution, revealing that APC4 comprises a WD40 domain split by a long α-helical domain. In the context of the APC/C (fitted to a 3.6-Å cryo-EM map), regions of APC4 disordered in the crystal become ordered through contacts with APC5.\",\n      \"method\": \"Protein crystallography, cryo-EM fitting\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with cryo-EM validation\",\n      \"pmids\": [\"26343760\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"HCMV protein UL21a induces degradation of APC1 in addition to APC4 and APC5. In uninfected cells, depletion of any one of APC1, APC4, APC5, or APC8 triggers a cellular mechanism that specifically reduces levels of all three platform subunits (APC1, APC4, APC5), revealing an interdependency in platform subcomplex stability.\",\n      \"method\": \"siRNA knockdown, Western blot, viral mutant infection\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — siRNA knockdown with consistent readout across multiple subunits; single lab\",\n      \"pmids\": [\"25903336\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"APC4 and APC5 are part of the APC/C platform subcomplex (with APC1 and APC15) that connects the catalytic module to the TPR lobe. The WD40 domain of APC1 mediates coactivator-induced conformational changes that expose the UbcH10-binding site, with APC4/APC5 providing structural scaffolding for this allosteric transition.\",\n      \"method\": \"Crystal structure of APC1 N-terminal domain, cryo-EM of APC/C-Cdh1 deletion mutant, in vitro ubiquitination assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — structure plus biochemical reconstitution with deletion mutant\",\n      \"pmids\": [\"27601667\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"APC4 is the major SUMO target within the APC/C during mitosis in human cells, with SUMO acceptor lysines at positions 772 and 798. SUMOylation of APC4 peaks during mitosis and is critical for timely APC/C activation and anaphase onset, and increases APC/C ubiquitylation activity toward a subset of substrates including KIF18B.\",\n      \"method\": \"SUMO machinery knockdown, site-directed mutagenesis of APC4 lysine residues, ubiquitination assays, live-cell mitotic timing assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mutagenesis of specific sites with functional readouts; replicated in independent study (PMID:29517484)\",\n      \"pmids\": [\"29549242\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"SUMOylation of the APC4 subunit peaks during mitosis and is critical for timely APC/C activation and anaphase onset. A functionally important SUMO-interacting motif (SIM) was identified in the cullin-homology domain of APC2, located near the APC4 SUMOylation sites, suggesting a SUMO-SIM interaction mechanism that regulates APC/C activity.\",\n      \"method\": \"Site-directed mutagenesis of APC4 SUMOylation sites, APC2 SIM identification and mutagenesis, cell-based mitotic progression assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mutagenesis of specific sites with functional readouts in two independent labs (PMID:29549242 and 29517484)\",\n      \"pmids\": [\"29517484\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Cryo-EM structural analysis of SUMOylated APC/C revealed that SUMOylation of APC4 causes a substantial rearrangement of the WHB domain of APC2 (APC2WHB). This repositioning reduces APC/CCdc20 affinity for the mitotic checkpoint complex (MCC), attenuating MCC-mediated suppression and allowing more efficient substrate ubiquitination when the SAC is silenced.\",\n      \"method\": \"Reconstitution of SUMOylated APC/C, cryo-EM, biochemical APC/C activity assays, MCC binding assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structure plus biochemical reconstitution revealing mechanistic basis\",\n      \"pmids\": [\"33789095\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Accumulated lactate inhibits the SUMO protease SENP1 by forming a complex with zinc in the SENP1 active site. SENP1 inhibition stabilizes SUMOylation of APC4 at two specific residues, which drives UBE2C binding to the APC/C, stimulating timed degradation of cell cycle proteins and efficient mitotic exit in proliferating human cells.\",\n      \"method\": \"Systematic proteome-wide lactate-binding screen, biochemical SENP1 inhibition assays, APC4 SUMOylation site mutagenesis, UBE2C co-immunoprecipitation, mitotic exit assays\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — multiple orthogonal methods including biochemical reconstitution, mutagenesis, and functional cell assays; 177 citations\",\n      \"pmids\": [\"36921622\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"In Pqbp1-conditional knockout mice with microcephaly, Apc4 was identified as a hub gene in the network of APC/C-related genes whose expression is disrupted. Exogenous Apc4 expression rescued cell cycle length, proliferation, and cell phenotypes of NSPCs caused by Pqbp1-cKO, demonstrating that Apc4 is a functionally limiting node in APC/C-mediated M-phase progression in neural stem progenitor cells.\",\n      \"method\": \"Conditional knockout mouse model, gene expression profiling, AAV-mediated exogenous Apc4 rescue, cell cycle analysis\",\n      \"journal\": \"Molecular psychiatry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — rescue experiment in vivo; mechanistic detail is partial (hub role, not direct mechanism)\",\n      \"pmids\": [\"25070536\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Deletion of Anapc4 specifically in neurons (conditional KO) revealed that APC/C controls USP1 protein levels in neurons and regulates the number of primary neurites exiting somata. APC4 SUMOylation was found not to impact primary neurite number or USP1 levels, distinguishing the structural APC4 scaffolding function from its SUMOylation-dependent mitotic role.\",\n      \"method\": \"Conditional knockout of Anapc4 in mouse neurons, Western blot for substrates, neurite morphology analysis, comparison with coactivator (Cdh1/Cdc20) perturbations\",\n      \"journal\": \"Frontiers in molecular neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean genetic KO of core subunit with defined cellular phenotype; single lab\",\n      \"pmids\": [\"38932933\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Homozygous missense variants in CDC23 (APC8) that cause female infertility in humans lead to decreased CDC23 and APC4 protein levels and accumulation of securin and cyclin B1 in mouse oocytes, demonstrating that APC4 protein stability depends on the integrity of the APC/C platform and is required for oocyte maturation.\",\n      \"method\": \"Patient variant identification, Cdc23 knockin mice, Western blot for APC4 and substrates, oocyte maturation phenotyping\",\n      \"journal\": \"Human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo mouse model with substrate accumulation readout; establishes APC4 co-dependency\",\n      \"pmids\": [\"37768355\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ANAPC4 (APC4) is a core scaffolding subunit of the APC/C ubiquitin ligase whose WD40 and α-helical domains form part of the platform subcomplex (with APC1 and APC5) that bridges the catalytic module (APC2/APC11) to the TPR coactivator-binding lobe; its protein stability is interdependent with other platform subunits, and its SUMOylation at K772/K798—regulated by the SENP1 SUMO protease (itself inhibited by lactate) and promoted by CDK1—drives UBE2C recruitment and APC/C activation, with the resulting structural rearrangement of APC2's WHB domain attenuating MCC inhibition to ensure timely anaphase onset.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"APC4 (ANAPC4) is a core scaffolding subunit of the anaphase-promoting complex/cyclosome (APC/C), an essential E3 ubiquitin ligase that drives metaphase-to-anaphase transition and mitotic exit by targeting key cell-cycle regulators for proteasomal destruction. APC4 forms, together with APC1 and APC5, the platform subcomplex that bridges the catalytic module (APC2/APC11) to the TPR lobe, and its structural integrity is required for APC/C assembly—loss of any single platform subunit triggers co-degradation of all three [PMID:12956947, PMID:25903336, PMID:26343760]. APC4 contains a WD40 domain that undergoes mitotically timed SUMOylation at K772 and K798; this modification repositions the APC2 WHB domain to reduce affinity for the mitotic checkpoint complex (MCC), promotes UBE2C recruitment, and stimulates APC/C ubiquitin ligase activity required for timely anaphase onset, a process further regulated by lactate-mediated inhibition of the SUMO protease SENP1 [PMID:29549242, PMID:29517484, PMID:33789095, PMID:36921622]. Beyond mitosis, APC4 functions in neuronal morphogenesis where it mediates APC/C-dependent ubiquitylation of USP1 to control neurite number, independently of its SUMOylation [PMID:38932933].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Identification of APC4 as a novel APC/C subunit established that the human anaphase-promoting complex contains additional, previously unknown components beyond those characterized in yeast.\",\n      \"evidence\": \"Biochemical purification and molecular cloning of human APC subunits\",\n      \"pmids\": [\"9469815\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No function assigned beyond complex membership\",\n        \"No structural information on APC4\",\n        \"Relationship to other APC subunits undefined\"\n      ]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Genetic analysis in fission yeast (Cut20) and C. elegans (EMB-30) demonstrated that APC4 orthologs are essential for the metaphase-to-anaphase transition, placing APC4 function downstream of the spindle assembly checkpoint.\",\n      \"evidence\": \"Temperature-sensitive mutants in S. pombe and loss-of-function/epistasis analysis in C. elegans\",\n      \"pmids\": [\"10526233\", \"10749938\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Biochemical mechanism by which APC4 contributes to APC/C activity unknown\",\n        \"Position within APC/C architecture unresolved\"\n      ]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Reconstitution of human APC subcomplexes revealed that APC4 forms a platform subcomplex with APC1 and APC5 that connects the catalytic APC2/APC11 module to the TPR subunits but cannot itself recruit co-activators or ubiquitinate substrates, defining APC4 as a structural scaffold rather than a catalytic participant.\",\n      \"evidence\": \"Biochemical reconstitution of human APC subcomplexes with in vitro ubiquitination and CDH1-binding assays\",\n      \"pmids\": [\"12956947\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Atomic-level contacts between APC4 and neighboring subunits unresolved\",\n        \"Regulatory modifications of APC4 unknown\"\n      ]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Discovery that HCMV targets APC4 and APC5 for proteasome-dependent degradation, later shown to be mediated by the viral protein pUL21a, revealed that the APC/C platform is a viral vulnerability and that platform subunit stability is interdependent—depletion of any one platform subunit triggers co-degradation of all three.\",\n      \"evidence\": \"Western blot and proteasome inhibitor experiments during HCMV infection; pUL21a sufficiency and PR-AA mutagenesis; siRNA knockdown of individual subunits in uninfected cells\",\n      \"pmids\": [\"20686030\", \"22792066\", \"25903336\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"E3 ligase responsible for cellular co-degradation of platform subunits not identified\",\n        \"Structural basis of pUL21a interaction with APC4/APC5 not determined\"\n      ]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Cryo-EM and crystallographic studies progressively resolved APC4's architecture—a WD40 domain split by a helical insertion—and its position within the platform, showing how it coordinates the catalytic module relative to co-activator binding sites.\",\n      \"evidence\": \"Cryo-EM pseudo-atomic model of APC/C; X-ray crystal structure of Apc4 fitted into 3.6-Å EM map\",\n      \"pmids\": [\"21307936\", \"26343760\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Post-translational modifications not visualized in these structures\",\n        \"Dynamic conformational changes during substrate engagement not captured\"\n      ]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Structural and biochemical dissection of the platform showed that the APC1 WD40 domain within the APC4-containing platform mediates a co-activator-induced conformational change required for UbcH10 binding and ubiquitination initiation, placing APC4 in the structural context of catalytic activation.\",\n      \"evidence\": \"Cryo-EM of APC/C-Cdh1 with APC1 WD40 deletion; in vitro ubiquitination assays\",\n      \"pmids\": [\"27601667\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Direct conformational contribution of APC4 versus APC1 WD40 not separated\",\n        \"Role of APC4 modifications in this conformational switch not tested\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Two independent studies established that APC4 is the major SUMO target in the APC/C, with SUMOylation at K772/K798 peaking in mitosis and being critical for timely APC/C activation and anaphase onset; a SUMO-interacting motif in APC2 was identified as the functional reader of this modification.\",\n      \"evidence\": \"SUMO site mapping by mass spectrometry, K772R/K798R mutagenesis, mitotic timing assays in human cells and Xenopus egg extracts, co-immunoprecipitation\",\n      \"pmids\": [\"29549242\", \"29517484\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"SUMO E3 ligase responsible for APC4 modification not identified\",\n        \"Structural mechanism by which SUMOylation activates APC/C not yet resolved\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Cryo-EM of SUMOylated APC/C revealed the structural mechanism: SUMO on APC4 displaces the APC2 WHB domain, reducing MCC affinity and thereby attenuating spindle checkpoint–mediated inhibition to allow efficient substrate ubiquitination.\",\n      \"evidence\": \"Reconstituted SUMOylated APC/C, cryo-EM structure, MCC-binding and ubiquitination assays\",\n      \"pmids\": [\"33789095\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"How SUMOylation is reversed during mitotic exit not addressed\",\n        \"Whether WHB repositioning also affects Ube2S-dependent chain elongation not tested\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Lactate was identified as an endogenous metabolic timer of APC4 SUMOylation: lactate inhibits the SUMO protease SENP1 by complexing with zinc in its active site, stabilizing APC4 SUMOylation at mitotic entry to drive UBE2C recruitment and APC/C activation, while pathological lactate accumulation causes mitotic slippage.\",\n      \"evidence\": \"Proteome-wide lactate interaction screen, biochemical SENP1 inhibition assays, APC4 SUMO mutagenesis, UBE2C co-immunoprecipitation, mitotic timing assays in human cells\",\n      \"pmids\": [\"36921622\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether other SENP family members also contribute to APC4 deSUMOylation not determined\",\n        \"Relevance of lactate-SENP1-APC4 axis in non-cancer contexts not established\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"APC4 protein stability depends on other APC/C subunits in vivo: in a mouse model of CDC23 dysfunction, APC4 levels were reduced alongside CDC23, and oocytes accumulated securin and cyclin B1, linking APC4/platform integrity to oocyte maturation.\",\n      \"evidence\": \"Cdc23 Y329C knockin mice, Western blot for APC4 and APC/C substrates in oocytes\",\n      \"pmids\": [\"37768355\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether APC4 reduction is cause or consequence of APC/C disassembly not fully distinguished\",\n        \"Human relevance of CDC23-dependent APC4 instability not directly tested\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Conditional APC4 knockout in neurons revealed a non-mitotic role: APC/C ubiquitylates USP1 to control neurite number, and this function does not require APC4 SUMOylation, demonstrating that APC4's scaffolding role extends beyond cell-cycle regulation.\",\n      \"evidence\": \"Conditional APC4 knockout in cultured mouse neurons, morphometric analysis, SUMO-site mutant rescue\",\n      \"pmids\": [\"38932933\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether USP1 is a direct APC/C substrate (degron and co-activator dependency) not defined\",\n        \"In vivo neuronal phenotype of APC4 loss not characterized\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The SUMO E3 ligase that modifies APC4, the precise dynamics of APC4 deSUMOylation during mitotic exit, and the full spectrum of non-mitotic APC4 functions (including neuronal and developmental roles) remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Identity of the SUMO E3 ligase for APC4 unknown\",\n        \"Structural basis of lactate-stabilized APC4 SUMOylation effect on APC/C conformation not visualized\",\n        \"Comprehensive identification of APC/C substrates dependent on APC4 integrity in non-dividing cells lacking\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [1, 4, 5, 13]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [6, 7, 8, 9, 14]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [2, 6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [2, 3, 6, 7, 8, 9]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 6, 8, 9, 14]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [14]}\n    ],\n    \"complexes\": [\n      \"APC/C\"\n    ],\n    \"partners\": [\n      \"ANAPC1\",\n      \"ANAPC5\",\n      \"ANAPC2\",\n      \"ANAPC11\",\n      \"CDC20\",\n      \"FZR1\",\n      \"UBE2C\",\n      \"SENP1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"ANAPC4 (APC4) is a core scaffolding subunit of the anaphase-promoting complex/cyclosome (APC/C), a multi-subunit E3 ubiquitin ligase essential for metaphase-to-anaphase transition and mitotic exit. APC4 contains a split WD40 domain interrupted by a long α-helical domain and, together with APC1 and APC5, forms the platform subcomplex that bridges the catalytic module (APC2/APC11) to the TPR coactivator-binding lobe; these platform subunits exhibit co-dependent protein stability [PMID:12956947, PMID:26343760, PMID:25903336]. APC4 is the principal SUMO target within the APC/C during mitosis: SUMOylation at K772 and K798—promoted by CDK1 activity and stabilized by lactate-mediated inhibition of the SUMO protease SENP1—drives UBE2C recruitment, repositions the APC2 WHB domain to attenuate mitotic checkpoint complex (MCC) inhibition, and is required for timely anaphase onset [PMID:29549242, PMID:29517484, PMID:33789095, PMID:36921622]. Beyond cell division, APC4 functions in postmitotic neurons where its scaffolding role—independent of SUMOylation—controls USP1 levels and primary neurite number [PMID:38932933].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Identification and cloning of APC4 as a novel subunit of the human APC/C established it as a structurally distinct component lacking homology to known functional domains, opening the question of what role it plays in the complex.\",\n      \"evidence\": \"Biochemical purification and molecular cloning of four new APC/C subunits from human cells\",\n      \"pmids\": [\"9469815\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No functional data for APC4 beyond complex membership\", \"Domain architecture unknown\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Genetic studies in fission yeast (Cut20) and C. elegans (EMB-30) demonstrated that APC4 orthologs are essential for the metaphase-to-anaphase transition, establishing a conserved requirement for this subunit in APC/C-dependent cell division across eukaryotes.\",\n      \"evidence\": \"Temperature-sensitive mutants in S. pombe and loss-of-function alleles in C. elegans with metaphase arrest phenotypes and genetic epistasis with spindle checkpoint components\",\n      \"pmids\": [\"10526233\", \"10749938\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of APC4 contribution to APC/C activity not resolved\", \"Direct biochemical function not addressed\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Biochemical reconstitution revealed that APC4 forms a subcomplex with APC1 and APC5 that supports multiubiquitin chain formation but cannot bind coactivators, defining APC4's role as a scaffold bridging the catalytic and coactivator-binding modules.\",\n      \"evidence\": \"Fractionation of APC/C subcomplexes with in vitro ubiquitination and CDH1 binding assays\",\n      \"pmids\": [\"12956947\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structural information on how APC4 interfaces with adjacent modules\", \"APC4 contacts with APC2/APC11 not mapped\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Cryo-EM and crystal structures provided the first pseudo-atomic model of APC4 within the assembled APC/C, showing it coordinates the platform that juxtaposes the catalytic and TPR modules, and later revealing a split WD40 domain stabilized by contacts with APC5.\",\n      \"evidence\": \"Recombinant APC/C reconstitution, electron microscopy with docked crystal/homology models; subsequent APC4 crystal structure at atomic resolution fitted to 3.6-Å cryo-EM map\",\n      \"pmids\": [\"21307936\", \"26343760\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Flexible regions of APC4 not resolved in crystal structure\", \"Structural basis for how APC4 communicates with the catalytic module remained unclear\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"The discovery that HCMV protein pUL21a directly targets APC4 and APC5 for proteasomal degradation, and that siRNA knockdown of any single platform subunit destabilizes all three (APC1/APC4/APC5), revealed an interdependent stability mechanism within the platform subcomplex.\",\n      \"evidence\": \"Viral mutant genetics, co-immunoprecipitation, proteasome inhibitor assays, siRNA knockdown of individual subunits\",\n      \"pmids\": [\"20686030\", \"22792066\", \"25903336\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of co-dependent degradation not identified\", \"Whether platform destabilization also affects catalytic subunit levels not tested\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"In a Pqbp1-knockout microcephaly model, APC4 was identified as a functionally limiting node whose exogenous expression rescued neural stem progenitor cell cycle defects, linking APC4 dosage to brain development.\",\n      \"evidence\": \"Conditional knockout mouse model, AAV-mediated APC4 rescue, cell cycle analysis in NSPCs\",\n      \"pmids\": [\"25070536\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct mechanism by which reduced APC4 causes NSPC phenotypes not established\", \"Whether APC4 is the sole limiting subunit in this context unclear\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Two independent studies established that APC4 is the major SUMO target on the APC/C during mitosis, with K772 and K798 as the acceptor sites, and that this modification is required for timely APC/C activation and anaphase onset; a SUMO-interacting motif on APC2 was identified as the likely intramolecular receptor.\",\n      \"evidence\": \"Site-directed mutagenesis of APC4 SUMO sites and APC2 SIM, live-cell mitotic timing assays, ubiquitination assays in two independent laboratories\",\n      \"pmids\": [\"29549242\", \"29517484\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of SUMO-SIM interaction within the complex not resolved\", \"Upstream regulation of APC4 SUMOylation kinetics not fully defined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Cryo-EM of SUMOylated APC/C revealed that APC4 SUMOylation repositions the APC2 WHB domain, reducing MCC affinity and thereby attenuating spindle assembly checkpoint inhibition—providing the structural mechanism for how SUMOylation activates APC/C at anaphase onset.\",\n      \"evidence\": \"Reconstituted SUMOylated APC/C, cryo-EM, MCC binding and ubiquitination assays\",\n      \"pmids\": [\"33789095\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether WHB repositioning also affects non-MCC substrates differentially is unknown\", \"In vivo validation of the conformational change not performed\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Lactate was identified as a physiological regulator of APC4 SUMOylation through inhibition of the SUMO protease SENP1 via zinc complexation, connecting cellular metabolic state to APC/C activation timing and mitotic exit.\",\n      \"evidence\": \"Proteome-wide lactate-binding screen, SENP1 inhibition assays, APC4 SUMOylation site mutagenesis, UBE2C co-immunoprecipitation, mitotic exit assays\",\n      \"pmids\": [\"36921622\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether lactate-SENP1 axis acts on APC4 SUMOylation in all tissue contexts is untested\", \"Quantitative relationship between lactate concentration and mitotic timing not established in vivo\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Conditional deletion of Anapc4 in postmitotic neurons demonstrated a SUMOylation-independent scaffolding function controlling USP1 levels and neurite number, distinguishing APC4's structural role from its mitosis-specific SUMOylation-dependent regulatory function.\",\n      \"evidence\": \"Neuron-specific Anapc4 conditional knockout in mice, comparison of SUMOylation-deficient and null phenotypes, neurite morphology quantification\",\n      \"pmids\": [\"38932933\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether APC4 scaffolding controls USP1 through direct ubiquitination or indirectly is unresolved\", \"Coactivator dependence (Cdh1 vs Cdc20) for the neuronal phenotype not fully clarified\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key open questions include the structural basis of the APC4-SUMO/APC2-SIM interaction at atomic resolution, identification of the full substrate repertoire whose ubiquitination specifically depends on APC4 SUMOylation, and the molecular mechanism by which APC4 loss destabilizes APC1/APC5 and whether this involves a dedicated quality-control pathway.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No atomic-resolution structure of SUMO-modified APC4 in complex with APC2 SIM\", \"Full set of SUMOylation-dependent APC/C substrates not cataloged\", \"Degradation pathway for orphan platform subunits not identified\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [3, 6, 8, 10]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [3, 10, 13]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [11, 12]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [6, 14]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0, 1, 3, 11, 12, 13, 14]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [3, 11, 13, 14]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [15, 16]}\n    ],\n    \"complexes\": [\n      \"APC/C\"\n    ],\n    \"partners\": [\n      \"ANAPC1\",\n      \"ANAPC5\",\n      \"ANAPC2\",\n      \"CDC23\",\n      \"UBE2C\",\n      \"SENP1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}