{"gene":"ANAPC16","run_date":"2026-04-28T17:12:37","timeline":{"discoveries":[{"year":2010,"finding":"APC16 was identified as a bona fide subunit of the human APC/C E3 ubiquitin ligase. It is present in APC/C complexes throughout the cell cycle, its depletion phenocopies depletion of other APC/C subunits, and it is required for APC/C activity toward mitotic substrates. Sequence homologues exist in metazoans but not fungi, and the C. elegans gene K10D2.4 and D. rerio zgc:110659 were identified as functional equivalents.","method":"Tandem-affinity purification, co-immunoprecipitation, RNAi knockdown with mitotic substrate ubiquitination assays, cross-species functional complementation","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 — reciprocal co-IP, clean KD with defined cellular phenotype and biochemical activity readout, replicated across species","pmids":["20392738"],"is_preprint":false},{"year":2010,"finding":"APC16 was discovered as a previously unknown, evolutionarily conserved subunit of the anaphase-promoting complex through systematic TAP-MS of human protein complexes, alongside identification of other novel APC/C subunits essential for chromosome segregation.","method":"Gene tagging on bacterial artificial chromosomes, tandem-affinity purification coupled to mass spectrometry, protein localization","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 — systematic TAP-MS with independent validation, large-scale study","pmids":["20360068"],"is_preprint":false},{"year":2011,"finding":"The C. elegans gene emb-1 encodes the APC16 ortholog (K10D2.4). Temperature-sensitive emb-1 mutants arrest as one-cell embryos in metaphase of meiosis I, phenotypically indistinguishable from depletion of other APC/C subunits. The emb-1 phenotype is enhanced in double mutants with known APC/C subunits and suppressed by known APC/C suppressors, establishing epistatic placement of APC16 within the APC/C pathway. emb-1/APC16 is also required for mitotic proliferation of the germline.","method":"Temperature-sensitive mutant analysis, genetic epistasis (double mutant combinations with APC/C subunits and suppressors), germline proliferation assays","journal":"Genetics","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis with multiple APC/C subunits and suppressors, defined meiotic and mitotic phenotypes","pmids":["21775471"],"is_preprint":false},{"year":2014,"finding":"Crystal structures of human APC3Δloop alone and in complex with the C-terminal domain of APC16 were determined. The structures show that one APC16 binds asymmetrically to the symmetric APC3 homodimer. APC16 recruits APC7 to APC3. APC3's C-terminal domain is rearranged in the full APC/C assembly relative to the isolated subcomplex. Biochemical mapping defined the basis for APC3 assembly with APC16 and APC7, and APC3's loop phosphorylation and IR tail binding surfaces were tested for roles in APC/C-catalyzed ubiquitination.","method":"X-ray crystallography, biochemical interaction mapping (pulldowns, mutagenesis), in vitro ubiquitination assays","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 — crystal structure combined with mutagenesis and in vitro ubiquitination assay","pmids":["25490258"],"is_preprint":false},{"year":2014,"finding":"Cryo-EM reconstruction of the human APC/C at 7.4 Å resolution determined the complete secondary structural architecture and definitive location of all 20 APC/C subunits within the 1.2 MDa assembly, including APC16, revealing the positions and folds of structurally uncharacterized subunits.","method":"Cryo-electron microscopy reconstruction at 7.4 Å resolution","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 — near-atomic cryo-EM structure of the full complex","pmids":["25043029"],"is_preprint":false},{"year":2015,"finding":"Atomic structures of APC/C-coactivator complexes by cryo-EM defined the architecture of all APC/C subunits including APC16 within the Arc Lamp subcomplex, and revealed the mechanism of Emi1-mediated inhibition and the initiating ubiquitination reaction with UbcH10-ubiquitin.","method":"Cryo-electron microscopy atomic structure determination","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 — atomic-resolution cryo-EM structure with functional validation","pmids":["26083744"],"is_preprint":false},{"year":2016,"finding":"Cryo-EM and biochemical analysis revealed that APC3's heavily phosphorylated loop recruits Cdk-cyclin-Cks to enable hyperphosphorylation of an auto-inhibitory segment of Apc1, which relieves auto-inhibition of the APC/C to allow Cdc20 binding. APC16 is part of the Arc Lamp containing APC3, contextualizing the role of the APC3 loop phosphorylation in overall APC/C activation.","method":"Cryo-electron microscopy, biochemical phosphorylation assays, mutagenesis","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 — cryo-EM structure combined with biochemical analysis and mutagenesis","pmids":["27120157"],"is_preprint":false}],"current_model":"ANAPC16 is a metazoan-specific subunit of the anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase that resides in the Arc Lamp subcomplex, where it binds asymmetrically to the APC3 homodimer (via its C-terminal domain, as defined by crystal structure) to recruit APC7 to APC3, is required throughout the cell cycle for APC/C ubiquitination activity toward mitotic substrates, and is essential for meiotic and mitotic progression in metazoans."},"narrative":{"teleology":[{"year":2010,"claim":"The identity of ANAPC16 as a bona fide APC/C subunit was established, answering whether the holoenzyme contained additional uncharacterized components; its depletion abolished APC/C ubiquitination activity and phenocopied loss of core subunits, demonstrating functional necessity.","evidence":"TAP-MS identification, reciprocal co-IP, RNAi knockdown with mitotic substrate ubiquitination readouts, and cross-species complementation in human, C. elegans, and zebrafish cells","pmids":["20392738","20360068"],"confidence":"High","gaps":["Binding interface between APC16 and other APC/C subunits was unresolved","Whether APC16 has a specific role beyond structural integrity of the complex was unknown"]},{"year":2011,"claim":"Genetic epistasis in C. elegans placed APC16 definitively within the APC/C pathway, revealing that it is required for both meiotic progression and mitotic germline proliferation — extending the functional requirement beyond somatic mitosis.","evidence":"Temperature-sensitive emb-1/APC16 mutants, double mutant combinations with known APC/C subunits and suppressors, meiosis I arrest and germline proliferation assays","pmids":["21775471"],"confidence":"High","gaps":["Whether APC16 contributes to substrate recognition or only to complex assembly was unknown","Structural basis of APC16 integration into the holoenzyme remained unresolved"]},{"year":2014,"claim":"Crystal structure and cryo-EM resolved how APC16 integrates into the APC/C: one APC16 binds asymmetrically to the symmetric APC3 homodimer via its C-terminal domain and recruits APC7, defining a specific architectural role within the Arc Lamp subcomplex.","evidence":"X-ray crystallography of APC3–APC16 C-terminal domain complex, 7.4 Å cryo-EM reconstruction of the full 1.2 MDa APC/C, mutagenesis, and in vitro ubiquitination assays","pmids":["25490258","25043029"],"confidence":"High","gaps":["How APC16's asymmetric binding relates to the asymmetric catalytic cycle of the APC/C was not established","Whether APC16 influences substrate selectivity or only holoenzyme assembly was unresolved"]},{"year":2015,"claim":"Atomic-resolution cryo-EM of APC/C–coactivator complexes placed APC16 in full mechanistic context, revealing the overall architecture that enables Emi1-mediated inhibition and UbcH10-dependent ubiquitin transfer.","evidence":"Atomic-resolution cryo-EM of APC/C–Cdh1–Emi1 and APC/C–UbcH10–Ub complexes","pmids":["26083744"],"confidence":"High","gaps":["Direct contribution of APC16 to catalytic steps (E2 positioning, substrate handoff) versus purely structural scaffolding was not dissected"]},{"year":2016,"claim":"The phosphorylation-driven activation mechanism of APC/C was elucidated in the context of the Arc Lamp subcomplex containing APC16: Cdk–cyclin–Cks recruited by the APC3 phospho-loop hyperphosphorylates an auto-inhibitory segment of APC1 to enable Cdc20 binding.","evidence":"Cryo-EM, biochemical phosphorylation assays, and mutagenesis of APC3 loop and APC1 auto-inhibitory segment","pmids":["27120157"],"confidence":"High","gaps":["Whether APC16 modulates the phosphorylation-dependent conformational switch in APC3/APC1 is unknown","No post-translational modifications of APC16 itself have been functionally characterized"]},{"year":null,"claim":"It remains unknown whether APC16 plays any role beyond structural scaffolding — for example, whether it directly influences substrate recognition, E2 engagement, or processivity of polyubiquitin chain elongation.","evidence":"","pmids":[],"confidence":"High","gaps":["No separation-of-function mutations distinguishing APC16's architectural role from potential regulatory roles have been reported","No post-translational regulation of APC16 has been characterized","Functional consequence of the asymmetric APC16 stoichiometry on the symmetric APC3 homodimer is unexplained"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,3]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[1]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0,2,6]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,3,5]}],"complexes":["APC/C"],"partners":["ANAPC3","ANAPC7"],"other_free_text":[]},"mechanistic_narrative":"ANAPC16 is a metazoan-specific subunit of the anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase that controls mitotic and meiotic progression by ubiquitinating cell-cycle substrates. Present in APC/C complexes throughout the cell cycle, ANAPC16 resides in the Arc Lamp subcomplex where its C-terminal domain binds asymmetrically to the APC3 homodimer and recruits APC7 to APC3, as defined by crystal structure and cryo-EM [PMID:25490258, PMID:26083744]. Depletion of ANAPC16 phenocopies loss of other APC/C subunits, abolishing APC/C ubiquitination activity toward mitotic substrates in human cells, and temperature-sensitive mutants of the C. elegans ortholog emb-1 arrest in meiosis I metaphase and fail in germline mitotic proliferation [PMID:20392738, PMID:21775471]. ANAPC16 is conserved across metazoans but absent in fungi, and cross-species complementation confirms functional equivalence of vertebrate and invertebrate orthologs [PMID:20392738]."},"prefetch_data":{"uniprot":{"accession":"Q96DE5","full_name":"Anaphase-promoting complex subunit 16","aliases":["Cyclosome subunit 16"],"length_aa":110,"mass_kda":11.7,"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:20360068). 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:20360068). The APC/C complex catalyzes assembly of branched 'Lys-11'-/'Lys-48'-linked branched ubiquitin chains on target proteins (PubMed:29033132)","subcellular_location":"Cytoplasm; Nucleus; Chromosome, centromere, kinetochore","url":"https://www.uniprot.org/uniprotkb/Q96DE5/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ANAPC16","classification":"Not Classified","n_dependent_lines":4,"n_total_lines":1208,"dependency_fraction":0.0033112582781456954},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000166295","cell_line_id":"CID000222","localizations":[{"compartment":"cytoplasmic","grade":3},{"compartment":"nucleoplasm","grade":3},{"compartment":"cytoskeleton","grade":1}],"interactors":[{"gene":"NEK2","stoichiometry":10.0},{"gene":"CDC27","stoichiometry":10.0},{"gene":"ANAPC5","stoichiometry":10.0},{"gene":"ANAPC13","stoichiometry":10.0},{"gene":"ANAPC1","stoichiometry":10.0},{"gene":"ANAPC10","stoichiometry":10.0},{"gene":"ANAPC4","stoichiometry":10.0},{"gene":"ANAPC7","stoichiometry":10.0},{"gene":"CDC23","stoichiometry":10.0},{"gene":"CDC26","stoichiometry":10.0}],"url":"https://opencell.sf.czbiohub.org/target/CID000222","total_profiled":1310},"omim":[{"mim_id":"613427","title":"ANAPHASE-PROMOTING COMPLEX, SUBUNIT 16; ANAPC16","url":"https://www.omim.org/entry/613427"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ANAPC16"},"hgnc":{"alias_symbol":["bA570G20.3","FLJ33728","APC16","CENP-27"],"prev_symbol":["C10orf104"]},"alphafold":{"accession":"Q96DE5","domains":[{"cath_id":"1.20.5","chopping":"48-94","consensus_level":"medium","plddt":93.7343,"start":48,"end":94}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96DE5","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96DE5-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96DE5-F1-predicted_aligned_error_v6.png","plddt_mean":71.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ANAPC16","jax_strain_url":"https://www.jax.org/strain/search?query=ANAPC16"},"sequence":{"accession":"Q96DE5","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96DE5.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96DE5/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96DE5"}},"corpus_meta":[{"pmid":"25490258","id":"PMC_25490258","title":"Structure of an APC3-APC16 complex: insights into assembly of the anaphase-promoting complex/cyclosome.","date":"2014","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/25490258","citation_count":31,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"20392738","id":"PMC_20392738","title":"APC16 is a conserved subunit of the anaphase-promoting complex/cyclosome.","date":"2010","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/20392738","citation_count":26,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"32286337","id":"PMC_32286337","title":"Alkyl-carbon chain length of two distinct compounds and derivatives are key determinants of their anti-Acanthamoeba activities.","date":"2020","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/32286337","citation_count":13,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"21775471","id":"PMC_21775471","title":"emb-1 encodes the APC16 subunit of the Caenorhabditis elegans anaphase-promoting complex.","date":"2011","source":"Genetics","url":"https://pubmed.ncbi.nlm.nih.gov/21775471","citation_count":12,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"33479685","id":"PMC_33479685","title":"Drug combinations as effective anti-leishmanials against drug resistant Leishmania mexicana.","date":"2020","source":"RSC medicinal chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/33479685","citation_count":8,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"39774722","id":"PMC_39774722","title":"A genome-wide scan of non-coding RNAs and enhancers for refractive error and myopia.","date":"2025","source":"Human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/39774722","citation_count":2,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"35924985","id":"PMC_35924985","title":"Correlations of expression of nuclear and mitochondrial genes in triploid fish.","date":"2022","source":"G3 (Bethesda, Md.)","url":"https://pubmed.ncbi.nlm.nih.gov/35924985","citation_count":0,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"12477932","id":"PMC_12477932","title":"Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences.","date":"2002","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/12477932","citation_count":1479,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"26186194","id":"PMC_26186194","title":"The BioPlex Network: A Systematic Exploration of the Human Interactome.","date":"2015","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/26186194","citation_count":1118,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"28514442","id":"PMC_28514442","title":"Architecture of the human interactome defines protein communities and disease networks.","date":"2017","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/28514442","citation_count":1085,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"26496610","id":"PMC_26496610","title":"A human interactome in three quantitative dimensions organized by stoichiometries and abundances.","date":"2015","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/26496610","citation_count":1015,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"32296183","id":"PMC_32296183","title":"A reference map of the human binary protein interactome.","date":"2020","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/32296183","citation_count":849,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"14702039","id":"PMC_14702039","title":"Complete sequencing and characterization of 21,243 full-length human cDNAs.","date":"2003","source":"Nature genetics","url":"https://pubmed.ncbi.nlm.nih.gov/14702039","citation_count":754,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"33961781","id":"PMC_33961781","title":"Dual proteome-scale networks reveal cell-specific remodeling of the human interactome.","date":"2021","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/33961781","citation_count":705,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"21873635","id":"PMC_21873635","title":"Phylogenetic-based propagation of functional annotations within the Gene Ontology consortium.","date":"2011","source":"Briefings in bioinformatics","url":"https://pubmed.ncbi.nlm.nih.gov/21873635","citation_count":656,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"15489334","id":"PMC_15489334","title":"The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).","date":"2004","source":"Genome research","url":"https://pubmed.ncbi.nlm.nih.gov/15489334","citation_count":438,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"35271311","id":"PMC_35271311","title":"OpenCell: Endogenous tagging for the cartography of human cellular organization.","date":"2022","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/35271311","citation_count":432,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"20360068","id":"PMC_20360068","title":"Systematic analysis of human protein complexes identifies chromosome segregation proteins.","date":"2010","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/20360068","citation_count":421,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"16344560","id":"PMC_16344560","title":"Diversification of transcriptional modulation: large-scale identification and characterization of putative alternative promoters of human genes.","date":"2005","source":"Genome research","url":"https://pubmed.ncbi.nlm.nih.gov/16344560","citation_count":409,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"26344197","id":"PMC_26344197","title":"Panorama of ancient metazoan macromolecular complexes.","date":"2015","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/26344197","citation_count":407,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"20813266","id":"PMC_20813266","title":"The protein composition of mitotic chromosomes determined using multiclassifier combinatorial proteomics.","date":"2010","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/20813266","citation_count":266,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"29033132","id":"PMC_29033132","title":"Assembly and Function of Heterotypic Ubiquitin Chains in Cell-Cycle and Protein Quality Control.","date":"2017","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/29033132","citation_count":255,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"15678131","id":"PMC_15678131","title":"The anaphase-promoting complex: a key factor in the regulation of cell cycle.","date":"2005","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/15678131","citation_count":211,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"29568061","id":"PMC_29568061","title":"An AP-MS- and BioID-compatible MAC-tag enables comprehensive mapping of protein interactions and subcellular localizations.","date":"2018","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/29568061","citation_count":201,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"26083744","id":"PMC_26083744","title":"Atomic structure of the APC/C and its mechanism of protein ubiquitination.","date":"2015","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/26083744","citation_count":187,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"23508102","id":"PMC_23508102","title":"Quantitative dissection and stoichiometry determination of the human SET1/MLL histone methyltransferase complexes.","date":"2013","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/23508102","citation_count":180,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"25043029","id":"PMC_25043029","title":"Molecular architecture and mechanism of the anaphase-promoting complex.","date":"2014","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/25043029","citation_count":175,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"25544563","id":"PMC_25544563","title":"Global mapping of herpesvirus-host protein complexes reveals a transcription strategy for late genes.","date":"2014","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/25544563","citation_count":173,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"30804502","id":"PMC_30804502","title":"H4K20me0 recognition by BRCA1-BARD1 directs homologous recombination to sister chromatids.","date":"2019","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/30804502","citation_count":162,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"27120157","id":"PMC_27120157","title":"Molecular mechanism of APC/C activation by mitotic phosphorylation.","date":"2016","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/27120157","citation_count":154,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"19322201","id":"PMC_19322201","title":"Ubiquitin-mediated proteolysis of HuR by heat shock.","date":"2009","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/19322201","citation_count":142,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"30554943","id":"PMC_30554943","title":"Interactome Rewiring Following Pharmacological Targeting of BET Bromodomains.","date":"2018","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/30554943","citation_count":136,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"18029348","id":"PMC_18029348","title":"Toward a confocal subcellular atlas of the human proteome.","date":"2007","source":"Molecular & cellular proteomics : MCP","url":"https://pubmed.ncbi.nlm.nih.gov/18029348","citation_count":114,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"34591612","id":"PMC_34591612","title":"A protein interaction landscape of breast cancer.","date":"2021","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/34591612","citation_count":111,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"22990118","id":"PMC_22990118","title":"Charting the landscape of tandem BRCT domain-mediated protein interactions.","date":"2012","source":"Science signaling","url":"https://pubmed.ncbi.nlm.nih.gov/22990118","citation_count":92,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"22193957","id":"PMC_22193957","title":"BubR1 blocks substrate recruitment to the APC/C in a KEN-box-dependent manner.","date":"2011","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/22193957","citation_count":90,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"29229926","id":"PMC_29229926","title":"Cell cycle-dependent phosphorylation regulates RECQL4 pathway choice and ubiquitination in DNA double-strand break repair.","date":"2017","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/29229926","citation_count":89,"is_preprint":false,"source_track":"gene2pubmed"}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":4897,"output_tokens":1172,"usd":0.016136},"stage2":{"model":"claude-opus-4-6","input_tokens":4391,"output_tokens":1263,"usd":0.080295},"total_usd":0.243819,"stage1_batch_id":"msgbatch_01TqNM6p6FsoMBT2jzQTwKBr","stage2_batch_id":"msgbatch_01Ya473eco8sTHbvRymQaU8o","note":"batch pricing = 50% of standard","round2_stage1":{"model":"claude-sonnet-4-6","input_tokens":17089,"output_tokens":1794,"usd":0.039088},"round2_rules_fired":"R3","round2_stage2":{"model":"claude-opus-4-6","input_tokens":5300,"output_tokens":1828,"usd":0.1083}},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2014,\n      \"finding\": \"Crystal structure of APC3Δloop alone and in complex with the C-terminal domain of APC16 revealed that one APC16 binds asymmetrically to the symmetric APC3 homodimer, and that APC16 recruits APC7 to APC3 within the APC/C Arc Lamp subcomplex.\",\n      \"method\": \"X-ray crystallography, biochemical mapping, mutagenesis of APC3 surfaces\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with functional biochemical validation and mutagenesis\",\n      \"pmids\": [\"25490258\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"APC3's loop region (>200 residues, heavily phosphorylated during mitosis) and its IR-tail binding cleft (which recruits CDC20/CDH1 coactivators) were shown to be important for APC/C-catalyzed ubiquitination, placing APC16 in the context of APC3-mediated regulation of coactivator binding.\",\n      \"method\": \"In vitro ubiquitination assays, mutagenesis of APC3 IR-tail binding surface\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstituted ubiquitination assay with mutagenesis in a single rigorous study\",\n      \"pmids\": [\"25490258\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"APC16 was identified as a bona fide subunit of the human APC/C, present throughout the cell cycle, required for APC/C ubiquitin ligase activity toward mitotic substrates, and found in association with mitotic checkpoint complex protein complexes by tandem-affinity purification.\",\n      \"method\": \"Tandem-affinity purification, co-immunoprecipitation, siRNA depletion with mitotic substrate ubiquitination assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-purification plus functional KD phenotype with defined substrate readout, replicated across organisms\",\n      \"pmids\": [\"20392738\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"APC16 depletion phenocopies depletion of other APC/C subunits, demonstrating a direct role in APC/C-dependent cell cycle progression and chromosome segregation.\",\n      \"method\": \"siRNA knockdown with cell cycle and mitotic phenotype analysis\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean KD with defined cellular phenotype but single laboratory\",\n      \"pmids\": [\"20392738\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"The C. elegans gene emb-1 (K10D2.4) encodes the APC16 ortholog; temperature-sensitive emb-1 mutants arrest in metaphase of meiosis I, phenocopying loss of other APC/C subunits, and emb-1 is also required for mitotic germline proliferation. Genetic epistasis showed emb-1 phenotype is enhanced by APC/C subunit mutations and suppressed by APC/C suppressor mutations.\",\n      \"method\": \"Genetic epistasis (double mutant analysis), temperature-sensitive allele characterization, C. elegans embryo arrest assay\",\n      \"journal\": \"Genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with multiple allele combinations confirming pathway position\",\n      \"pmids\": [\"21775471\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ANAPC16 (APC16) is a conserved metazoan-specific subunit of the APC/C E3 ubiquitin ligase that binds asymmetrically to the APC3 homodimer via its C-terminal domain, thereby recruiting APC7 to the Arc Lamp subcomplex; it is required throughout the cell cycle for APC/C integrity and ubiquitin ligase activity toward mitotic substrates, and loss of APC16 in human cells or its C. elegans ortholog EMB-1 causes metaphase arrest phenocopying loss of core APC/C subunits.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2010,\n      \"finding\": \"APC16 was identified as a bona fide subunit of the human APC/C E3 ubiquitin ligase. It is present in APC/C complexes throughout the cell cycle, its depletion phenocopies depletion of other APC/C subunits, and it is required for APC/C activity toward mitotic substrates. Sequence homologues exist in metazoans but not fungi, and the C. elegans gene K10D2.4 and D. rerio zgc:110659 were identified as functional equivalents.\",\n      \"method\": \"Tandem-affinity purification, co-immunoprecipitation, RNAi knockdown with mitotic substrate ubiquitination assays, cross-species functional complementation\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP, clean KD with defined cellular phenotype and biochemical activity readout, replicated across species\",\n      \"pmids\": [\"20392738\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"APC16 was discovered as a previously unknown, evolutionarily conserved subunit of the anaphase-promoting complex through systematic TAP-MS of human protein complexes, alongside identification of other novel APC/C subunits essential for chromosome segregation.\",\n      \"method\": \"Gene tagging on bacterial artificial chromosomes, tandem-affinity purification coupled to mass spectrometry, protein localization\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — systematic TAP-MS with independent validation, large-scale study\",\n      \"pmids\": [\"20360068\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"The C. elegans gene emb-1 encodes the APC16 ortholog (K10D2.4). Temperature-sensitive emb-1 mutants arrest as one-cell embryos in metaphase of meiosis I, phenotypically indistinguishable from depletion of other APC/C subunits. The emb-1 phenotype is enhanced in double mutants with known APC/C subunits and suppressed by known APC/C suppressors, establishing epistatic placement of APC16 within the APC/C pathway. emb-1/APC16 is also required for mitotic proliferation of the germline.\",\n      \"method\": \"Temperature-sensitive mutant analysis, genetic epistasis (double mutant combinations with APC/C subunits and suppressors), germline proliferation assays\",\n      \"journal\": \"Genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with multiple APC/C subunits and suppressors, defined meiotic and mitotic phenotypes\",\n      \"pmids\": [\"21775471\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Crystal structures of human APC3Δloop alone and in complex with the C-terminal domain of APC16 were determined. The structures show that one APC16 binds asymmetrically to the symmetric APC3 homodimer. APC16 recruits APC7 to APC3. APC3's C-terminal domain is rearranged in the full APC/C assembly relative to the isolated subcomplex. Biochemical mapping defined the basis for APC3 assembly with APC16 and APC7, and APC3's loop phosphorylation and IR tail binding surfaces were tested for roles in APC/C-catalyzed ubiquitination.\",\n      \"method\": \"X-ray crystallography, biochemical interaction mapping (pulldowns, mutagenesis), in vitro ubiquitination assays\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure combined with mutagenesis and in vitro ubiquitination assay\",\n      \"pmids\": [\"25490258\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Cryo-EM reconstruction of the human APC/C at 7.4 Å resolution determined the complete secondary structural architecture and definitive location of all 20 APC/C subunits within the 1.2 MDa assembly, including APC16, revealing the positions and folds of structurally uncharacterized subunits.\",\n      \"method\": \"Cryo-electron microscopy reconstruction at 7.4 Å resolution\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — near-atomic cryo-EM structure of the full complex\",\n      \"pmids\": [\"25043029\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Atomic structures of APC/C-coactivator complexes by cryo-EM defined the architecture of all APC/C subunits including APC16 within the Arc Lamp subcomplex, and revealed the mechanism of Emi1-mediated inhibition and the initiating ubiquitination reaction with UbcH10-ubiquitin.\",\n      \"method\": \"Cryo-electron microscopy atomic structure determination\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — atomic-resolution cryo-EM structure with functional validation\",\n      \"pmids\": [\"26083744\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Cryo-EM and biochemical analysis revealed that APC3's heavily phosphorylated loop recruits Cdk-cyclin-Cks to enable hyperphosphorylation of an auto-inhibitory segment of Apc1, which relieves auto-inhibition of the APC/C to allow Cdc20 binding. APC16 is part of the Arc Lamp containing APC3, contextualizing the role of the APC3 loop phosphorylation in overall APC/C activation.\",\n      \"method\": \"Cryo-electron microscopy, biochemical phosphorylation assays, mutagenesis\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structure combined with biochemical analysis and mutagenesis\",\n      \"pmids\": [\"27120157\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ANAPC16 is a metazoan-specific subunit of the anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase that resides in the Arc Lamp subcomplex, where it binds asymmetrically to the APC3 homodimer (via its C-terminal domain, as defined by crystal structure) to recruit APC7 to APC3, is required throughout the cell cycle for APC/C ubiquitination activity toward mitotic substrates, and is essential for meiotic and mitotic progression in metazoans.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"ANAPC16 (APC16) is a conserved metazoan-specific subunit of the anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase, required for APC/C integrity and ubiquitin ligase activity throughout the cell cycle [PMID:20392738]. Crystallographic analysis shows that one APC16 molecule binds asymmetrically to the symmetric APC3 homodimer via its C-terminal domain, thereby recruiting APC7 into the APC/C Arc Lamp subcomplex [PMID:25490258]. Depletion of APC16 in human cells causes metaphase arrest phenocopying loss of core APC/C subunits, and the C. elegans ortholog EMB-1 is similarly required for meiotic and mitotic progression, as demonstrated by temperature-sensitive alleles and genetic epistasis with other APC/C components [PMID:20392738, PMID:21775471].\",\n  \"teleology\": [\n    {\n      \"year\": 2010,\n      \"claim\": \"Identification of APC16 as a bona fide APC/C subunit resolved the question of whether the complex contained additional small components, establishing that APC16 is present throughout the cell cycle, required for ubiquitin ligase activity toward mitotic substrates, and associated with mitotic checkpoint complex proteins.\",\n      \"evidence\": \"Tandem-affinity purification, co-immunoprecipitation, siRNA depletion with in vitro ubiquitination assays in human cells\",\n      \"pmids\": [\"20392738\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis for APC16 incorporation into the APC/C was unknown\",\n        \"Whether APC16 has organism-specific functions beyond ubiquitin ligase support was not addressed\"\n      ]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Genetic characterization of the C. elegans ortholog EMB-1 confirmed that APC16 function is conserved in metazoans and placed it firmly within the APC/C pathway by epistasis, resolving the question of whether APC16 loss-of-function phenocopies other APC/C subunit mutations across species.\",\n      \"evidence\": \"Temperature-sensitive allele characterization and double-mutant epistasis analysis in C. elegans embryos\",\n      \"pmids\": [\"21775471\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Which APC/C subunit APC16 directly contacts was not determined\",\n        \"Whether APC16 participates in coactivator recruitment was unknown\"\n      ]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Crystal structures of APC3 alone and complexed with the APC16 C-terminal domain resolved the architectural question of how APC16 integrates into the APC/C, revealing asymmetric binding to the APC3 homodimer and a bridging role in recruiting APC7 to the Arc Lamp subcomplex.\",\n      \"evidence\": \"X-ray crystallography of APC3Δloop–APC16 complex with mutagenesis validation and in vitro ubiquitination assays\",\n      \"pmids\": [\"25490258\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Why only one APC16 binds the symmetric APC3 dimer (basis for asymmetry) is not fully explained\",\n        \"How APC16 binding influences APC3 phosphorylation-dependent regulation during mitosis is unclear\",\n        \"No direct structural data on the APC16–APC7 interface\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unknown whether APC16 contributes to substrate specificity or coactivator (CDC20/CDH1) recruitment beyond its structural scaffolding role, and whether its loss has consequences distinct from general APC/C disassembly.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No reconstituted APC/C lacking only APC16 has been tested for substrate-specific effects\",\n        \"Post-translational modifications of APC16 itself have not been characterized\",\n        \"No disease-associated mutations in ANAPC16 have been functionally validated\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [3, 4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [2, 3, 4]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 2]}\n    ],\n    \"complexes\": [\"APC/C\"],\n    \"partners\": [\"APC3\", \"APC7\"],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"ANAPC16 is a metazoan-specific subunit of the anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase that controls mitotic and meiotic progression by ubiquitinating cell-cycle substrates. Present in APC/C complexes throughout the cell cycle, ANAPC16 resides in the Arc Lamp subcomplex where its C-terminal domain binds asymmetrically to the APC3 homodimer and recruits APC7 to APC3, as defined by crystal structure and cryo-EM [PMID:25490258, PMID:26083744]. Depletion of ANAPC16 phenocopies loss of other APC/C subunits, abolishing APC/C ubiquitination activity toward mitotic substrates in human cells, and temperature-sensitive mutants of the C. elegans ortholog emb-1 arrest in meiosis I metaphase and fail in germline mitotic proliferation [PMID:20392738, PMID:21775471]. ANAPC16 is conserved across metazoans but absent in fungi, and cross-species complementation confirms functional equivalence of vertebrate and invertebrate orthologs [PMID:20392738].\",\n  \"teleology\": [\n    {\n      \"year\": 2010,\n      \"claim\": \"The identity of ANAPC16 as a bona fide APC/C subunit was established, answering whether the holoenzyme contained additional uncharacterized components; its depletion abolished APC/C ubiquitination activity and phenocopied loss of core subunits, demonstrating functional necessity.\",\n      \"evidence\": \"TAP-MS identification, reciprocal co-IP, RNAi knockdown with mitotic substrate ubiquitination readouts, and cross-species complementation in human, C. elegans, and zebrafish cells\",\n      \"pmids\": [\"20392738\", \"20360068\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Binding interface between APC16 and other APC/C subunits was unresolved\",\n        \"Whether APC16 has a specific role beyond structural integrity of the complex was unknown\"\n      ]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Genetic epistasis in C. elegans placed APC16 definitively within the APC/C pathway, revealing that it is required for both meiotic progression and mitotic germline proliferation — extending the functional requirement beyond somatic mitosis.\",\n      \"evidence\": \"Temperature-sensitive emb-1/APC16 mutants, double mutant combinations with known APC/C subunits and suppressors, meiosis I arrest and germline proliferation assays\",\n      \"pmids\": [\"21775471\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether APC16 contributes to substrate recognition or only to complex assembly was unknown\",\n        \"Structural basis of APC16 integration into the holoenzyme remained unresolved\"\n      ]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Crystal structure and cryo-EM resolved how APC16 integrates into the APC/C: one APC16 binds asymmetrically to the symmetric APC3 homodimer via its C-terminal domain and recruits APC7, defining a specific architectural role within the Arc Lamp subcomplex.\",\n      \"evidence\": \"X-ray crystallography of APC3–APC16 C-terminal domain complex, 7.4 Å cryo-EM reconstruction of the full 1.2 MDa APC/C, mutagenesis, and in vitro ubiquitination assays\",\n      \"pmids\": [\"25490258\", \"25043029\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"How APC16's asymmetric binding relates to the asymmetric catalytic cycle of the APC/C was not established\",\n        \"Whether APC16 influences substrate selectivity or only holoenzyme assembly was unresolved\"\n      ]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Atomic-resolution cryo-EM of APC/C–coactivator complexes placed APC16 in full mechanistic context, revealing the overall architecture that enables Emi1-mediated inhibition and UbcH10-dependent ubiquitin transfer.\",\n      \"evidence\": \"Atomic-resolution cryo-EM of APC/C–Cdh1–Emi1 and APC/C–UbcH10–Ub complexes\",\n      \"pmids\": [\"26083744\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Direct contribution of APC16 to catalytic steps (E2 positioning, substrate handoff) versus purely structural scaffolding was not dissected\"\n      ]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"The phosphorylation-driven activation mechanism of APC/C was elucidated in the context of the Arc Lamp subcomplex containing APC16: Cdk–cyclin–Cks recruited by the APC3 phospho-loop hyperphosphorylates an auto-inhibitory segment of APC1 to enable Cdc20 binding.\",\n      \"evidence\": \"Cryo-EM, biochemical phosphorylation assays, and mutagenesis of APC3 loop and APC1 auto-inhibitory segment\",\n      \"pmids\": [\"27120157\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether APC16 modulates the phosphorylation-dependent conformational switch in APC3/APC1 is unknown\",\n        \"No post-translational modifications of APC16 itself have been functionally characterized\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unknown whether APC16 plays any role beyond structural scaffolding — for example, whether it directly influences substrate recognition, E2 engagement, or processivity of polyubiquitin chain elongation.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No separation-of-function mutations distinguishing APC16's architectural role from potential regulatory roles have been reported\",\n        \"No post-translational regulation of APC16 has been characterized\",\n        \"Functional consequence of the asymmetric APC16 stoichiometry on the symmetric APC3 homodimer is unexplained\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0, 2, 6]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 3, 5]}\n    ],\n    \"complexes\": [\n      \"APC/C\"\n    ],\n    \"partners\": [\n      \"ANAPC3\",\n      \"ANAPC7\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}