{"gene":"FBXO43","run_date":"2026-06-09T23:54:43","timeline":{"discoveries":[{"year":2005,"finding":"Emi2/XErp1/FBXO43 is a stable APC/C inhibitor in CSF-arrested Xenopus eggs that prevents parthenogenesis by maintaining metaphase II arrest; it is rapidly degraded in a Polo-like kinase 1 (Plk1)-dependent manner following calcium-mediated egg activation.","method":"Xenopus egg extracts, antibody depletion, exogenous protein addition, inhibitor experiments; crossreactivity of anti-Emi1 antibodies identified endogenous Emi2","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal identification in Xenopus egg system, multiple functional assays (CSF arrest, calcium activation, Plk1 dependency), replicated in parallel studies","pmids":["15753281"],"is_preprint":false},{"year":2014,"finding":"The zinc-binding region (ZBR) domain of Emi2/FBXO43 inhibits APC/C by two distinct mechanisms: (1) disrupting the association of the coactivator Cdc20 with the APC/C core complex, and (2) inhibiting in vitro ubiquitin chain elongation catalyzed by the APC/C cullin-RING ligase module (ANAPC2-ANAPC11) together with UBE2C/E2C. The post-ZBR (PZ) region additionally interacts with the cullin subunit ANAPC2. Key residues in the ZBR domain mediating each mechanism were identified by mutagenesis and transplantation into the paralog Emi1/FBXO5 ZBR.","method":"In vitro ubiquitylation assay, co-immunoprecipitation in HEK293T cells, domain mutagenesis, structural analyses (NMR/structural characterization of ZBR domain), domain transplantation experiments","journal":"FEBS open bio","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution of ubiquitylation, active-site mutagenesis, structural analysis, and cell-based co-IP in a single study with multiple orthogonal methods","pmids":["25161877"],"is_preprint":false},{"year":2021,"finding":"In mouse testicular protein extracts, FBXO43 directly interacts with three APC/C subunits (ANAPC2, ANAPC8, and ANAPC10), but no interaction was detected between FBXO43 and SKP1, indicating FBXO43 functions as a direct APC/C inhibitor rather than as an SCF complex component in male meiosis.","method":"Co-immunoprecipitation from mouse testicular protein extracts; immunostaining of human testicular tissue; whole-exome sequencing identifying loss-of-function mutation causing meiotic arrest at early diplotene of prophase I","journal":"Clinical genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP identifying specific APC/C subunit interactions, supported by human genetic loss-of-function data showing meiotic arrest, single lab","pmids":["34595750"],"is_preprint":false},{"year":2021,"finding":"Homozygous loss-of-function variants in FBXO43 reduce protein levels of FBXO43 and its downstream target Cyclin B1 in HEK293T cells, and reduce the ability of exogenous human FBXO43 to rescue parthenogenetic activation phenotype in Fbxo43 knockdown mouse oocytes, establishing Cyclin B1 stabilization as a downstream effector of FBXO43 in oocyte meiotic arrest.","method":"Western blotting in HEK293T cells expressing variant constructs; complementary RNA injection rescue assay in Fbxo43 knockdown mouse oocytes","journal":"Human reproduction (Oxford, England)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional rescue assay in mouse oocytes combined with cell-based protein level assay, two orthogonal methods, single lab","pmids":["34052850"],"is_preprint":false},{"year":2022,"finding":"In Xenopus multiciliated cell (MCC) progenitors, emi2/fbxo43 expression is upregulated after cell cycle exit and transiently inhibits APC/C-Cdh1 activity, which is required for phosphorylation and activation of Plk1; this emi2-APC/C-plk1 axis promotes centriole disengagement, apical migration, and maturation into basal bodies during centriole amplification, and subsequently down-regulates gene expression required for centriole amplification after differentiation is complete.","method":"Xenopus MCC differentiation system, genetic epistasis (loss-of-function and rescue experiments), phosphorylation assays, centriole amplification and basal body formation phenotypic readouts, gene expression analysis","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis establishing pathway order (emi2→APC/C-Cdh1→Plk1→centriole amplification), multiple orthogonal phenotypic readouts, functional rescue experiments","pmids":["35363516"],"is_preprint":false},{"year":2023,"finding":"FBXO43 interacts with Cyclin D1 (CCND1) and promotes its stability through polyubiquitination, leading to increased HCC cell proliferation, migration, and epithelial-mesenchymal transition (EMT); knockdown of CCND1 blocks FBXO43-mediated cell proliferation and metastasis.","method":"Co-immunoprecipitation assay, in vivo ubiquitination assay, functional rescue experiments with CCND1 knockdown, MTT/EdU/colony formation/Transwell assays","journal":"Frontiers in oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus in vivo ubiquitination assay plus functional rescue, two orthogonal mechanistic methods, single lab","pmids":["36937431"],"is_preprint":false},{"year":2023,"finding":"FBXO43 depletion reduces expression of UBE2C (a p53 ubiquitin-conjugating enzyme), suppresses proteasomal degradation of p53, and inhibits HCC cell proliferation and invasion; METTL3 and IGF2BP2 act as m6A writer and reader respectively to stabilize FBXO43 mRNA, and ectopic FBXO43 expression rescues the inhibitory effects of METTL3/IGF2BP2 depletion.","method":"siRNA knockdown, Western blotting, overexpression rescue experiments, m6A modification analysis (METTL3/IGF2BP2 correlation and functional assays), cell proliferation and invasion assays","journal":"Cancers","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional epistasis via rescue experiments, protein level measurements, and m6A writer/reader identification; single lab, multiple orthogonal methods","pmids":["36765911"],"is_preprint":false},{"year":2024,"finding":"FBXO43 interacts with AKT1-phosphorylated SKP2, reducing SKP2 auto-ubiquitylation and subsequent proteasomal degradation, thereby stabilizing SKP2 and promoting cell cycle progression in cancer cells (non-small cell lung cancer, hepatocellular carcinoma, sarcoma).","method":"Co-immunoprecipitation, ubiquitylation assays, AKT1 phosphorylation experiments, loss-of-function (knockdown/knockout) with cell cycle phenotypic readout","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with phosphorylation dependency and ubiquitylation assay, single lab, multiple cancer cell line contexts","pmids":["38604312"],"is_preprint":false},{"year":2021,"finding":"FBXO43 interacts with PCNA in breast cancer cells as identified by mass spectrometry and confirmed by co-immunoprecipitation; overexpression of PCNA significantly reverses the inhibitory effects of FBXO43 knockdown on BC cell proliferation, migration, and invasion.","method":"Mass spectrometry identification of interacting proteins, co-immunoprecipitation (Co-IP), PCNA overexpression rescue experiments, xenograft mouse model","journal":"Journal of translational medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus MS plus functional rescue assay, single lab, multiple orthogonal methods","pmids":["34645483"],"is_preprint":false}],"current_model":"FBXO43 (Emi2) is a direct inhibitor of the anaphase-promoting complex/cyclosome (APC/C) that maintains metaphase II arrest in oocytes (cytostatic factor activity) through its ZBR domain, which both displaces the coactivator Cdc20 and inhibits UBE2C-mediated ubiquitin chain elongation on APC/C substrates including Cyclin B1; it is activated/degraded in a Plk1-dependent manner upon calcium signaling at fertilization, and additionally functions in post-mitotic multiciliated cell progenitors via an emi2-APC/C-Cdh1-Plk1 axis to enable centriole amplification, while in somatic/cancer contexts it stabilizes substrates including SKP2, Cyclin D1, and PCNA to promote cell cycle progression."},"narrative":{"mechanistic_narrative":"FBXO43 (Emi2/XErp1) is a direct inhibitor of the anaphase-promoting complex/cyclosome (APC/C) that enforces meiotic metaphase arrest by blocking the destruction of APC/C substrates such as Cyclin B1 [PMID:15753281, PMID:34052850]. Its zinc-binding region (ZBR) domain achieves inhibition by two distinct mechanisms: displacing the coactivator Cdc20 from the APC/C core and blocking UBE2C-driven ubiquitin chain elongation by the ANAPC2–ANAPC11 catalytic module, while an adjacent post-ZBR region docks onto the cullin subunit ANAPC2 [PMID:25161877]. Consistent with a direct-inhibitor rather than SCF role, FBXO43 contacts APC/C subunits ANAPC2, ANAPC8, and ANAPC10 but not SKP1 [PMID:34595750]. This APC/C-restraining activity maintains meiotic arrest, and loss-of-function variants that destabilize FBXO43 and lower Cyclin B1 cause meiotic arrest and parthenogenetic activation, linking FBXO43 to human meiotic/reproductive failure [PMID:34595750, PMID:34052850]. Beyond classical meiosis, transient FBXO43-mediated inhibition of APC/C-Cdh1 is required to activate Plk1 and drive centriole amplification in multiciliated cell progenitors [PMID:35363516]. In somatic and cancer contexts FBXO43 instead stabilizes pro-proliferative factors—Cyclin D1, AKT1-phosphorylated SKP2, and PCNA—to promote cell cycle progression, proliferation, and invasion [PMID:36937431, PMID:38604312, PMID:34645483].","teleology":[{"year":2005,"claim":"Established FBXO43/Emi2 as the stable APC/C inhibitor responsible for cytostatic-factor metaphase II arrest and showed how fertilization releases the brake, answering what holds the egg arrested and how it is reversed.","evidence":"Antibody depletion, exogenous protein add-back, and Plk1-inhibitor experiments in Xenopus egg extracts","pmids":["15753281"],"confidence":"High","gaps":["Molecular mechanism of APC/C inhibition not yet resolved","Did not define the domain or residues required for inhibition"]},{"year":2014,"claim":"Resolved the dual molecular mechanism of inhibition—Cdc20 displacement plus blockade of UBE2C-mediated chain elongation by the ZBR, with cullin docking by the post-ZBR region—explaining how a single domain suppresses APC/C catalysis.","evidence":"In vitro ubiquitylation reconstitution, ZBR mutagenesis and transplantation into the Emi1/FBXO5 ZBR, structural characterization, and co-IP in HEK293T","pmids":["25161877"],"confidence":"High","gaps":["No full structure of the FBXO43–APC/C complex","How calcium/Plk1 signaling relieves this inhibition mechanistically not addressed"]},{"year":2021,"claim":"Confirmed in mammals that FBXO43 acts as a direct APC/C inhibitor rather than an SCF substrate-receptor by mapping subunit contacts and tying loss-of-function to human meiotic arrest, extending the Xenopus model to human male and female meiosis.","evidence":"Co-IP from mouse testis showing ANAPC2/ANAPC8/ANAPC10 but not SKP1 binding; whole-exome sequencing of patients; oocyte rescue and HEK293T variant Western blots establishing Cyclin B1 as the effector","pmids":["34595750","34052850"],"confidence":"Medium","gaps":["Single-lab Co-IP without reciprocal in vivo validation","Stoichiometry and dynamics of FBXO43–APC/C engagement during meiotic progression not defined"]},{"year":2022,"claim":"Revealed a non-meiotic role in which transient FBXO43 inhibition of APC/C-Cdh1 activates Plk1 to drive centriole amplification in multiciliated cell progenitors, showing the emi2–APC/C axis is repurposed after cell-cycle exit.","evidence":"Genetic epistasis (loss-of-function and rescue), phosphorylation assays, and centriole/basal-body phenotypic readouts in the Xenopus MCC differentiation system","pmids":["35363516"],"confidence":"High","gaps":["Direct biochemical demonstration of FBXO43-Cdh1 inhibition not shown in this system","Mechanism timing the switch from inhibition to down-regulation unclear"]},{"year":2021,"claim":"Began characterizing somatic/oncogenic FBXO43 by identifying PCNA as an interactor whose abundance is required for FBXO43-driven proliferation and invasion, opening a cancer-context function distinct from APC/C inhibition.","evidence":"Mass spectrometry, co-IP, PCNA overexpression rescue, and xenograft model in breast cancer cells","pmids":["34645483"],"confidence":"Medium","gaps":["Whether FBXO43 directly ubiquitinates/stabilizes PCNA not established","Single-lab finding without independent replication"]},{"year":2023,"claim":"Extended the oncogenic role by showing FBXO43 promotes polyubiquitination and stabilization of Cyclin D1 and influences UBE2C/p53 turnover, with m6A regulation (METTL3/IGF2BP2) controlling FBXO43 mRNA, framing how FBXO43 is regulated and how it drives proliferation in HCC.","evidence":"Co-IP, in vivo ubiquitination assays, CCND1/UBE2C knockdown rescue, and m6A writer/reader functional assays in HCC cells","pmids":["36937431","36765911"],"confidence":"Medium","gaps":["Apparent stabilizing ubiquitination contrasts with the APC/C-inhibitory role and is not mechanistically reconciled","Direct enzymatic relationship between FBXO43 and these substrates not reconstituted"]},{"year":2024,"claim":"Identified AKT1-phosphorylated SKP2 as an FBXO43 partner whose auto-ubiquitylation FBXO43 suppresses, linking phospho-dependent substrate recognition to cell-cycle progression across multiple cancer types.","evidence":"Co-IP, AKT1 phosphorylation experiments, ubiquitylation assays, and loss-of-function cell-cycle readouts in NSCLC, HCC, and sarcoma lines","pmids":["38604312"],"confidence":"Medium","gaps":["Single-lab study","How FBXO43 reduces SKP2 auto-ubiquitylation mechanistically not defined"]},{"year":null,"claim":"How FBXO43 reconciles its APC/C-inhibitory function in meiosis with substrate-stabilizing roles in somatic cancer cells, and what governs its context-specific partner selection, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified biochemical model linking APC/C inhibition and Cyclin D1/SKP2/PCNA stabilization","No structure of FBXO43 bound to its somatic partners","Tissue-specific regulation of which activity dominates is unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,1,2]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[5,7]}],"localization":[{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[4]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0,3,7]},{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[2,3]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[5,6,7,8]}],"complexes":[],"partners":["ANAPC2","ANAPC8","ANAPC10","CDC20","UBE2C","CCND1","SKP2","PCNA"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q4G163","full_name":"F-box only protein 43","aliases":["Endogenous meiotic inhibitor 2"],"length_aa":708,"mass_kda":78.4,"function":"Required to establish and maintain the arrest of oocytes at the second meiotic metaphase until fertilization. Acts by inhibiting the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase. Probably recognizes and binds to some phosphorylated proteins and promotes their ubiquitination and degradation (PubMed:34052850, PubMed:34595750). Plays a vital role in modulating the ubiquitilation of CCNB1 and CDK1 during gametogenesis","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q4G163/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/FBXO43","classification":"Not Classified","n_dependent_lines":37,"n_total_lines":1208,"dependency_fraction":0.030629139072847682},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/FBXO43","total_profiled":1310},"omim":[{"mim_id":"619697","title":"OOCYTE/ZYGOTE/EMBRYO MATURATION ARREST 12; OZEMA12","url":"https://www.omim.org/entry/619697"},{"mim_id":"619696","title":"SPERMATOGENIC FAILURE 64; SPGF64","url":"https://www.omim.org/entry/619696"},{"mim_id":"615774","title":"OOCYTE/ZYGOTE/EMBRYO MATURATION ARREST 1; OZEMA1","url":"https://www.omim.org/entry/615774"},{"mim_id":"609110","title":"F-BOX ONLY PROTEIN 43; FBXO43","url":"https://www.omim.org/entry/609110"},{"mim_id":"258150","title":"SPERMATOGENIC FAILURE 1; SPGF1","url":"https://www.omim.org/entry/258150"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Cytosol","reliability":"Approved"}],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in single","driving_tissues":[{"tissue":"testis","ntpm":3.0}],"url":"https://www.proteinatlas.org/search/FBXO43"},"hgnc":{"alias_symbol":["Fbx43"],"prev_symbol":[]},"alphafold":{"accession":"Q4G163","domains":[{"cath_id":"-","chopping":"496-564","consensus_level":"medium","plddt":89.4012,"start":496,"end":564},{"cath_id":"2.20.25.20","chopping":"622-680","consensus_level":"medium","plddt":91.1066,"start":622,"end":680}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q4G163","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q4G163-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q4G163-F1-predicted_aligned_error_v6.png","plddt_mean":52.34},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=FBXO43","jax_strain_url":"https://www.jax.org/strain/search?query=FBXO43"},"sequence":{"accession":"Q4G163","fasta_url":"https://rest.uniprot.org/uniprotkb/Q4G163.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q4G163/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q4G163"}},"corpus_meta":[{"pmid":"15753281","id":"PMC_15753281","title":"A role for the anaphase-promoting complex inhibitor Emi2/XErp1, a homolog of early mitotic inhibitor 1, in cytostatic factor arrest of Xenopus eggs.","date":"2005","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/15753281","citation_count":138,"is_preprint":false},{"pmid":"34052850","id":"PMC_34052850","title":"FBXO43 variants in patients with female infertility characterized by early embryonic arrest.","date":"2021","source":"Human reproduction (Oxford, England)","url":"https://pubmed.ncbi.nlm.nih.gov/34052850","citation_count":41,"is_preprint":false},{"pmid":"35900055","id":"PMC_35900055","title":"Gene mutations impede oocyte maturation, fertilization, and early embryonic development.","date":"2022","source":"BioEssays : news and reviews in molecular, cellular and developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/35900055","citation_count":28,"is_preprint":false},{"pmid":"30878252","id":"PMC_30878252","title":"A novel homozygous FBXO43 mutation associated with male infertility and teratozoospermia in a consanguineous Chinese family.","date":"2019","source":"Fertility and sterility","url":"https://pubmed.ncbi.nlm.nih.gov/30878252","citation_count":23,"is_preprint":false},{"pmid":"27079378","id":"PMC_27079378","title":"Screening of whole genome sequences identified high-impact variants for stallion fertility.","date":"2016","source":"BMC genomics","url":"https://pubmed.ncbi.nlm.nih.gov/27079378","citation_count":23,"is_preprint":false},{"pmid":"33523351","id":"PMC_33523351","title":"Differential expression analysis and identification of sex-related genes by gonad transcriptome sequencing in estradiol-treated and non-treated Ussuri catfish Pseudobagrus ussuriensis.","date":"2021","source":"Fish physiology and biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/33523351","citation_count":20,"is_preprint":false},{"pmid":"25161877","id":"PMC_25161877","title":"The zinc-binding region (ZBR) fragment of Emi2 can inhibit APC/C by targeting its association with the coactivator Cdc20 and UBE2C-mediated ubiquitylation.","date":"2014","source":"FEBS open bio","url":"https://pubmed.ncbi.nlm.nih.gov/25161877","citation_count":20,"is_preprint":false},{"pmid":"36765911","id":"PMC_36765911","title":"F-Box Protein 43, Stabilized by N6-Methyladenosine Methylation, Enhances Hepatocellular Carcinoma Cell Growth and Invasion via Promoting p53 Degradation in a Ubiquitin Conjugating Enzyme E2 C-Dependent Manner.","date":"2023","source":"Cancers","url":"https://pubmed.ncbi.nlm.nih.gov/36765911","citation_count":17,"is_preprint":false},{"pmid":"34201347","id":"PMC_34201347","title":"A Novel Signature of CCNF-Associated E3 Ligases Collaborate and Counter Each Other in Breast Cancer.","date":"2021","source":"Cancers","url":"https://pubmed.ncbi.nlm.nih.gov/34201347","citation_count":17,"is_preprint":false},{"pmid":"39417902","id":"PMC_39417902","title":"Genetic etiological spectrum of sperm morphological abnormalities.","date":"2024","source":"Journal of assisted reproduction and genetics","url":"https://pubmed.ncbi.nlm.nih.gov/39417902","citation_count":17,"is_preprint":false},{"pmid":"35363516","id":"PMC_35363516","title":"Emi2 enables centriole amplification during multiciliated cell differentiation.","date":"2022","source":"Science advances","url":"https://pubmed.ncbi.nlm.nih.gov/35363516","citation_count":15,"is_preprint":false},{"pmid":"34595750","id":"PMC_34595750","title":"A homozygous loss-of-function mutation in FBXO43 causes human non-obstructive azoospermia.","date":"2021","source":"Clinical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/34595750","citation_count":14,"is_preprint":false},{"pmid":"32754194","id":"PMC_32754194","title":"Integration of GWAS and eQTL Analysis to Identify Risk Loci and Susceptibility Genes for Gastric Cancer.","date":"2020","source":"Frontiers in genetics","url":"https://pubmed.ncbi.nlm.nih.gov/32754194","citation_count":14,"is_preprint":false},{"pmid":"38604312","id":"PMC_38604312","title":"FBXO43 promotes cell cycle progression in cancer cells through stabilizing SKP2.","date":"2024","source":"Cancer letters","url":"https://pubmed.ncbi.nlm.nih.gov/38604312","citation_count":12,"is_preprint":false},{"pmid":"34645483","id":"PMC_34645483","title":"Downregulation of the FBXO43 gene inhibits tumor growth in human breast cancer by limiting its interaction with PCNA.","date":"2021","source":"Journal of translational 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Study.","date":"2026","source":"Genes","url":"https://pubmed.ncbi.nlm.nih.gov/41898836","citation_count":0,"is_preprint":false},{"pmid":"41828918","id":"PMC_41828918","title":"Transcriptome Analysis of Different Stages in the Early Ovarian Development of the Greater Amberjack (Seriola dumerili).","date":"2026","source":"Animals : an open access journal from MDPI","url":"https://pubmed.ncbi.nlm.nih.gov/41828918","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":16365,"output_tokens":2658,"usd":0.044483,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9875,"output_tokens":3119,"usd":0.063675,"stage2_stop_reason":"end_turn"},"total_usd":0.108158,"stage1_batch_id":"msgbatch_01AsR7g8dGTEiEw9U83bJeWn","stage2_batch_id":"msgbatch_01YV5ZyxtFNUZEjxRcpd9jzb","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2005,\n      \"finding\": \"Emi2/XErp1/FBXO43 is a stable APC/C inhibitor in CSF-arrested Xenopus eggs that prevents parthenogenesis by maintaining metaphase II arrest; it is rapidly degraded in a Polo-like kinase 1 (Plk1)-dependent manner following calcium-mediated egg activation.\",\n      \"method\": \"Xenopus egg extracts, antibody depletion, exogenous protein addition, inhibitor experiments; crossreactivity of anti-Emi1 antibodies identified endogenous Emi2\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal identification in Xenopus egg system, multiple functional assays (CSF arrest, calcium activation, Plk1 dependency), replicated in parallel studies\",\n      \"pmids\": [\"15753281\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The zinc-binding region (ZBR) domain of Emi2/FBXO43 inhibits APC/C by two distinct mechanisms: (1) disrupting the association of the coactivator Cdc20 with the APC/C core complex, and (2) inhibiting in vitro ubiquitin chain elongation catalyzed by the APC/C cullin-RING ligase module (ANAPC2-ANAPC11) together with UBE2C/E2C. The post-ZBR (PZ) region additionally interacts with the cullin subunit ANAPC2. Key residues in the ZBR domain mediating each mechanism were identified by mutagenesis and transplantation into the paralog Emi1/FBXO5 ZBR.\",\n      \"method\": \"In vitro ubiquitylation assay, co-immunoprecipitation in HEK293T cells, domain mutagenesis, structural analyses (NMR/structural characterization of ZBR domain), domain transplantation experiments\",\n      \"journal\": \"FEBS open bio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution of ubiquitylation, active-site mutagenesis, structural analysis, and cell-based co-IP in a single study with multiple orthogonal methods\",\n      \"pmids\": [\"25161877\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In mouse testicular protein extracts, FBXO43 directly interacts with three APC/C subunits (ANAPC2, ANAPC8, and ANAPC10), but no interaction was detected between FBXO43 and SKP1, indicating FBXO43 functions as a direct APC/C inhibitor rather than as an SCF complex component in male meiosis.\",\n      \"method\": \"Co-immunoprecipitation from mouse testicular protein extracts; immunostaining of human testicular tissue; whole-exome sequencing identifying loss-of-function mutation causing meiotic arrest at early diplotene of prophase I\",\n      \"journal\": \"Clinical genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP identifying specific APC/C subunit interactions, supported by human genetic loss-of-function data showing meiotic arrest, single lab\",\n      \"pmids\": [\"34595750\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Homozygous loss-of-function variants in FBXO43 reduce protein levels of FBXO43 and its downstream target Cyclin B1 in HEK293T cells, and reduce the ability of exogenous human FBXO43 to rescue parthenogenetic activation phenotype in Fbxo43 knockdown mouse oocytes, establishing Cyclin B1 stabilization as a downstream effector of FBXO43 in oocyte meiotic arrest.\",\n      \"method\": \"Western blotting in HEK293T cells expressing variant constructs; complementary RNA injection rescue assay in Fbxo43 knockdown mouse oocytes\",\n      \"journal\": \"Human reproduction (Oxford, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional rescue assay in mouse oocytes combined with cell-based protein level assay, two orthogonal methods, single lab\",\n      \"pmids\": [\"34052850\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In Xenopus multiciliated cell (MCC) progenitors, emi2/fbxo43 expression is upregulated after cell cycle exit and transiently inhibits APC/C-Cdh1 activity, which is required for phosphorylation and activation of Plk1; this emi2-APC/C-plk1 axis promotes centriole disengagement, apical migration, and maturation into basal bodies during centriole amplification, and subsequently down-regulates gene expression required for centriole amplification after differentiation is complete.\",\n      \"method\": \"Xenopus MCC differentiation system, genetic epistasis (loss-of-function and rescue experiments), phosphorylation assays, centriole amplification and basal body formation phenotypic readouts, gene expression analysis\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis establishing pathway order (emi2→APC/C-Cdh1→Plk1→centriole amplification), multiple orthogonal phenotypic readouts, functional rescue experiments\",\n      \"pmids\": [\"35363516\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"FBXO43 interacts with Cyclin D1 (CCND1) and promotes its stability through polyubiquitination, leading to increased HCC cell proliferation, migration, and epithelial-mesenchymal transition (EMT); knockdown of CCND1 blocks FBXO43-mediated cell proliferation and metastasis.\",\n      \"method\": \"Co-immunoprecipitation assay, in vivo ubiquitination assay, functional rescue experiments with CCND1 knockdown, MTT/EdU/colony formation/Transwell assays\",\n      \"journal\": \"Frontiers in oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus in vivo ubiquitination assay plus functional rescue, two orthogonal mechanistic methods, single lab\",\n      \"pmids\": [\"36937431\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"FBXO43 depletion reduces expression of UBE2C (a p53 ubiquitin-conjugating enzyme), suppresses proteasomal degradation of p53, and inhibits HCC cell proliferation and invasion; METTL3 and IGF2BP2 act as m6A writer and reader respectively to stabilize FBXO43 mRNA, and ectopic FBXO43 expression rescues the inhibitory effects of METTL3/IGF2BP2 depletion.\",\n      \"method\": \"siRNA knockdown, Western blotting, overexpression rescue experiments, m6A modification analysis (METTL3/IGF2BP2 correlation and functional assays), cell proliferation and invasion assays\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional epistasis via rescue experiments, protein level measurements, and m6A writer/reader identification; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"36765911\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"FBXO43 interacts with AKT1-phosphorylated SKP2, reducing SKP2 auto-ubiquitylation and subsequent proteasomal degradation, thereby stabilizing SKP2 and promoting cell cycle progression in cancer cells (non-small cell lung cancer, hepatocellular carcinoma, sarcoma).\",\n      \"method\": \"Co-immunoprecipitation, ubiquitylation assays, AKT1 phosphorylation experiments, loss-of-function (knockdown/knockout) with cell cycle phenotypic readout\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with phosphorylation dependency and ubiquitylation assay, single lab, multiple cancer cell line contexts\",\n      \"pmids\": [\"38604312\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"FBXO43 interacts with PCNA in breast cancer cells as identified by mass spectrometry and confirmed by co-immunoprecipitation; overexpression of PCNA significantly reverses the inhibitory effects of FBXO43 knockdown on BC cell proliferation, migration, and invasion.\",\n      \"method\": \"Mass spectrometry identification of interacting proteins, co-immunoprecipitation (Co-IP), PCNA overexpression rescue experiments, xenograft mouse model\",\n      \"journal\": \"Journal of translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus MS plus functional rescue assay, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"34645483\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"FBXO43 (Emi2) is a direct inhibitor of the anaphase-promoting complex/cyclosome (APC/C) that maintains metaphase II arrest in oocytes (cytostatic factor activity) through its ZBR domain, which both displaces the coactivator Cdc20 and inhibits UBE2C-mediated ubiquitin chain elongation on APC/C substrates including Cyclin B1; it is activated/degraded in a Plk1-dependent manner upon calcium signaling at fertilization, and additionally functions in post-mitotic multiciliated cell progenitors via an emi2-APC/C-Cdh1-Plk1 axis to enable centriole amplification, while in somatic/cancer contexts it stabilizes substrates including SKP2, Cyclin D1, and PCNA to promote cell cycle progression.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"FBXO43 (Emi2/XErp1) is a direct inhibitor of the anaphase-promoting complex/cyclosome (APC/C) that enforces meiotic metaphase arrest by blocking the destruction of APC/C substrates such as Cyclin B1 [#0, #3]. Its zinc-binding region (ZBR) domain achieves inhibition by two distinct mechanisms: displacing the coactivator Cdc20 from the APC/C core and blocking UBE2C-driven ubiquitin chain elongation by the ANAPC2–ANAPC11 catalytic module, while an adjacent post-ZBR region docks onto the cullin subunit ANAPC2 [#1]. Consistent with a direct-inhibitor rather than SCF role, FBXO43 contacts APC/C subunits ANAPC2, ANAPC8, and ANAPC10 but not SKP1 [#2]. This APC/C-restraining activity maintains meiotic arrest, and loss-of-function variants that destabilize FBXO43 and lower Cyclin B1 cause meiotic arrest and parthenogenetic activation, linking FBXO43 to human meiotic/reproductive failure [#2, #3]. Beyond classical meiosis, transient FBXO43-mediated inhibition of APC/C-Cdh1 is required to activate Plk1 and drive centriole amplification in multiciliated cell progenitors [#4]. In somatic and cancer contexts FBXO43 instead stabilizes pro-proliferative factors—Cyclin D1, AKT1-phosphorylated SKP2, and PCNA—to promote cell cycle progression, proliferation, and invasion [#5, #7, #8].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Established FBXO43/Emi2 as the stable APC/C inhibitor responsible for cytostatic-factor metaphase II arrest and showed how fertilization releases the brake, answering what holds the egg arrested and how it is reversed.\",\n      \"evidence\": \"Antibody depletion, exogenous protein add-back, and Plk1-inhibitor experiments in Xenopus egg extracts\",\n      \"pmids\": [\"15753281\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism of APC/C inhibition not yet resolved\", \"Did not define the domain or residues required for inhibition\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Resolved the dual molecular mechanism of inhibition—Cdc20 displacement plus blockade of UBE2C-mediated chain elongation by the ZBR, with cullin docking by the post-ZBR region—explaining how a single domain suppresses APC/C catalysis.\",\n      \"evidence\": \"In vitro ubiquitylation reconstitution, ZBR mutagenesis and transplantation into the Emi1/FBXO5 ZBR, structural characterization, and co-IP in HEK293T\",\n      \"pmids\": [\"25161877\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No full structure of the FBXO43–APC/C complex\", \"How calcium/Plk1 signaling relieves this inhibition mechanistically not addressed\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Confirmed in mammals that FBXO43 acts as a direct APC/C inhibitor rather than an SCF substrate-receptor by mapping subunit contacts and tying loss-of-function to human meiotic arrest, extending the Xenopus model to human male and female meiosis.\",\n      \"evidence\": \"Co-IP from mouse testis showing ANAPC2/ANAPC8/ANAPC10 but not SKP1 binding; whole-exome sequencing of patients; oocyte rescue and HEK293T variant Western blots establishing Cyclin B1 as the effector\",\n      \"pmids\": [\"34595750\", \"34052850\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab Co-IP without reciprocal in vivo validation\", \"Stoichiometry and dynamics of FBXO43–APC/C engagement during meiotic progression not defined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Revealed a non-meiotic role in which transient FBXO43 inhibition of APC/C-Cdh1 activates Plk1 to drive centriole amplification in multiciliated cell progenitors, showing the emi2–APC/C axis is repurposed after cell-cycle exit.\",\n      \"evidence\": \"Genetic epistasis (loss-of-function and rescue), phosphorylation assays, and centriole/basal-body phenotypic readouts in the Xenopus MCC differentiation system\",\n      \"pmids\": [\"35363516\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct biochemical demonstration of FBXO43-Cdh1 inhibition not shown in this system\", \"Mechanism timing the switch from inhibition to down-regulation unclear\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Began characterizing somatic/oncogenic FBXO43 by identifying PCNA as an interactor whose abundance is required for FBXO43-driven proliferation and invasion, opening a cancer-context function distinct from APC/C inhibition.\",\n      \"evidence\": \"Mass spectrometry, co-IP, PCNA overexpression rescue, and xenograft model in breast cancer cells\",\n      \"pmids\": [\"34645483\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether FBXO43 directly ubiquitinates/stabilizes PCNA not established\", \"Single-lab finding without independent replication\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Extended the oncogenic role by showing FBXO43 promotes polyubiquitination and stabilization of Cyclin D1 and influences UBE2C/p53 turnover, with m6A regulation (METTL3/IGF2BP2) controlling FBXO43 mRNA, framing how FBXO43 is regulated and how it drives proliferation in HCC.\",\n      \"evidence\": \"Co-IP, in vivo ubiquitination assays, CCND1/UBE2C knockdown rescue, and m6A writer/reader functional assays in HCC cells\",\n      \"pmids\": [\"36937431\", \"36765911\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Apparent stabilizing ubiquitination contrasts with the APC/C-inhibitory role and is not mechanistically reconciled\", \"Direct enzymatic relationship between FBXO43 and these substrates not reconstituted\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified AKT1-phosphorylated SKP2 as an FBXO43 partner whose auto-ubiquitylation FBXO43 suppresses, linking phospho-dependent substrate recognition to cell-cycle progression across multiple cancer types.\",\n      \"evidence\": \"Co-IP, AKT1 phosphorylation experiments, ubiquitylation assays, and loss-of-function cell-cycle readouts in NSCLC, HCC, and sarcoma lines\",\n      \"pmids\": [\"38604312\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab study\", \"How FBXO43 reduces SKP2 auto-ubiquitylation mechanistically not defined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How FBXO43 reconciles its APC/C-inhibitory function in meiosis with substrate-stabilizing roles in somatic cancer cells, and what governs its context-specific partner selection, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified biochemical model linking APC/C inhibition and Cyclin D1/SKP2/PCNA stabilization\", \"No structure of FBXO43 bound to its somatic partners\", \"Tissue-specific regulation of which activity dominates is unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [5, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0, 3, 7]},\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [5, 6, 7, 8]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"ANAPC2\", \"ANAPC8\", \"ANAPC10\", \"CDC20\", \"UBE2C\", \"CCND1\", \"SKP2\", \"PCNA\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}