{"gene":"FBXO24","run_date":"2026-06-09T23:54:43","timeline":{"discoveries":[{"year":2015,"finding":"SCF-FBXO24 acts as a ubiquitin E3 ligase that polyubiquitinates NDPK-A at K85, targeting it for proteasomal degradation. The interaction requires NDPK-A residues L55 and K56; K56 acetylation by GCN5 impairs FBXO24 binding and stabilizes NDPK-A, while GCN5 depletion accelerates NDPK-A degradation. FBXO24 silencing or expression of an acetylation mimic increases NDPK-A lifespan and impairs cell migration and wound healing.","method":"Co-immunoprecipitation, in vitro ubiquitination assay, site-directed mutagenesis (K85R, K56Q acetylation mimic), cycloheximide chase, GCN5 knockdown, wound-healing/migration assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro ubiquitination assay plus mutagenesis at multiple sites, GCN5 writer identified, functional phenotype confirmed; single lab but multiple orthogonal methods","pmids":["25582197"],"is_preprint":false},{"year":2020,"finding":"FBXO24 binds PRMT6 at residues 270–275 and mediates K48-linked polyubiquitination of PRMT6 at K369, targeting it for proteasomal degradation. A K369R PRMT6 mutant is resistant to degradation, and PRMT6 K369R overexpression drives cell cycle progression and proliferation. FBXO24 overexpression or PRMT6 knockout inhibits cell proliferation, migration, and invasion in H1299 cells.","method":"Co-immunoprecipitation, ubiquitination assay, site-directed mutagenesis (K369R), cycloheximide chase, cell proliferation/migration/invasion assays, FBXO24 overexpression and PRMT6 knockout","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, mutagenesis, functional cellular readouts; single lab","pmids":["32828318"],"is_preprint":false},{"year":2023,"finding":"FBXO24 functions as an E3 ligase that ubiquitinates LSD1 (KDM1A), targeting it for proteasomal degradation. Identified via unbiased siRNA screen of all human SCF family E3 ligases; FBXO24 inhibits LSD1-induced tumorigenesis and acts as a tumor suppressor in breast cancer cells.","method":"Unbiased siRNA screen targeting SCF E3 ligases, Co-immunoprecipitation, ubiquitination assay, FBXO24 knockdown/overexpression with LSD1 protein stability and tumorigenesis readouts","journal":"Molecular cancer research : MCR","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — unbiased screen plus biochemical ubiquitination assay plus functional tumorigenesis readout; single lab","pmids":["37540490"],"is_preprint":false},{"year":2024,"finding":"FBXO24 interacts with splicing factors SRSF2, SRSF3, and SRSF9 to modulate alternative splicing of mRNAs in round spermatids. FBXO24 genetic knockout in mice causes widespread aberrant splicing events affecting genes critical for sperm formation, leading to abnormal histone retention, incomplete axonemes, oversized chromatoid body, and abnormal mitochondrial coiling, resulting in male sterility. Additionally, FBXO24 interacts with MIWI and SCF subunits to mediate K48-linked polyubiquitination and degradation of MIWI, and FBXO24 depletion causes aberrant piRNA production.","method":"Co-immunoprecipitation (FBXO24 with SRSF2/3/9 and MIWI), genetic knockout mice, RNA-seq (alternative splicing analysis), ubiquitination assay (K48-linked), sperm morphology/motility analysis, piRNA profiling","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo KO mouse model with multiple phenotypic readouts, Co-IP for multiple interaction partners, ubiquitination assay; multiple orthogonal methods in single rigorous study","pmids":["38470475"],"is_preprint":false},{"year":2024,"finding":"FBXO24 ubiquitinates IPO5 (importin-5) in male germ cells. FBXO24 knockout in mice causes abnormal accumulation of RNAs, IPO5, and KPNB1 in sperm flagella and formation of aberrant membraneless electron-dense RNP granules, leading to malformed flagellar structures, impaired sperm motility, and male infertility. IPO5 and KPNB1 associate with stress granules upon oxidative stress or proteasome inhibition, suggesting FBXO24-driven IPO5 degradation prevents aberrant granule accumulation.","method":"FBXO24 knockout mice, in vivo ubiquitination assay for IPO5, electron microscopy, immunofluorescence/localization of RNP proteins, proteasome inhibitor and oxidative stress treatments","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo KO mouse, ubiquitination assay, electron microscopy, multiple functional readouts; peer-reviewed and replicated across two independent publications (PMID 39163107 and preprint 37986737)","pmids":["39163107","37986737"],"is_preprint":false},{"year":2024,"finding":"FBXO24 targets FOXK2 transcription factor for polyubiquitylation and proteasomal degradation in lung epithelia. FOXK2 binds FBXO24 through its carboxyl terminus (aa 428–478), undergoes multisite polyubiquitylation, and is degraded in the nucleus. FOXK2 localizes to mitochondria, and its depletion or expression of C-terminal deletion mutants impairs mitochondrial function. In experimental bacterial pneumonia, Fbxo24 heterozygous mice show preserved FOXK2 levels and mitochondrial function compared to WT littermates.","method":"Co-immunoprecipitation, ubiquitination assay, deletion mutagenesis of FOXK2 C-terminus, cycloheximide chase, mitochondrial function assays, Fbxo24 heterozygous mouse model with bacterial pneumonia","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, in vivo ubiquitination, domain mutagenesis, in vivo mouse validation, mitochondrial functional readout; single lab with multiple orthogonal methods","pmids":["38735474"],"is_preprint":false},{"year":2025,"finding":"FBXO24 mediates K6-linked polyubiquitylation of SLC25A26 (mitochondrial S-adenosylmethionine transporter) at K31, targeting it for degradation. In Fbxo24 knockout mice, elevated SLC25A26 causes mitochondrial fragmentation, disorganized mitochondrial clustering, reduced mitochondrial membrane potential, elevated ROS, and suppressed glycolysis and oxidative phosphorylation, leading to decreased ATP production, sperm motility defects, and male infertility.","method":"Fbxo24 knockout mice, quantitative proteomics to identify substrates, in vitro/in vivo ubiquitination assay with linkage analysis (K6), site-directed mutagenesis (K31R), mitochondrial function assays (membrane potential, ROS, ATP production, glycolysis/OXPHOS)","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — quantitative proteomics substrate identification, ubiquitination assay with linkage specificity, mutagenesis, in vivo KO mouse with multiple functional readouts; single lab but multiple orthogonal methods","pmids":["40657752"],"is_preprint":false},{"year":2025,"finding":"FBXO24 (as part of SCF complex) ubiquitinates FoxP1 via K48- and K63-linked polyubiquitination, co-localizing with FoxP1 in the nucleus of lung epithelial cells. FBXO24 depletion stabilizes FoxP1 and reverses FoxP1-loss-induced unfolded protein response and cell death. Fbxo24 KO mice show elevated FoxP1 in lung and heart and reduced UPR after cigarette smoke exposure. Reciprocally, FoxP1 protein binds the FBXO24 promoter to suppress FBXO24 transcription.","method":"Co-immunoprecipitation, nuclear co-localization (imaging), FBXO24 knockdown/KO with FoxP1 stability and UPR readouts, Fbxo24 KO mouse model, FBXO24 promoter binding assay for FoxP1","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, nuclear co-localization, in vivo KO mouse, functional UPR readout, promoter binding assay; preprint, single lab, multiple methods","pmids":["40501863"],"is_preprint":true}],"current_model":"FBXO24 is the substrate-recognition subunit of an SCF (Skp1-Cullin-F-box) ubiquitin E3 ligase complex that polyubiquitinates multiple substrates—including NDPK-A (K85, regulated by GCN5-mediated K56 acetylation), PRMT6 (K369), LSD1, FOXK2, MIWI, IPO5, SLC25A26 (K6-linked at K31), and FoxP1—to drive their proteasomal degradation, thereby controlling cell proliferation, migration, mitochondrial function, and male fertility; in the testis it additionally interacts with splicing factors SRSF2/3/9 to regulate alternative splicing during spermiogenesis."},"narrative":{"mechanistic_narrative":"FBXO24 is the substrate-recognition subunit of an SCF (Skp1-Cullin-F-box) ubiquitin E3 ligase that selects a diverse set of nuclear and metabolic substrates for polyubiquitination and proteasomal degradation, thereby governing cell proliferation, migration, mitochondrial function, and male fertility [PMID:25582197, PMID:40657752]. Through direct binding it ubiquitinates the nucleoside diphosphate kinase NDPK-A at K85—an event antagonized by GCN5-mediated K56 acetylation that blocks FBXO24 recognition [PMID:25582197]—as well as the methyltransferase PRMT6 (K48-linked at K369) [PMID:32828318], the demethylase LSD1 [PMID:37540490], and the transcription factors FOXK2 and FoxP1 [PMID:38735474, PMID:40501863], coupling its E3 activity to control of proliferation, migration, and the unfolded protein response. In the testis FBXO24 is essential for spermiogenesis: it both degrades the piRNA pathway protein MIWI via K48-linked ubiquitination and interacts with the splicing factors SRSF2, SRSF3, and SRSF9 to control alternative splicing in round spermatids, and its loss causes aberrant histone retention, defective axonemes, and male sterility [PMID:38470475]. Its degradation of the importin IPO5 and of the mitochondrial S-adenosylmethionine transporter SLC25A26 (K6-linked at K31) maintains normal sperm flagellar architecture and mitochondrial bioenergetics, with loss of either leading to motility defects and infertility [PMID:39163107, PMID:37986737, PMID:40657752].","teleology":[{"year":2015,"claim":"Established FBXO24 as a functional SCF E3 ligase by identifying its first substrate and a writer-controlled recognition switch, answering whether FBXO24 directs targeted protein degradation.","evidence":"Co-IP, in vitro ubiquitination, site-directed mutagenesis (K85R, acetyl-mimic K56Q), cycloheximide chase and GCN5 knockdown in a migration model","pmids":["25582197"],"confidence":"High","gaps":["SCF subunit composition (Cullin/Skp1 identity) not defined","physiological context of NDPK-A regulation beyond migration unaddressed"]},{"year":2020,"claim":"Extended the substrate repertoire to a chromatin-modifying enzyme, showing FBXO24 restrains proliferation via K48-linked degradation of PRMT6.","evidence":"Reciprocal Co-IP, ubiquitination assay, K369R mutant, proliferation/migration/invasion assays in H1299 cells with FBXO24 overexpression and PRMT6 knockout","pmids":["32828318"],"confidence":"Medium","gaps":["binding interface mapped only to PRMT6 residues 270-275 without structural detail","single cell line and single lab"]},{"year":2023,"claim":"An unbiased screen of SCF E3 ligases placed FBXO24 as the ligase for LSD1, framing it as a tumor suppressor in breast cancer.","evidence":"Genome-scale siRNA screen of SCF E3 ligases, Co-IP, ubiquitination assay, FBXO24 knockdown/overexpression with tumorigenesis readouts","pmids":["37540490"],"confidence":"Medium","gaps":["ubiquitin linkage type and target lysine on LSD1 not defined","in vivo validation limited"]},{"year":2024,"claim":"In vivo knockout established a non-degradative, splicing-regulatory role for FBXO24 alongside MIWI degradation, defining its essential function in spermiogenesis.","evidence":"Fbxo24 KO mice, Co-IP with SRSF2/3/9 and MIWI, RNA-seq splicing analysis, K48-linked ubiquitination assay, sperm morphology and piRNA profiling","pmids":["38470475"],"confidence":"High","gaps":["mechanism by which FBXO24 influences SRSF-dependent splicing is unresolved","whether splicing role is ligase-dependent unclear"]},{"year":2024,"claim":"Showed FBXO24-driven degradation of IPO5 prevents aberrant RNP granule accumulation in sperm flagella, linking the ligase to flagellar architecture and motility.","evidence":"Fbxo24 KO mice, in vivo IPO5 ubiquitination assay, electron microscopy, RNP protein localization, proteasome-inhibitor and oxidative-stress treatments","pmids":["39163107","37986737"],"confidence":"High","gaps":["target lysine and linkage type on IPO5 not specified","direct contribution of KPNB1 ubiquitination unclear"]},{"year":2024,"claim":"Demonstrated FBXO24 degrades FOXK2 in the nucleus to control mitochondrial function, with relevance to bacterial pneumonia.","evidence":"Reciprocal Co-IP, in vivo ubiquitination, FOXK2 C-terminal deletion mapping, cycloheximide chase, mitochondrial assays, Fbxo24 heterozygous pneumonia mouse model","pmids":["38735474"],"confidence":"High","gaps":["target lysines on FOXK2 not enumerated","connection between nuclear degradation and mitochondrial FOXK2 pool incompletely resolved"]},{"year":2025,"claim":"Identified an atypical K6-linked ubiquitination of the mitochondrial SAM transporter SLC25A26, mechanistically linking FBXO24 to sperm bioenergetics.","evidence":"Fbxo24 KO mice, quantitative proteomics, ubiquitination assay with K6 linkage analysis, K31R mutagenesis, mitochondrial membrane potential/ROS/ATP/OXPHOS readouts","pmids":["40657752"],"confidence":"High","gaps":["functional consequence of K6 linkage versus canonical chains not dissected","whether SLC25A26 is degraded via proteasome given non-K48 linkage unclear"]},{"year":2025,"claim":"Revealed a reciprocal regulatory loop in which FBXO24 ubiquitinates FoxP1 to modulate the unfolded protein response while FoxP1 represses FBXO24 transcription.","evidence":"Co-IP, nuclear co-localization imaging, FBXO24 KO/knockdown with FoxP1 stability and UPR readouts, Fbxo24 KO mice, FBXO24 promoter binding assay (preprint)","pmids":["40501863"],"confidence":"Medium","gaps":["preprint, not peer-reviewed","FoxP1 target lysines and relative roles of K48 versus K63 chains undefined"]},{"year":null,"claim":"The composition of the FBXO24 SCF complex and the structural basis distinguishing its broad substrate set remain undefined.","evidence":"","pmids":[],"confidence":"Low","gaps":["no structural model of FBXO24-substrate recognition","Cullin/Skp1/Rbx partners not directly characterized in the corpus","rules governing K6/K48/K63 linkage choice across substrates unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,2,3,4,5,6]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,1,6]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[7]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[5,7]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1,5,6]},{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[3,4,6]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[3]}],"complexes":["SCF (Skp1-Cullin-F-box) E3 ubiquitin ligase"],"partners":["NDPK-A","PRMT6","LSD1","MIWI","IPO5","FOXK2","FOXP1","SRSF2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O75426","full_name":"F-box only protein 24","aliases":[],"length_aa":580,"mass_kda":64.9,"function":"Substrate recognition component of an SCF (SKP1-CUL1-F-box protein) E3 ubiquitin-protein ligase complex that mediates the ubiquitination and subsequent proteasomal degradation of target proteins. Specifically recognizes the deacetylated form of nucleoside diphosphate kinase A/NME1 and targets it for ubiquitin-mediated degradation (PubMed:25582197). Functions as a key regulator of cell proliferation by targeting PRMT6 for ubiquitination and proteasomal degradation, leading to cell cycle inhibition (PubMed:32828318). Also mediates 'Lys-6'-linked polyubiquitination of the mitochondrial S-adenosylmethionine transporter SLC25A26, targeting it for degradation and thereby maintaining mitochondrial function during spermiogenesis (By similarity). Promotes ubiquitination and degradation of mitochondrial aspartyl-tRNA synthetase DARS2, contributing to immunosuppressive activity (PubMed:39039092). In the nucleus, targets FOXK2 for ubiquitin-mediated degradation, a process that critically modulates mitochondrial respiration and cellular bioenergetics in lung epithelial cells (PubMed:38735474)","subcellular_location":"Mitochondrion; Nucleus","url":"https://www.uniprot.org/uniprotkb/O75426/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/FBXO24","classification":"Not Classified","n_dependent_lines":5,"n_total_lines":1208,"dependency_fraction":0.0041390728476821195},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/FBXO24","total_profiled":1310},"omim":[{"mim_id":"609097","title":"F-BOX ONLY PROTEIN 24; FBXO24","url":"https://www.omim.org/entry/609097"},{"mim_id":"156490","title":"NME/NM23 NUCLEOSIDE DIPHOSPHATE KINASE 1; NME1","url":"https://www.omim.org/entry/156490"},{"mim_id":"154360","title":"MALTASE-GLUCOAMYLASE; MGAM","url":"https://www.omim.org/entry/154360"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"testis","ntpm":77.1}],"url":"https://www.proteinatlas.org/search/FBXO24"},"hgnc":{"alias_symbol":["FBX24"],"prev_symbol":[]},"alphafold":{"accession":"O75426","domains":[{"cath_id":"1.20.1280.50","chopping":"39-110","consensus_level":"medium","plddt":84.1772,"start":39,"end":110},{"cath_id":"-","chopping":"343-457","consensus_level":"medium","plddt":91.8403,"start":343,"end":457},{"cath_id":"-","chopping":"515-562","consensus_level":"high","plddt":76.7727,"start":515,"end":562}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O75426","model_url":"https://alphafold.ebi.ac.uk/files/AF-O75426-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O75426-F1-predicted_aligned_error_v6.png","plddt_mean":76.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=FBXO24","jax_strain_url":"https://www.jax.org/strain/search?query=FBXO24"},"sequence":{"accession":"O75426","fasta_url":"https://rest.uniprot.org/uniprotkb/O75426.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O75426/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O75426"}},"corpus_meta":[{"pmid":"25582197","id":"PMC_25582197","title":"The ubiquitin E3 ligase SCF-FBXO24 recognizes deacetylated nucleoside diphosphate kinase A to enhance its degradation.","date":"2015","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/25582197","citation_count":24,"is_preprint":false},{"pmid":"37540490","id":"PMC_37540490","title":"FBXO24 Suppresses Breast Cancer Tumorigenesis by Targeting LSD1 for Ubiquitination.","date":"2023","source":"Molecular cancer research : MCR","url":"https://pubmed.ncbi.nlm.nih.gov/37540490","citation_count":12,"is_preprint":false},{"pmid":"32828318","id":"PMC_32828318","title":"SCF-FBXO24 regulates cell proliferation by mediating ubiquitination and degradation of PRMT6.","date":"2020","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/32828318","citation_count":12,"is_preprint":false},{"pmid":"38470475","id":"PMC_38470475","title":"FBXO24 modulates mRNA alternative splicing and MIWI degradation and is required for normal sperm formation and male fertility.","date":"2024","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/38470475","citation_count":9,"is_preprint":false},{"pmid":"39163107","id":"PMC_39163107","title":"FBXO24 deletion causes abnormal accumulation of membraneless electron-dense granules in sperm flagella and male infertility.","date":"2024","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/39163107","citation_count":7,"is_preprint":false},{"pmid":"38735474","id":"PMC_38735474","title":"FOXK2 targeting by the SCF-E3 ligase subunit FBXO24 for ubiquitin mediated degradation modulates mitochondrial respiration.","date":"2024","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/38735474","citation_count":7,"is_preprint":false},{"pmid":"40657752","id":"PMC_40657752","title":"FBXO24 targets SLC25A26 for K6-linked polyubiquitylation to maintain mitochondrial function during spermiogenesis.","date":"2025","source":"Development (Cambridge, England)","url":"https://pubmed.ncbi.nlm.nih.gov/40657752","citation_count":5,"is_preprint":false},{"pmid":"40501863","id":"PMC_40501863","title":"Reciprocal Regulation Between the SCFFBXO24 Ubiquitin E3 Ligase and FoxP1 Protein.","date":"2025","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/40501863","citation_count":0,"is_preprint":false},{"pmid":"37986737","id":"PMC_37986737","title":"FBXO24 deletion causes abnormal accumulation of membraneless electron-dense granules in sperm flagella and male infertility.","date":"2024","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/37986737","citation_count":0,"is_preprint":false},{"pmid":"42168344","id":"PMC_42168344","title":"Hsa-circ_0081481-miR 3960-FBXO24 regulatory axis in non-obstructive azoospermia identifies potential biomarkers of spermatogenic failure.","date":"2026","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/42168344","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.05.23.653845","title":"Reciprocal Regulation Between the SCF  <sup>FBXO24</sup>  Ubiquitin E3 Ligase and FoxP1 Protein","date":"2025-05-28","source":"bioRxiv","url":"https://doi.org/10.1101/2025.05.23.653845","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7913,"output_tokens":2838,"usd":0.033155,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10035,"output_tokens":3150,"usd":0.064463,"stage2_stop_reason":"end_turn"},"total_usd":0.097618,"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\": 2015,\n      \"finding\": \"SCF-FBXO24 acts as a ubiquitin E3 ligase that polyubiquitinates NDPK-A at K85, targeting it for proteasomal degradation. The interaction requires NDPK-A residues L55 and K56; K56 acetylation by GCN5 impairs FBXO24 binding and stabilizes NDPK-A, while GCN5 depletion accelerates NDPK-A degradation. FBXO24 silencing or expression of an acetylation mimic increases NDPK-A lifespan and impairs cell migration and wound healing.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ubiquitination assay, site-directed mutagenesis (K85R, K56Q acetylation mimic), cycloheximide chase, GCN5 knockdown, wound-healing/migration assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro ubiquitination assay plus mutagenesis at multiple sites, GCN5 writer identified, functional phenotype confirmed; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"25582197\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"FBXO24 binds PRMT6 at residues 270–275 and mediates K48-linked polyubiquitination of PRMT6 at K369, targeting it for proteasomal degradation. A K369R PRMT6 mutant is resistant to degradation, and PRMT6 K369R overexpression drives cell cycle progression and proliferation. FBXO24 overexpression or PRMT6 knockout inhibits cell proliferation, migration, and invasion in H1299 cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, site-directed mutagenesis (K369R), cycloheximide chase, cell proliferation/migration/invasion assays, FBXO24 overexpression and PRMT6 knockout\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, mutagenesis, functional cellular readouts; single lab\",\n      \"pmids\": [\"32828318\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"FBXO24 functions as an E3 ligase that ubiquitinates LSD1 (KDM1A), targeting it for proteasomal degradation. Identified via unbiased siRNA screen of all human SCF family E3 ligases; FBXO24 inhibits LSD1-induced tumorigenesis and acts as a tumor suppressor in breast cancer cells.\",\n      \"method\": \"Unbiased siRNA screen targeting SCF E3 ligases, Co-immunoprecipitation, ubiquitination assay, FBXO24 knockdown/overexpression with LSD1 protein stability and tumorigenesis readouts\",\n      \"journal\": \"Molecular cancer research : MCR\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — unbiased screen plus biochemical ubiquitination assay plus functional tumorigenesis readout; single lab\",\n      \"pmids\": [\"37540490\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"FBXO24 interacts with splicing factors SRSF2, SRSF3, and SRSF9 to modulate alternative splicing of mRNAs in round spermatids. FBXO24 genetic knockout in mice causes widespread aberrant splicing events affecting genes critical for sperm formation, leading to abnormal histone retention, incomplete axonemes, oversized chromatoid body, and abnormal mitochondrial coiling, resulting in male sterility. Additionally, FBXO24 interacts with MIWI and SCF subunits to mediate K48-linked polyubiquitination and degradation of MIWI, and FBXO24 depletion causes aberrant piRNA production.\",\n      \"method\": \"Co-immunoprecipitation (FBXO24 with SRSF2/3/9 and MIWI), genetic knockout mice, RNA-seq (alternative splicing analysis), ubiquitination assay (K48-linked), sperm morphology/motility analysis, piRNA profiling\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo KO mouse model with multiple phenotypic readouts, Co-IP for multiple interaction partners, ubiquitination assay; multiple orthogonal methods in single rigorous study\",\n      \"pmids\": [\"38470475\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"FBXO24 ubiquitinates IPO5 (importin-5) in male germ cells. FBXO24 knockout in mice causes abnormal accumulation of RNAs, IPO5, and KPNB1 in sperm flagella and formation of aberrant membraneless electron-dense RNP granules, leading to malformed flagellar structures, impaired sperm motility, and male infertility. IPO5 and KPNB1 associate with stress granules upon oxidative stress or proteasome inhibition, suggesting FBXO24-driven IPO5 degradation prevents aberrant granule accumulation.\",\n      \"method\": \"FBXO24 knockout mice, in vivo ubiquitination assay for IPO5, electron microscopy, immunofluorescence/localization of RNP proteins, proteasome inhibitor and oxidative stress treatments\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo KO mouse, ubiquitination assay, electron microscopy, multiple functional readouts; peer-reviewed and replicated across two independent publications (PMID 39163107 and preprint 37986737)\",\n      \"pmids\": [\"39163107\", \"37986737\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"FBXO24 targets FOXK2 transcription factor for polyubiquitylation and proteasomal degradation in lung epithelia. FOXK2 binds FBXO24 through its carboxyl terminus (aa 428–478), undergoes multisite polyubiquitylation, and is degraded in the nucleus. FOXK2 localizes to mitochondria, and its depletion or expression of C-terminal deletion mutants impairs mitochondrial function. In experimental bacterial pneumonia, Fbxo24 heterozygous mice show preserved FOXK2 levels and mitochondrial function compared to WT littermates.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, deletion mutagenesis of FOXK2 C-terminus, cycloheximide chase, mitochondrial function assays, Fbxo24 heterozygous mouse model with bacterial pneumonia\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, in vivo ubiquitination, domain mutagenesis, in vivo mouse validation, mitochondrial functional readout; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"38735474\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"FBXO24 mediates K6-linked polyubiquitylation of SLC25A26 (mitochondrial S-adenosylmethionine transporter) at K31, targeting it for degradation. In Fbxo24 knockout mice, elevated SLC25A26 causes mitochondrial fragmentation, disorganized mitochondrial clustering, reduced mitochondrial membrane potential, elevated ROS, and suppressed glycolysis and oxidative phosphorylation, leading to decreased ATP production, sperm motility defects, and male infertility.\",\n      \"method\": \"Fbxo24 knockout mice, quantitative proteomics to identify substrates, in vitro/in vivo ubiquitination assay with linkage analysis (K6), site-directed mutagenesis (K31R), mitochondrial function assays (membrane potential, ROS, ATP production, glycolysis/OXPHOS)\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — quantitative proteomics substrate identification, ubiquitination assay with linkage specificity, mutagenesis, in vivo KO mouse with multiple functional readouts; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"40657752\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"FBXO24 (as part of SCF complex) ubiquitinates FoxP1 via K48- and K63-linked polyubiquitination, co-localizing with FoxP1 in the nucleus of lung epithelial cells. FBXO24 depletion stabilizes FoxP1 and reverses FoxP1-loss-induced unfolded protein response and cell death. Fbxo24 KO mice show elevated FoxP1 in lung and heart and reduced UPR after cigarette smoke exposure. Reciprocally, FoxP1 protein binds the FBXO24 promoter to suppress FBXO24 transcription.\",\n      \"method\": \"Co-immunoprecipitation, nuclear co-localization (imaging), FBXO24 knockdown/KO with FoxP1 stability and UPR readouts, Fbxo24 KO mouse model, FBXO24 promoter binding assay for FoxP1\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, nuclear co-localization, in vivo KO mouse, functional UPR readout, promoter binding assay; preprint, single lab, multiple methods\",\n      \"pmids\": [\"40501863\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"FBXO24 is the substrate-recognition subunit of an SCF (Skp1-Cullin-F-box) ubiquitin E3 ligase complex that polyubiquitinates multiple substrates—including NDPK-A (K85, regulated by GCN5-mediated K56 acetylation), PRMT6 (K369), LSD1, FOXK2, MIWI, IPO5, SLC25A26 (K6-linked at K31), and FoxP1—to drive their proteasomal degradation, thereby controlling cell proliferation, migration, mitochondrial function, and male fertility; in the testis it additionally interacts with splicing factors SRSF2/3/9 to regulate alternative splicing during spermiogenesis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"FBXO24 is the substrate-recognition subunit of an SCF (Skp1-Cullin-F-box) ubiquitin E3 ligase that selects a diverse set of nuclear and metabolic substrates for polyubiquitination and proteasomal degradation, thereby governing cell proliferation, migration, mitochondrial function, and male fertility [#0, #6]. Through direct binding it ubiquitinates the nucleoside diphosphate kinase NDPK-A at K85—an event antagonized by GCN5-mediated K56 acetylation that blocks FBXO24 recognition [#0]—as well as the methyltransferase PRMT6 (K48-linked at K369) [#1], the demethylase LSD1 [#2], and the transcription factors FOXK2 and FoxP1 [#5, #7], coupling its E3 activity to control of proliferation, migration, and the unfolded protein response. In the testis FBXO24 is essential for spermiogenesis: it both degrades the piRNA pathway protein MIWI via K48-linked ubiquitination and interacts with the splicing factors SRSF2, SRSF3, and SRSF9 to control alternative splicing in round spermatids, and its loss causes aberrant histone retention, defective axonemes, and male sterility [#3]. Its degradation of the importin IPO5 and of the mitochondrial S-adenosylmethionine transporter SLC25A26 (K6-linked at K31) maintains normal sperm flagellar architecture and mitochondrial bioenergetics, with loss of either leading to motility defects and infertility [#4, #6].\",\n  \"teleology\": [\n    {\n      \"year\": 2015,\n      \"claim\": \"Established FBXO24 as a functional SCF E3 ligase by identifying its first substrate and a writer-controlled recognition switch, answering whether FBXO24 directs targeted protein degradation.\",\n      \"evidence\": \"Co-IP, in vitro ubiquitination, site-directed mutagenesis (K85R, acetyl-mimic K56Q), cycloheximide chase and GCN5 knockdown in a migration model\",\n      \"pmids\": [\"25582197\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"SCF subunit composition (Cullin/Skp1 identity) not defined\", \"physiological context of NDPK-A regulation beyond migration unaddressed\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Extended the substrate repertoire to a chromatin-modifying enzyme, showing FBXO24 restrains proliferation via K48-linked degradation of PRMT6.\",\n      \"evidence\": \"Reciprocal Co-IP, ubiquitination assay, K369R mutant, proliferation/migration/invasion assays in H1299 cells with FBXO24 overexpression and PRMT6 knockout\",\n      \"pmids\": [\"32828318\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"binding interface mapped only to PRMT6 residues 270-275 without structural detail\", \"single cell line and single lab\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"An unbiased screen of SCF E3 ligases placed FBXO24 as the ligase for LSD1, framing it as a tumor suppressor in breast cancer.\",\n      \"evidence\": \"Genome-scale siRNA screen of SCF E3 ligases, Co-IP, ubiquitination assay, FBXO24 knockdown/overexpression with tumorigenesis readouts\",\n      \"pmids\": [\"37540490\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"ubiquitin linkage type and target lysine on LSD1 not defined\", \"in vivo validation limited\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"In vivo knockout established a non-degradative, splicing-regulatory role for FBXO24 alongside MIWI degradation, defining its essential function in spermiogenesis.\",\n      \"evidence\": \"Fbxo24 KO mice, Co-IP with SRSF2/3/9 and MIWI, RNA-seq splicing analysis, K48-linked ubiquitination assay, sperm morphology and piRNA profiling\",\n      \"pmids\": [\"38470475\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"mechanism by which FBXO24 influences SRSF-dependent splicing is unresolved\", \"whether splicing role is ligase-dependent unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Showed FBXO24-driven degradation of IPO5 prevents aberrant RNP granule accumulation in sperm flagella, linking the ligase to flagellar architecture and motility.\",\n      \"evidence\": \"Fbxo24 KO mice, in vivo IPO5 ubiquitination assay, electron microscopy, RNP protein localization, proteasome-inhibitor and oxidative-stress treatments\",\n      \"pmids\": [\"39163107\", \"37986737\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"target lysine and linkage type on IPO5 not specified\", \"direct contribution of KPNB1 ubiquitination unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstrated FBXO24 degrades FOXK2 in the nucleus to control mitochondrial function, with relevance to bacterial pneumonia.\",\n      \"evidence\": \"Reciprocal Co-IP, in vivo ubiquitination, FOXK2 C-terminal deletion mapping, cycloheximide chase, mitochondrial assays, Fbxo24 heterozygous pneumonia mouse model\",\n      \"pmids\": [\"38735474\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"target lysines on FOXK2 not enumerated\", \"connection between nuclear degradation and mitochondrial FOXK2 pool incompletely resolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified an atypical K6-linked ubiquitination of the mitochondrial SAM transporter SLC25A26, mechanistically linking FBXO24 to sperm bioenergetics.\",\n      \"evidence\": \"Fbxo24 KO mice, quantitative proteomics, ubiquitination assay with K6 linkage analysis, K31R mutagenesis, mitochondrial membrane potential/ROS/ATP/OXPHOS readouts\",\n      \"pmids\": [\"40657752\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"functional consequence of K6 linkage versus canonical chains not dissected\", \"whether SLC25A26 is degraded via proteasome given non-K48 linkage unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Revealed a reciprocal regulatory loop in which FBXO24 ubiquitinates FoxP1 to modulate the unfolded protein response while FoxP1 represses FBXO24 transcription.\",\n      \"evidence\": \"Co-IP, nuclear co-localization imaging, FBXO24 KO/knockdown with FoxP1 stability and UPR readouts, Fbxo24 KO mice, FBXO24 promoter binding assay (preprint)\",\n      \"pmids\": [\"40501863\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"preprint, not peer-reviewed\", \"FoxP1 target lysines and relative roles of K48 versus K63 chains undefined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The composition of the FBXO24 SCF complex and the structural basis distinguishing its broad substrate set remain undefined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"no structural model of FBXO24-substrate recognition\", \"Cullin/Skp1/Rbx partners not directly characterized in the corpus\", \"rules governing K6/K48/K63 linkage choice across substrates unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 2, 3, 4, 5, 6]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 1, 6]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [5, 7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 5, 6]},\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [3, 4, 6]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"complexes\": [\"SCF (Skp1-Cullin-F-box) E3 ubiquitin ligase\"],\n    \"partners\": [\"NDPK-A\", \"PRMT6\", \"LSD1\", \"MIWI\", \"IPO5\", \"FOXK2\", \"FOXP1\", \"SRSF2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":4,"faith_total":4,"faith_pct":100.0}}