{"gene":"FBXO15","run_date":"2026-06-09T23:54:43","timeline":{"discoveries":[{"year":2003,"finding":"Fbxo15 is a direct transcriptional target of Oct3/4 and Sox2: an 18-bp enhancer element ~500 bp upstream of the Fbxo15 transcription start site contains an octamer-like motif and an adjacent Sox-binding motif; cooperative binding of Oct3/4 and Sox2 to this enhancer was demonstrated by gel mobility shift assay, and point mutation of either motif abolished enhancer activity.","method":"Reporter gene (luciferase) assay, gel mobility shift assay (EMSA), site-directed mutagenesis, Oct3/4 inducible knockout in ES cells","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro binding (EMSA) combined with mutagenesis and reporter assays in a single rigorous study, replicated conceptually by Okumura-Nakanishi et al. 2004","pmids":["12665572"],"is_preprint":false},{"year":2003,"finding":"Fbxo15 knockout mice are viable, fertile, and show no gross developmental defects; Fbxo15-null ES cells are normal in morphology, proliferation, and differentiation, demonstrating that Fbxo15 is dispensable for ES cell self-renewal and mouse development.","method":"Homologous recombination knockout (beta-galactosidase knock-in), phenotypic analysis of null mice and ES cells","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean germline knockout with multiple phenotypic readouts in a single focused study","pmids":["12665572"],"is_preprint":false},{"year":2004,"finding":"Oct-3/4 and Sox2 cooperatively regulate Fbxo15 expression through an octamer-like and Sox-binding motif in the Fbxo15 enhancer, placing Fbxo15 in a broader regulatory circuit controlled by the Oct-3/4·Sox2 complex alongside Fgf-4, Utf1, and Sox2 genes.","method":"EMSA with ES-derived cell extracts, luciferase reporter assay, inducible Oct-3/4 knockout in ES cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — independent replication of Oct3/4·Sox2 cooperative binding to Fbxo15 enhancer using EMSA and reporter assays in a separate lab","pmids":["15557334"],"is_preprint":false},{"year":2013,"finding":"FBXO15 functions as an F-box protein within an SCF(Fbxo15) E3 ubiquitin ligase complex (with Skp1/Cullin1) to ubiquitinate P-glycoprotein (ABCB1), targeting it for proteasomal degradation; co-precipitated P-gp and identified Ube2r1/Cdc34 as the cognate E2 ubiquitin-conjugating enzyme; exogenous FBXO15 enhanced P-gp ubiquitination while FBXO15 knockdown suppressed ubiquitination and increased P-gp protein without affecting mRNA.","method":"Co-immunoprecipitation, MALDI-TOF mass spectrometry, exogenous overexpression, siRNA knockdown, proteasome inhibitor (MG132) treatment","journal":"Cancer science","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, gain- and loss-of-function, E2 partner identification, replicated across multiple orthogonal methods in one study and confirmed in follow-up (PMID:27786305)","pmids":["23465077"],"is_preprint":false},{"year":2014,"finding":"FBXO15 mediates proteasomal degradation of cardiolipin synthase 1 (CLS1) in lung epithelia during S. aureus infection; PINK1 kinase binds CLS1, phosphorylates Thr219 to create a phosphodegron recognized by FBXO15, and FBXO15 interaction with CLS1 leads to its ubiquitin-mediated disposal, disrupting cardiolipin synthesis and mitochondrial function.","method":"Co-immunoprecipitation, site-directed mutagenesis (Thr219), overexpression and knockdown of FBXO15 and PINK1, Pink1 knockout mice, proteasome inhibitor assays","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP, phospho-mutant mutagenesis, KO mouse model with defined phenotype, and pathway placement via multiple orthogonal methods","pmids":["24703837"],"is_preprint":false},{"year":2016,"finding":"RSK1 phosphorylates Thr162 on Ube2r1, destabilizing Ube2r1 via self-ubiquitination and thereby protecting P-gp from SCF(Fbxo15)-mediated ubiquitination; FBXO15 knockdown (along with Ube2r1 knockdown) blocked MEK inhibitor-induced P-gp downregulation, confirming FBXO15 is required for P-gp degradation downstream of the MAPK pathway.","method":"siRNA knockdown, phospho-site mutagenesis (Thr162 UBE2R1), proteasome inhibitor rescue, drug sensitivity and rhodamine 123 accumulation assays","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — single lab, multiple orthogonal methods but FBXO15's role is confirmed indirectly through epistasis/knockdown rather than direct biochemical reconstitution","pmids":["27786305"],"is_preprint":false},{"year":2017,"finding":"Fbxo15 recognizes and ubiquitinates KBP (encoded by Kif1bp) in a manner dependent on acetylation of KBP Lys501 by GCN5L1 (using TDH-derived acetyl-CoA), thereby limiting mitochondrial biogenesis in mouse embryonic stem cells; loss of KBP degradation causes unscheduled increase in mitochondria, enhanced respiration and ROS, and impaired proliferation.","method":"Mass spectrometry-based interactome (affinity purification-MS), Co-immunoprecipitation, acetylation mutagenesis (Lys501), Fbxo15 knockdown/rescue, mitochondrial mass and respiration assays, embryoid body formation","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — AP-MS substrate identification with mutagenesis validation, multiple orthogonal methods (Co-IP, metabolic assays, genetic rescue) in a single rigorous study","pmids":["28319092"],"is_preprint":false},{"year":2020,"finding":"Oroxylin A induces transcription of FBXO15, and activated FBXO15 protein binds to and mediates proteasomal degradation of CHOP (C/EBP-homologous protein) in CLP sepsis mice, relieving immunoparalysis by reducing CHOP-driven anti-inflammatory signaling.","method":"Co-immunoprecipitation, FBXO15 overexpression/knockdown, in vivo CLP mouse model, cytokine measurements, proteasome pathway assays","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and in vivo model with defined phenotype, single lab, mechanistic pathway placement but limited biochemical reconstitution detail in abstract","pmids":["33159144"],"is_preprint":false},{"year":2024,"finding":"Gastrodin binds directly to FBXO15 (identified by molecular docking, molecular dynamics simulations, and microscale thermophoresis) and inhibits FBXO15-P-gp interaction, reducing P-gp ubiquitination and proteasomal degradation at the blood-brain barrier, thereby increasing P-gp expression and Aβ clearance in APP/PS1 mice.","method":"Molecular docking, molecular dynamics simulations, microscale thermophoresis (MST), co-immunoprecipitation, western blot, in vivo APP/PS1 mouse model, Morris Water Maze","journal":"Phytomedicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding measured by MST, Co-IP for mechanism, and in vivo validation, but single lab and limited mutagenesis/reconstitution detail","pmids":["39541666"],"is_preprint":false}],"current_model":"FBXO15 (FBX15) is an F-box protein that forms SCF(FBXO15) E3 ubiquitin ligase complexes and targets specific substrates—including P-glycoprotein/ABCB1 (with E2 partner Ube2r1), cardiolipin synthase 1 (via a PINK1-generated phosphodegron at Thr219), KBP/Kif1bp (after GCN5L1-dependent acetylation of Lys501), and CHOP—for ubiquitin-proteasomal degradation; in embryonic stem cells its transcription is directly activated by cooperative Oct3/4–Sox2 binding to an 18-bp enhancer, and it restrains mitochondrial biogenesis by degrading KBP, while being dispensable for ES cell self-renewal and mouse development."},"narrative":{"mechanistic_narrative":"FBXO15 is an F-box protein that assembles into an SCF(FBXO15) E3 ubiquitin ligase complex (with Skp1 and Cullin1) to direct selective substrates toward proteasomal degradation [PMID:23465077]. It targets a structurally diverse set of substrates whose recognition is frequently gated by post-translational modification: P-glycoprotein/ABCB1, ubiquitinated using the E2 enzyme Ube2r1/Cdc34 [PMID:23465077]; cardiolipin synthase 1, recognized via a PINK1-generated phosphodegron at Thr219 during bacterial infection [PMID:24703837]; the KBP protein (Kif1bp), recognized after GCN5L1-dependent acetylation of Lys501 [PMID:28319092]; and the transcription factor CHOP [PMID:33159144]. Through degradation of KBP, FBXO15 restrains mitochondrial biogenesis in embryonic stem cells, with its loss causing unscheduled mitochondrial expansion, elevated respiration and ROS, and impaired proliferation [PMID:28319092]. Its degradation of P-gp is embedded in MAPK signaling, where RSK1 phosphorylation of Ube2r1 modulates the pathway, and FBXO15 is required for MEK-inhibitor-induced P-gp downregulation [PMID:27786305]. In embryonic stem cells, Fbxo15 transcription is directly activated by cooperative Oct3/4–Sox2 binding to an 18-bp enhancer upstream of its transcription start site, placing it within the pluripotency regulatory circuit [PMID:12665572, PMID:15557334]; despite this, Fbxo15 is dispensable for ES cell self-renewal and mouse development [PMID:12665572].","teleology":[{"year":2003,"claim":"Established that Fbxo15 is a direct transcriptional output of the core pluripotency factors, answering how this gene is wired into the ES cell program.","evidence":"Luciferase reporter and EMSA with site-directed mutagenesis plus inducible Oct3/4 knockout in ES cells, defining an 18-bp Oct3/4–Sox2 enhancer","pmids":["12665572"],"confidence":"High","gaps":["Does not define a molecular function for the FBXO15 protein itself","Enhancer mapping does not address the protein's biochemical role"]},{"year":2003,"claim":"Determined the loss-of-function consequence, showing Fbxo15 is not required for pluripotency or development despite its ES-cell-specific expression.","evidence":"Germline beta-galactosidase knock-in knockout mice and null ES cells with phenotypic analysis","pmids":["12665572"],"confidence":"High","gaps":["Lack of phenotype leaves the protein's molecular activity undefined","Possible functional redundancy not tested"]},{"year":2004,"claim":"Independently confirmed cooperative Oct3/4–Sox2 control of Fbxo15 and positioned it within a shared regulatory circuit with Fgf-4, Utf1 and Sox2.","evidence":"EMSA with ES-derived extracts and luciferase reporter assays in a separate lab","pmids":["15557334"],"confidence":"High","gaps":["Reinforces transcriptional regulation but adds no mechanism for the protein"]},{"year":2013,"claim":"Defined the core biochemical identity of FBXO15 as an SCF E3 ligase substrate-recognition subunit and identified its first substrate and cognate E2 enzyme.","evidence":"Reciprocal Co-IP, MALDI-TOF MS, gain/loss-of-function, and MG132 rescue identifying P-glycoprotein as substrate and Ube2r1/Cdc34 as E2","pmids":["23465077"],"confidence":"High","gaps":["Degron on P-gp not mapped","No structural model of the SCF(FBXO15)–substrate interaction"]},{"year":2014,"claim":"Revealed that FBXO15 substrate recognition can be gated by upstream kinase-generated phosphodegrons, linking it to mitochondrial lipid metabolism during infection.","evidence":"Co-IP, Thr219 phospho-mutant mutagenesis, PINK1 manipulation and Pink1 knockout mice, proteasome inhibitor assays in lung epithelia","pmids":["24703837"],"confidence":"High","gaps":["How FBXO15 distinguishes phosphorylated CLS1 structurally not defined","Physiological scope beyond S. aureus infection unclear"]},{"year":2016,"claim":"Placed FBXO15-mediated P-gp degradation downstream of MAPK signaling and showed the pathway is tuned by RSK1 phosphorylation of the E2 enzyme.","evidence":"siRNA knockdown, Thr162 UBE2R1 phospho-mutagenesis, proteasome rescue, and drug/rhodamine 123 accumulation assays","pmids":["27786305"],"confidence":"Medium","gaps":["FBXO15's role shown by epistasis/knockdown rather than direct reconstitution","Direct effect of RSK1 on FBXO15 itself not tested"]},{"year":2017,"claim":"Connected FBXO15 to control of mitochondrial biogenesis in ES cells via acetylation-dependent recognition of the KBP substrate.","evidence":"AP-MS interactome, Co-IP, Lys501 acetylation mutagenesis, GCN5L1 link, knockdown/rescue, and mitochondrial mass/respiration assays","pmids":["28319092"],"confidence":"High","gaps":["Whether this reconciles with the lack of developmental phenotype not addressed","Structural basis for acetyl-degron recognition undefined"]},{"year":2020,"claim":"Extended FBXO15 substrate repertoire to the transcription factor CHOP, implicating it in resolving sepsis-associated immunoparalysis.","evidence":"Co-IP, overexpression/knockdown, CLP mouse model and cytokine measurements following Oroxylin A induction","pmids":["33159144"],"confidence":"Medium","gaps":["CHOP degron not mapped","Limited biochemical reconstitution of the ubiquitination event"]},{"year":2024,"claim":"Demonstrated the FBXO15–P-gp interaction is pharmacologically targetable, with direct ligand binding modulating P-gp levels at the blood-brain barrier.","evidence":"Molecular docking/MD simulations, MST direct binding, Co-IP, western blot, and APP/PS1 mouse behavioral assays with Gastrodin","pmids":["39541666"],"confidence":"Medium","gaps":["Binding site on FBXO15 not validated by mutagenesis","Single lab, limited reconstitution detail"]},{"year":null,"claim":"The structural basis by which a single F-box protein recognizes its diverse modified degrons (phospho-, acetyl-) and how its multiple substrate-specific roles are coordinated in vivo remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of SCF(FBXO15) bound to any substrate","Reconciliation of substrate diversity with the dispensable knockout phenotype not established","Tissue-specific substrate selection mechanisms unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[3,4,6]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[3]}],"localization":[],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[3,4,6]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,2]}],"complexes":["SCF(FBXO15) E3 ubiquitin ligase"],"partners":["SKP1","CUL1","UBE2R1","ABCB1","CLS1","KIF1BP","PINK1","DDIT3"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8NCQ5","full_name":"F-box only protein 15","aliases":[],"length_aa":510,"mass_kda":57.3,"function":"Substrate-recognition component of the SCF (SKP1-CUL1-F-box protein)-type E3 ubiquitin ligase complex","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q8NCQ5/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/FBXO15","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/FBXO15","total_profiled":1310},"omim":[{"mim_id":"609093","title":"F-BOX ONLY PROTEIN 15; FBXO15","url":"https://www.omim.org/entry/609093"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"fallopian tube","ntpm":3.8},{"tissue":"testis","ntpm":7.2}],"url":"https://www.proteinatlas.org/search/FBXO15"},"hgnc":{"alias_symbol":["MGC39671","FBX15"],"prev_symbol":[]},"alphafold":{"accession":"Q8NCQ5","domains":[{"cath_id":"1.20.1280","chopping":"78-127_143-176","consensus_level":"medium","plddt":80.5626,"start":78,"end":176},{"cath_id":"-","chopping":"184-253_262-326","consensus_level":"high","plddt":88.5604,"start":184,"end":326},{"cath_id":"-","chopping":"355-507","consensus_level":"high","plddt":88.8171,"start":355,"end":507}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8NCQ5","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8NCQ5-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8NCQ5-F1-predicted_aligned_error_v6.png","plddt_mean":76.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=FBXO15","jax_strain_url":"https://www.jax.org/strain/search?query=FBXO15"},"sequence":{"accession":"Q8NCQ5","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8NCQ5.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8NCQ5/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8NCQ5"}},"corpus_meta":[{"pmid":"17554338","id":"PMC_17554338","title":"Generation of germline-competent induced pluripotent stem cells.","date":"2007","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/17554338","citation_count":3166,"is_preprint":false},{"pmid":"22980981","id":"PMC_22980981","title":"Single-cell expression analyses during cellular reprogramming reveal an early stochastic and a late hierarchic phase.","date":"2012","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/22980981","citation_count":654,"is_preprint":false},{"pmid":"15557334","id":"PMC_15557334","title":"Oct-3/4 and Sox2 regulate Oct-3/4 gene in embryonic stem cells.","date":"2004","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15557334","citation_count":295,"is_preprint":false},{"pmid":"12665572","id":"PMC_12665572","title":"Fbx15 is a novel target of Oct3/4 but is dispensable for embryonic stem cell self-renewal and mouse development.","date":"2003","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/12665572","citation_count":201,"is_preprint":false},{"pmid":"23465077","id":"PMC_23465077","title":"FBXO15 regulates P-glycoprotein/ABCB1 expression through the ubiquitin--proteasome pathway in cancer cells.","date":"2013","source":"Cancer science","url":"https://pubmed.ncbi.nlm.nih.gov/23465077","citation_count":55,"is_preprint":false},{"pmid":"28319092","id":"PMC_28319092","title":"The TDH-GCN5L1-Fbxo15-KBP axis limits mitochondrial biogenesis in mouse embryonic stem cells.","date":"2017","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/28319092","citation_count":45,"is_preprint":false},{"pmid":"24703837","id":"PMC_24703837","title":"E3 ligase subunit Fbxo15 and PINK1 kinase regulate cardiolipin synthase 1 stability and mitochondrial function in pneumonia.","date":"2014","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/24703837","citation_count":43,"is_preprint":false},{"pmid":"20360837","id":"PMC_20360837","title":"Constitutive expression of pluripotency-associated genes in mesodermal progenitor cells (MPCs).","date":"2010","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/20360837","citation_count":40,"is_preprint":false},{"pmid":"27649508","id":"PMC_27649508","title":"SCF Ubiquitin Ligase F-box Protein Fbx15 Controls Nuclear Co-repressor Localization, Stress Response and Virulence of the Human Pathogen Aspergillus fumigatus.","date":"2016","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/27649508","citation_count":39,"is_preprint":false},{"pmid":"23258129","id":"PMC_23258129","title":"Leukemia inhibitory factor is essential for subventricular zone neural stem cell and progenitor homeostasis as revealed by a novel flow cytometric analysis.","date":"2012","source":"Developmental neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/23258129","citation_count":34,"is_preprint":false},{"pmid":"24376456","id":"PMC_24376456","title":"Gene-alcohol interactions identify several novel blood pressure loci including a promising locus near SLC16A9.","date":"2013","source":"Frontiers in genetics","url":"https://pubmed.ncbi.nlm.nih.gov/24376456","citation_count":32,"is_preprint":false},{"pmid":"27786305","id":"PMC_27786305","title":"RSK1 protects P-glycoprotein/ABCB1 against ubiquitin-proteasomal degradation by downregulating the ubiquitin-conjugating enzyme E2 R1.","date":"2016","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/27786305","citation_count":29,"is_preprint":false},{"pmid":"20499253","id":"PMC_20499253","title":"Genetic determinants of autism in individuals with deletions of 18q.","date":"2010","source":"Human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/20499253","citation_count":18,"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":"34347012","id":"PMC_34347012","title":"Ubiquitination-Related miRNA-mRNA Interaction Is a Potential Mechanism in the Progression of Retinoblastoma.","date":"2021","source":"Investigative ophthalmology & visual science","url":"https://pubmed.ncbi.nlm.nih.gov/34347012","citation_count":15,"is_preprint":false},{"pmid":"32302560","id":"PMC_32302560","title":"Developmental IL-6 Exposure Favors Production of PDGF-Responsive Multipotential Progenitors at the Expense of Neural Stem Cells and Other Progenitors.","date":"2020","source":"Stem cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/32302560","citation_count":13,"is_preprint":false},{"pmid":"39541666","id":"PMC_39541666","title":"Gastrodin reduces Aβ brain levels in an Alzheimer's disease mouse model by inhibiting P-glycoprotein ubiquitination.","date":"2024","source":"Phytomedicine : international journal of phytotherapy and phytopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/39541666","citation_count":11,"is_preprint":false},{"pmid":"33159144","id":"PMC_33159144","title":"Oroxylin A alleviates immunoparalysis of CLP mice by degrading CHOP through interacting with FBXO15.","date":"2020","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/33159144","citation_count":9,"is_preprint":false},{"pmid":"37249397","id":"PMC_37249397","title":"Evaluation of Genetic and Nongenetic Risk Factors for Degenerative Cervical Myelopathy.","date":"2023","source":"Spine","url":"https://pubmed.ncbi.nlm.nih.gov/37249397","citation_count":6,"is_preprint":false},{"pmid":"40457403","id":"PMC_40457403","title":"Genome-to-genome analysis reveals associations between human and mycobacterial genetic variation in tuberculosis patients from Tanzania.","date":"2025","source":"BMC medical genomics","url":"https://pubmed.ncbi.nlm.nih.gov/40457403","citation_count":3,"is_preprint":false},{"pmid":"41268268","id":"PMC_41268268","title":"F-box in breast cancer: mechanism of action and therapeutic potential.","date":"2025","source":"American journal of translational research","url":"https://pubmed.ncbi.nlm.nih.gov/41268268","citation_count":0,"is_preprint":false},{"pmid":"29381301","id":"PMC_29381301","title":"[Investigation of the liver DNA methylation profile of rats under the influence of hepatotoxicants of different nature].","date":"2016","source":"Voprosy pitaniia","url":"https://pubmed.ncbi.nlm.nih.gov/29381301","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":13965,"output_tokens":2635,"usd":0.04071,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9836,"output_tokens":3067,"usd":0.062927,"stage2_stop_reason":"end_turn"},"total_usd":0.103637,"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\": 2003,\n      \"finding\": \"Fbxo15 is a direct transcriptional target of Oct3/4 and Sox2: an 18-bp enhancer element ~500 bp upstream of the Fbxo15 transcription start site contains an octamer-like motif and an adjacent Sox-binding motif; cooperative binding of Oct3/4 and Sox2 to this enhancer was demonstrated by gel mobility shift assay, and point mutation of either motif abolished enhancer activity.\",\n      \"method\": \"Reporter gene (luciferase) assay, gel mobility shift assay (EMSA), site-directed mutagenesis, Oct3/4 inducible knockout in ES cells\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro binding (EMSA) combined with mutagenesis and reporter assays in a single rigorous study, replicated conceptually by Okumura-Nakanishi et al. 2004\",\n      \"pmids\": [\"12665572\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Fbxo15 knockout mice are viable, fertile, and show no gross developmental defects; Fbxo15-null ES cells are normal in morphology, proliferation, and differentiation, demonstrating that Fbxo15 is dispensable for ES cell self-renewal and mouse development.\",\n      \"method\": \"Homologous recombination knockout (beta-galactosidase knock-in), phenotypic analysis of null mice and ES cells\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean germline knockout with multiple phenotypic readouts in a single focused study\",\n      \"pmids\": [\"12665572\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Oct-3/4 and Sox2 cooperatively regulate Fbxo15 expression through an octamer-like and Sox-binding motif in the Fbxo15 enhancer, placing Fbxo15 in a broader regulatory circuit controlled by the Oct-3/4·Sox2 complex alongside Fgf-4, Utf1, and Sox2 genes.\",\n      \"method\": \"EMSA with ES-derived cell extracts, luciferase reporter assay, inducible Oct-3/4 knockout in ES cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — independent replication of Oct3/4·Sox2 cooperative binding to Fbxo15 enhancer using EMSA and reporter assays in a separate lab\",\n      \"pmids\": [\"15557334\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"FBXO15 functions as an F-box protein within an SCF(Fbxo15) E3 ubiquitin ligase complex (with Skp1/Cullin1) to ubiquitinate P-glycoprotein (ABCB1), targeting it for proteasomal degradation; co-precipitated P-gp and identified Ube2r1/Cdc34 as the cognate E2 ubiquitin-conjugating enzyme; exogenous FBXO15 enhanced P-gp ubiquitination while FBXO15 knockdown suppressed ubiquitination and increased P-gp protein without affecting mRNA.\",\n      \"method\": \"Co-immunoprecipitation, MALDI-TOF mass spectrometry, exogenous overexpression, siRNA knockdown, proteasome inhibitor (MG132) treatment\",\n      \"journal\": \"Cancer science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, gain- and loss-of-function, E2 partner identification, replicated across multiple orthogonal methods in one study and confirmed in follow-up (PMID:27786305)\",\n      \"pmids\": [\"23465077\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"FBXO15 mediates proteasomal degradation of cardiolipin synthase 1 (CLS1) in lung epithelia during S. aureus infection; PINK1 kinase binds CLS1, phosphorylates Thr219 to create a phosphodegron recognized by FBXO15, and FBXO15 interaction with CLS1 leads to its ubiquitin-mediated disposal, disrupting cardiolipin synthesis and mitochondrial function.\",\n      \"method\": \"Co-immunoprecipitation, site-directed mutagenesis (Thr219), overexpression and knockdown of FBXO15 and PINK1, Pink1 knockout mice, proteasome inhibitor assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP, phospho-mutant mutagenesis, KO mouse model with defined phenotype, and pathway placement via multiple orthogonal methods\",\n      \"pmids\": [\"24703837\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"RSK1 phosphorylates Thr162 on Ube2r1, destabilizing Ube2r1 via self-ubiquitination and thereby protecting P-gp from SCF(Fbxo15)-mediated ubiquitination; FBXO15 knockdown (along with Ube2r1 knockdown) blocked MEK inhibitor-induced P-gp downregulation, confirming FBXO15 is required for P-gp degradation downstream of the MAPK pathway.\",\n      \"method\": \"siRNA knockdown, phospho-site mutagenesis (Thr162 UBE2R1), proteasome inhibitor rescue, drug sensitivity and rhodamine 123 accumulation assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — single lab, multiple orthogonal methods but FBXO15's role is confirmed indirectly through epistasis/knockdown rather than direct biochemical reconstitution\",\n      \"pmids\": [\"27786305\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Fbxo15 recognizes and ubiquitinates KBP (encoded by Kif1bp) in a manner dependent on acetylation of KBP Lys501 by GCN5L1 (using TDH-derived acetyl-CoA), thereby limiting mitochondrial biogenesis in mouse embryonic stem cells; loss of KBP degradation causes unscheduled increase in mitochondria, enhanced respiration and ROS, and impaired proliferation.\",\n      \"method\": \"Mass spectrometry-based interactome (affinity purification-MS), Co-immunoprecipitation, acetylation mutagenesis (Lys501), Fbxo15 knockdown/rescue, mitochondrial mass and respiration assays, embryoid body formation\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — AP-MS substrate identification with mutagenesis validation, multiple orthogonal methods (Co-IP, metabolic assays, genetic rescue) in a single rigorous study\",\n      \"pmids\": [\"28319092\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Oroxylin A induces transcription of FBXO15, and activated FBXO15 protein binds to and mediates proteasomal degradation of CHOP (C/EBP-homologous protein) in CLP sepsis mice, relieving immunoparalysis by reducing CHOP-driven anti-inflammatory signaling.\",\n      \"method\": \"Co-immunoprecipitation, FBXO15 overexpression/knockdown, in vivo CLP mouse model, cytokine measurements, proteasome pathway assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and in vivo model with defined phenotype, single lab, mechanistic pathway placement but limited biochemical reconstitution detail in abstract\",\n      \"pmids\": [\"33159144\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Gastrodin binds directly to FBXO15 (identified by molecular docking, molecular dynamics simulations, and microscale thermophoresis) and inhibits FBXO15-P-gp interaction, reducing P-gp ubiquitination and proteasomal degradation at the blood-brain barrier, thereby increasing P-gp expression and Aβ clearance in APP/PS1 mice.\",\n      \"method\": \"Molecular docking, molecular dynamics simulations, microscale thermophoresis (MST), co-immunoprecipitation, western blot, in vivo APP/PS1 mouse model, Morris Water Maze\",\n      \"journal\": \"Phytomedicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding measured by MST, Co-IP for mechanism, and in vivo validation, but single lab and limited mutagenesis/reconstitution detail\",\n      \"pmids\": [\"39541666\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"FBXO15 (FBX15) is an F-box protein that forms SCF(FBXO15) E3 ubiquitin ligase complexes and targets specific substrates—including P-glycoprotein/ABCB1 (with E2 partner Ube2r1), cardiolipin synthase 1 (via a PINK1-generated phosphodegron at Thr219), KBP/Kif1bp (after GCN5L1-dependent acetylation of Lys501), and CHOP—for ubiquitin-proteasomal degradation; in embryonic stem cells its transcription is directly activated by cooperative Oct3/4–Sox2 binding to an 18-bp enhancer, and it restrains mitochondrial biogenesis by degrading KBP, while being dispensable for ES cell self-renewal and mouse development.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"FBXO15 is an F-box protein that assembles into an SCF(FBXO15) E3 ubiquitin ligase complex (with Skp1 and Cullin1) to direct selective substrates toward proteasomal degradation [#3]. It targets a structurally diverse set of substrates whose recognition is frequently gated by post-translational modification: P-glycoprotein/ABCB1, ubiquitinated using the E2 enzyme Ube2r1/Cdc34 [#3]; cardiolipin synthase 1, recognized via a PINK1-generated phosphodegron at Thr219 during bacterial infection [#4]; the KBP protein (Kif1bp), recognized after GCN5L1-dependent acetylation of Lys501 [#6]; and the transcription factor CHOP [#7]. Through degradation of KBP, FBXO15 restrains mitochondrial biogenesis in embryonic stem cells, with its loss causing unscheduled mitochondrial expansion, elevated respiration and ROS, and impaired proliferation [#6]. Its degradation of P-gp is embedded in MAPK signaling, where RSK1 phosphorylation of Ube2r1 modulates the pathway, and FBXO15 is required for MEK-inhibitor-induced P-gp downregulation [#5]. In embryonic stem cells, Fbxo15 transcription is directly activated by cooperative Oct3/4–Sox2 binding to an 18-bp enhancer upstream of its transcription start site, placing it within the pluripotency regulatory circuit [#0, #2]; despite this, Fbxo15 is dispensable for ES cell self-renewal and mouse development [#1].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Established that Fbxo15 is a direct transcriptional output of the core pluripotency factors, answering how this gene is wired into the ES cell program.\",\n      \"evidence\": \"Luciferase reporter and EMSA with site-directed mutagenesis plus inducible Oct3/4 knockout in ES cells, defining an 18-bp Oct3/4–Sox2 enhancer\",\n      \"pmids\": [\"12665572\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not define a molecular function for the FBXO15 protein itself\", \"Enhancer mapping does not address the protein's biochemical role\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Determined the loss-of-function consequence, showing Fbxo15 is not required for pluripotency or development despite its ES-cell-specific expression.\",\n      \"evidence\": \"Germline beta-galactosidase knock-in knockout mice and null ES cells with phenotypic analysis\",\n      \"pmids\": [\"12665572\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Lack of phenotype leaves the protein's molecular activity undefined\", \"Possible functional redundancy not tested\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Independently confirmed cooperative Oct3/4–Sox2 control of Fbxo15 and positioned it within a shared regulatory circuit with Fgf-4, Utf1 and Sox2.\",\n      \"evidence\": \"EMSA with ES-derived extracts and luciferase reporter assays in a separate lab\",\n      \"pmids\": [\"15557334\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reinforces transcriptional regulation but adds no mechanism for the protein\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defined the core biochemical identity of FBXO15 as an SCF E3 ligase substrate-recognition subunit and identified its first substrate and cognate E2 enzyme.\",\n      \"evidence\": \"Reciprocal Co-IP, MALDI-TOF MS, gain/loss-of-function, and MG132 rescue identifying P-glycoprotein as substrate and Ube2r1/Cdc34 as E2\",\n      \"pmids\": [\"23465077\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Degron on P-gp not mapped\", \"No structural model of the SCF(FBXO15)–substrate interaction\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Revealed that FBXO15 substrate recognition can be gated by upstream kinase-generated phosphodegrons, linking it to mitochondrial lipid metabolism during infection.\",\n      \"evidence\": \"Co-IP, Thr219 phospho-mutant mutagenesis, PINK1 manipulation and Pink1 knockout mice, proteasome inhibitor assays in lung epithelia\",\n      \"pmids\": [\"24703837\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How FBXO15 distinguishes phosphorylated CLS1 structurally not defined\", \"Physiological scope beyond S. aureus infection unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Placed FBXO15-mediated P-gp degradation downstream of MAPK signaling and showed the pathway is tuned by RSK1 phosphorylation of the E2 enzyme.\",\n      \"evidence\": \"siRNA knockdown, Thr162 UBE2R1 phospho-mutagenesis, proteasome rescue, and drug/rhodamine 123 accumulation assays\",\n      \"pmids\": [\"27786305\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"FBXO15's role shown by epistasis/knockdown rather than direct reconstitution\", \"Direct effect of RSK1 on FBXO15 itself not tested\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Connected FBXO15 to control of mitochondrial biogenesis in ES cells via acetylation-dependent recognition of the KBP substrate.\",\n      \"evidence\": \"AP-MS interactome, Co-IP, Lys501 acetylation mutagenesis, GCN5L1 link, knockdown/rescue, and mitochondrial mass/respiration assays\",\n      \"pmids\": [\"28319092\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this reconciles with the lack of developmental phenotype not addressed\", \"Structural basis for acetyl-degron recognition undefined\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Extended FBXO15 substrate repertoire to the transcription factor CHOP, implicating it in resolving sepsis-associated immunoparalysis.\",\n      \"evidence\": \"Co-IP, overexpression/knockdown, CLP mouse model and cytokine measurements following Oroxylin A induction\",\n      \"pmids\": [\"33159144\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"CHOP degron not mapped\", \"Limited biochemical reconstitution of the ubiquitination event\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstrated the FBXO15–P-gp interaction is pharmacologically targetable, with direct ligand binding modulating P-gp levels at the blood-brain barrier.\",\n      \"evidence\": \"Molecular docking/MD simulations, MST direct binding, Co-IP, western blot, and APP/PS1 mouse behavioral assays with Gastrodin\",\n      \"pmids\": [\"39541666\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Binding site on FBXO15 not validated by mutagenesis\", \"Single lab, limited reconstitution detail\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The structural basis by which a single F-box protein recognizes its diverse modified degrons (phospho-, acetyl-) and how its multiple substrate-specific roles are coordinated in vivo remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of SCF(FBXO15) bound to any substrate\", \"Reconciliation of substrate diversity with the dispensable knockout phenotype not established\", \"Tissue-specific substrate selection mechanisms unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [3, 4, 6]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [3, 4, 6]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 2]}\n    ],\n    \"complexes\": [\"SCF(FBXO15) E3 ubiquitin ligase\"],\n    \"partners\": [\"SKP1\", \"CUL1\", \"UBE2R1\", \"ABCB1\", \"CLS1\", \"KIF1BP\", \"PINK1\", \"DDIT3\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}