{"gene":"FBXO9","run_date":"2026-04-28T17:46:03","timeline":{"discoveries":[{"year":2021,"finding":"FBXO9 interacts with p53 and promotes its ubiquitination and degradation, thereby decreasing p53 protein stability in osteoclasts; zoledronic acid inhibits FBXO9, stabilizing p53 and promoting ferroptosis.","method":"Co-immunoprecipitation, knockdown/overexpression with functional readouts (Fe2+, ROS, MDA, GPX4, GSH levels)","journal":"PeerJ","confidence":"Medium","confidence_rationale":"Tier 3 — single lab, Co-IP plus functional rescue experiment","pmids":["35003915"],"is_preprint":false},{"year":2019,"finding":"FBXO9 acts as the substrate recognition component of the SCF E3 ubiquitin ligase complex; loss of FBXO9 in AML leads to increased proteasome activity, identifying a role for FBXO9 in regulating proteasome activity in hematopoietic malignancy.","method":"Conditional CRISPR/Cas9 knockout mouse model, quantitative mass spectrometry from primary tumors, in vitro proteasome activity assay","journal":"Cancers","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo KO + quantitative proteomics + functional proteasome assay, single lab","pmids":["31684170"],"is_preprint":false},{"year":2020,"finding":"FBXO9 functions as an SCF-type ubiquitin ligase for Neurog2, interacting with Neurog2 via its F-box motif to promote Neurog2 ubiquitination and destabilization, downstream of Sox10, thereby directing neural crest progenitors toward glial rather than neuronal fate in the dorsal root ganglia.","method":"Gain- and loss-of-function in avian NC progenitors, co-immunoprecipitation, ubiquitination assay, epistasis analysis (Sox10-Fbxo9-Neurog2 axis)","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (CoIP, ubiquitination assay, epistasis, gain/loss-of-function), moderate evidence","pmids":["32029586"],"is_preprint":false},{"year":2022,"finding":"FBXO9 directly targets FBXW7 for ubiquitination and proteasomal degradation in hepatocellular carcinoma, and is itself transcriptionally regulated by ZNF143, establishing a ZNF143-FBXO9-FBXW7 signaling axis.","method":"Loss- and gain-of-function experiments in vitro and in vivo, co-immunoprecipitation, ubiquitination assay, transcription factor binding assay","journal":"Frontiers in oncology","confidence":"Medium","confidence_rationale":"Tier 2 — reciprocal CoIP and ubiquitination assay, single lab","pmids":["35847937"],"is_preprint":false},{"year":2024,"finding":"FBXO9 ubiquitinates the V-ATPase catalytic subunit ATP6V1A, promoting its interaction with cytoplasmic chaperone HSPA8 and sequestration in the cytoplasm, thereby hindering V-ATPase assembly, reducing vesicular acidification, and suppressing pro-metastatic Wnt signaling in lung cancer.","method":"Immunoprecipitation, mass spectrometry, shRNA knockdown, CRISPR-KO, migration/clonogenic/sphere assays, ubiquitination assay, in vivo mouse model","journal":"Experimental hematology & oncology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (MS, CoIP, ubiquitination assay, KO, functional assays, in vivo), moderate evidence","pmids":["38486234"],"is_preprint":false},{"year":2024,"finding":"FBXO9 targets DPPA5 for ubiquitylation and proteasomal degradation; silencing FBXO9 stabilizes DPPA5, facilitating induction of cellular pluripotency.","method":"RNAi screen during reprogramming, knockdown with pluripotency functional readouts, proteasomal degradation assay","journal":"Stem cells (Dayton, Ohio)","confidence":"Medium","confidence_rationale":"Tier 3 — RNAi screen identification with functional follow-up, single lab","pmids":["38227647"],"is_preprint":false},{"year":2016,"finding":"The Drosophila FBXO9 ortholog CG5961 is required for proper neuronal tissue formation; altered expression in dopaminergic neurons reduces lifespan, and the protein domains are highly conserved with human FBXO9.","method":"Directed expression and RNAi knockdown in Drosophila eye and dopaminergic neurons (UAS-GAL4 system), domain conservation analysis","journal":"Genetics and molecular research : GMR","confidence":"Low","confidence_rationale":"Tier 3 — Drosophila ortholog, single method per phenotype, weak mechanistic resolution","pmids":["27173356"],"is_preprint":false},{"year":2025,"finding":"FBXO9, as part of the SCF-CRL1 complex, recognizes YAP through a conserved degron motif and facilitates K48-linked polyubiquitination at lysine K76, promoting proteasomal degradation; GSK-3β phosphorylation of YAP at Ser338 and Thr342 primes YAP for FBXO9 recognition, and Akt modulates this axis by regulating GSK-3β phosphorylation status.","method":"Co-immunoprecipitation, in vitro ubiquitination assay, site-directed mutagenesis (K76 and phosphorylation sites), pharmacological Akt inhibition, proteasome inhibitor experiments","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro ubiquitination assay with mutagenesis, CoIP, pharmacological validation, multiple orthogonal approaches in single study","pmids":["40902979"],"is_preprint":false},{"year":2026,"finding":"FBXO9 directly binds PD-L1 protein and promotes its ubiquitination and degradation, impairing PD-L1 maturation and tumor immune evasion in pancreatic cancer; FBXO9 overexpression suppresses tumor growth and promotes cytotoxic T cell activation in vivo.","method":"Co-immunoprecipitation, ubiquitination assay, in vivo tumor model with T cell functional readouts, gain-of-function experiments","journal":"Frontiers in immunology","confidence":"Medium","confidence_rationale":"Tier 2 — CoIP and ubiquitination assay with in vivo immune functional readout, single lab","pmids":["41646985"],"is_preprint":false}],"current_model":"FBXO9 is the substrate-recognition subunit of the SCF (SKP1-Cullin1-RBX1) E3 ubiquitin ligase complex that promotes proteasomal degradation of diverse substrates—including Neurog2, FBXW7, ATP6V1A, YAP (via GSK-3β-primed phosphodegron), DPPA5, PD-L1, and p53—thereby controlling cell fate decisions (neural crest gliogenesis, pluripotency), proteostasis (proteasome and V-ATPase activity), and oncogenic signaling, with substrate recognition mediated through its F-box motif and, for YAP, through phosphorylation-dependent degron recognition."},"narrative":{"teleology":[{"year":2016,"claim":"Initial evidence that the FBXO9 ortholog CG5961 is required for neuronal tissue integrity in Drosophila established that the gene has a conserved role in nervous system development, motivating functional studies of the mammalian protein.","evidence":"UAS-GAL4 directed expression and RNAi in Drosophila eye and dopaminergic neurons","pmids":["27173356"],"confidence":"Low","gaps":["Drosophila system only, no mammalian validation","no substrate identified","lifespan phenotype lacks mechanistic explanation"]},{"year":2019,"claim":"Demonstration that FBXO9 functions as an SCF E3 ligase subunit whose loss increases proteasome activity in AML established its role in proteostasis regulation in hematopoietic malignancy.","evidence":"Conditional CRISPR/Cas9 knockout mouse model with quantitative mass spectrometry and in vitro proteasome activity assay","pmids":["31684170"],"confidence":"Medium","gaps":["specific proteasomal substrates targeted by FBXO9 in AML not identified","mechanism linking FBXO9 loss to increased proteasome activity unknown"]},{"year":2020,"claim":"Identification of Neurog2 as a direct FBXO9 substrate linked FBXO9-mediated ubiquitination to a developmental cell-fate switch, showing that Sox10 induces FBXO9 to degrade Neurog2 and promote gliogenesis over neurogenesis in neural crest progenitors.","evidence":"Gain/loss-of-function in avian neural crest progenitors, co-immunoprecipitation, ubiquitination assay, epistasis analysis","pmids":["32029586"],"confidence":"High","gaps":["degron on Neurog2 not mapped","whether phosphorylation primes Neurog2 for FBXO9 recognition not tested"]},{"year":2021,"claim":"Discovery that FBXO9 ubiquitinates p53 and promotes its degradation expanded the substrate repertoire to a major tumor suppressor, linking FBXO9 to ferroptosis regulation in osteoclasts.","evidence":"Co-immunoprecipitation, knockdown/overexpression with functional readouts of ferroptosis markers","pmids":["35003915"],"confidence":"Medium","gaps":["single lab study without in vitro reconstitution","ubiquitination site on p53 not mapped","relationship to MDM2-mediated p53 degradation not addressed"]},{"year":2022,"claim":"Identification of FBXW7 as an FBXO9 substrate revealed an unusual E3-degrades-E3 regulatory circuit in hepatocellular carcinoma, with ZNF143 transcriptionally driving FBXO9 expression.","evidence":"Reciprocal co-immunoprecipitation, ubiquitination assay, gain/loss-of-function in vitro and in vivo, transcription factor binding assay","pmids":["35847937"],"confidence":"Medium","gaps":["degron on FBXW7 not mapped","functional consequences on FBXW7 substrate pool not fully characterized"]},{"year":2024,"claim":"Two studies expanded FBXO9 substrate repertoire to ATP6V1A and DPPA5: ubiquitination of ATP6V1A sequesters it with HSPA8 in the cytoplasm to impair V-ATPase assembly and Wnt signaling, while degradation of DPPA5 restricts pluripotency induction.","evidence":"Mass spectrometry, CRISPR-KO, in vivo mouse tumor model, ubiquitination assay (ATP6V1A); RNAi screen with reprogramming functional readouts (DPPA5)","pmids":["38486234","38227647"],"confidence":"High","gaps":["ubiquitination sites on ATP6V1A not mapped","DPPA5 interaction domain on FBXO9 not defined","whether HSPA8 sequestration is a general feature of FBXO9 substrate handling is unknown"]},{"year":2025,"claim":"Mechanistic dissection of YAP as an FBXO9 substrate revealed the first phosphodegron-dependent recognition mode for FBXO9: GSK-3β phosphorylates YAP at Ser338/Thr342 to create a degron recognized by FBXO9, which then catalyzes K48-linked polyubiquitination at K76.","evidence":"In vitro ubiquitination assay, site-directed mutagenesis of K76 and phosphorylation sites, pharmacological Akt inhibition, co-immunoprecipitation","pmids":["40902979"],"confidence":"High","gaps":["structural basis of FBXO9-phosphodegron interaction not determined","whether other FBXO9 substrates use analogous phosphodegrons is untested"]},{"year":2026,"claim":"Demonstration that FBXO9 ubiquitinates PD-L1 to block its maturation and promote antitumor T cell responses extended FBXO9 function to immune checkpoint regulation.","evidence":"Co-immunoprecipitation, ubiquitination assay, in vivo pancreatic tumor model with cytotoxic T cell functional readouts","pmids":["41646985"],"confidence":"Medium","gaps":["PD-L1 ubiquitination sites not mapped","whether FBXO9-mediated PD-L1 degradation is phosphorylation-dependent is unknown","single cancer type tested"]},{"year":null,"claim":"A unifying structural and biochemical framework for how FBXO9 discriminates among its many substrates — including whether phosphodegron recognition (established for YAP) is a general mechanism — remains to be defined.","evidence":"","pmids":[],"confidence":"High","gaps":["no crystal or cryo-EM structure of FBXO9 or an FBXO9–substrate complex","substrate degron motifs mapped only for YAP","in vivo physiological relevance established mainly for Neurog2 and ATP6V1A; other substrates lack genetic models"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,2,3,4,5,7,8]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[4]}],"pathway":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[2,7]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1,2,3,4,5,7,8]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[4,7]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[2]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[8]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[0]}],"complexes":["SCF (SKP1-CUL1-RBX1-FBXO9)"],"partners":["SKP1","CUL1","NEUROG2","FBXW7","ATP6V1A","YAP1","DPPA5","CD274"],"other_free_text":[]},"mechanistic_narrative":"FBXO9 is the substrate-recognition subunit of an SCF (SKP1–CUL1–RBX1) E3 ubiquitin ligase that targets a diverse set of substrates for proteasomal degradation, thereby governing cell fate, proteostasis, and oncogenic signaling. Through its F-box motif, FBXO9 ubiquitinates Neurog2 downstream of Sox10 to direct neural crest progenitors toward gliogenesis [PMID:32029586], ubiquitinates ATP6V1A to impair V-ATPase assembly and suppress pro-metastatic Wnt signaling [PMID:38486234], and targets the tumor suppressor FBXW7 for degradation in hepatocellular carcinoma [PMID:35847937]. FBXO9 also recognizes a GSK-3β-phosphorylated degron on YAP, catalyzing K48-linked polyubiquitination at K76 to promote YAP turnover in an Akt-modulated manner [PMID:40902979], and degrades PD-L1 to enhance antitumor immunity [PMID:41646985], DPPA5 to restrain pluripotency [PMID:38227647], and p53 to regulate ferroptosis susceptibility in osteoclasts [PMID:35003915]."},"prefetch_data":{"uniprot":{"accession":"Q9UK97","full_name":"F-box only protein 9","aliases":["Cross-immune reaction antigen 1","Renal carcinoma antigen NY-REN-57"],"length_aa":447,"mass_kda":52.3,"function":"Substrate recognition component of a SCF (SKP1-CUL1-F-box protein) E3 ubiquitin-protein ligase complex which mediates the ubiquitination and subsequent proteasomal degradation of target proteins and plays a role in several biological processes such as cell cycle, cell proliferation, or maintenance of chromosome stability (PubMed:23263282, PubMed:34480022). Ubiquitinates mTORC1-bound TTI1 and TELO2 when they are phosphorylated by CK2 following growth factor deprivation, leading to their degradation. In contrast, does not mediate ubiquitination of TTI1 and TELO2 when they are part of the mTORC2 complex. As a consequence, mTORC1 is inactivated to restrain cell growth and protein translation, while mTORC2 is the activated due to the relief of feedback inhibition by mTORC1 (PubMed:23263282). Plays a role in maintaining epithelial cell survival by regulating the turn-over of chromatin modulator PRMT4 through ubiquitination and degradation by the proteasomal pathway (PubMed:34480022). Regulates also PPARgamma stability by facilitating PPARgamma/PPARG ubiquitination and thereby plays a role in adipocyte differentiation (By similarity)","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q9UK97/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/FBXO9","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/FBXO9","total_profiled":1310},"omim":[{"mim_id":"609091","title":"F-BOX ONLY PROTEIN 9; FBXO9","url":"https://www.omim.org/entry/609091"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Centrosome","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/FBXO9"},"hgnc":{"alias_symbol":["FBX9","NY-REN-57"],"prev_symbol":[]},"alphafold":{"accession":"Q9UK97","domains":[{"cath_id":"-","chopping":"87-131","consensus_level":"high","plddt":89.6749,"start":87,"end":131},{"cath_id":"-","chopping":"162-261","consensus_level":"medium","plddt":87.3228,"start":162,"end":261},{"cath_id":"-","chopping":"269-360_378-440","consensus_level":"high","plddt":90.6852,"start":269,"end":440}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UK97","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UK97-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UK97-F1-predicted_aligned_error_v6.png","plddt_mean":77.44},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=FBXO9","jax_strain_url":"https://www.jax.org/strain/search?query=FBXO9"},"sequence":{"accession":"Q9UK97","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9UK97.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9UK97/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UK97"}},"corpus_meta":[{"pmid":"35003915","id":"PMC_35003915","title":"Zoledronic acid promotes osteoclasts ferroptosis by inhibiting FBXO9-mediated p53 ubiquitination and degradation.","date":"2021","source":"PeerJ","url":"https://pubmed.ncbi.nlm.nih.gov/35003915","citation_count":36,"is_preprint":false},{"pmid":"31684170","id":"PMC_31684170","title":"Loss of FBXO9 Enhances Proteasome Activity and Promotes Aggressiveness in Acute Myeloid Leukemia.","date":"2019","source":"Cancers","url":"https://pubmed.ncbi.nlm.nih.gov/31684170","citation_count":22,"is_preprint":false},{"pmid":"32029586","id":"PMC_32029586","title":"Fbxo9 functions downstream of Sox10 to determine neuron-glial fate choice in the dorsal root ganglia through Neurog2 destabilization.","date":"2020","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/32029586","citation_count":16,"is_preprint":false},{"pmid":"35847937","id":"PMC_35847937","title":"FBXO9 Mediates the Cancer-Promoting Effects of ZNF143 by Degrading FBXW7 and Facilitates Drug Resistance in Hepatocellular Carcinoma.","date":"2022","source":"Frontiers in oncology","url":"https://pubmed.ncbi.nlm.nih.gov/35847937","citation_count":12,"is_preprint":false},{"pmid":"38486234","id":"PMC_38486234","title":"Ubiquitin ligase subunit FBXO9 inhibits V-ATPase assembly and impedes lung cancer metastasis.","date":"2024","source":"Experimental hematology & oncology","url":"https://pubmed.ncbi.nlm.nih.gov/38486234","citation_count":11,"is_preprint":false},{"pmid":"38227647","id":"PMC_38227647","title":"Ubiquitin E3 Ligase FBXO9 Regulates Pluripotency by Targeting DPPA5 for Ubiquitylation and Degradation.","date":"2024","source":"Stem cells (Dayton, Ohio)","url":"https://pubmed.ncbi.nlm.nih.gov/38227647","citation_count":1,"is_preprint":false},{"pmid":"27173356","id":"PMC_27173356","title":"Altered expression of CG5961, a putative Drosophila melanogaster homologue of FBXO9, provides a new model of Parkinson disease.","date":"2016","source":"Genetics and molecular research : GMR","url":"https://pubmed.ncbi.nlm.nih.gov/27173356","citation_count":1,"is_preprint":false},{"pmid":"40902979","id":"PMC_40902979","title":"FBXO9 mediated the ubiquitination and degradation of YAP in a GSK-3β-dependent manner.","date":"2025","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/40902979","citation_count":0,"is_preprint":false},{"pmid":"41646985","id":"PMC_41646985","title":"FBXO9 promotes anti-tumor immunity via degradation of PD-L1 in pancreatic cancer.","date":"2026","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/41646985","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":6101,"output_tokens":2200,"usd":0.025652},"stage2":{"model":"claude-opus-4-6","input_tokens":5510,"output_tokens":2461,"usd":0.133612},"total_usd":0.159264,"stage1_batch_id":"msgbatch_0118RScg3WGaGkaZRtWZbtce","stage2_batch_id":"msgbatch_01Cpfzvtbj42aqp5oYZ5Te2s","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2021,\n      \"finding\": \"FBXO9 interacts with p53 and promotes its ubiquitination and degradation, thereby decreasing p53 protein stability in osteoclasts; zoledronic acid inhibits FBXO9, stabilizing p53 and promoting ferroptosis.\",\n      \"method\": \"Co-immunoprecipitation, knockdown/overexpression with functional readouts (Fe2+, ROS, MDA, GPX4, GSH levels)\",\n      \"journal\": \"PeerJ\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single lab, Co-IP plus functional rescue experiment\",\n      \"pmids\": [\"35003915\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"FBXO9 acts as the substrate recognition component of the SCF E3 ubiquitin ligase complex; loss of FBXO9 in AML leads to increased proteasome activity, identifying a role for FBXO9 in regulating proteasome activity in hematopoietic malignancy.\",\n      \"method\": \"Conditional CRISPR/Cas9 knockout mouse model, quantitative mass spectrometry from primary tumors, in vitro proteasome activity assay\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo KO + quantitative proteomics + functional proteasome assay, single lab\",\n      \"pmids\": [\"31684170\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"FBXO9 functions as an SCF-type ubiquitin ligase for Neurog2, interacting with Neurog2 via its F-box motif to promote Neurog2 ubiquitination and destabilization, downstream of Sox10, thereby directing neural crest progenitors toward glial rather than neuronal fate in the dorsal root ganglia.\",\n      \"method\": \"Gain- and loss-of-function in avian NC progenitors, co-immunoprecipitation, ubiquitination assay, epistasis analysis (Sox10-Fbxo9-Neurog2 axis)\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (CoIP, ubiquitination assay, epistasis, gain/loss-of-function), moderate evidence\",\n      \"pmids\": [\"32029586\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"FBXO9 directly targets FBXW7 for ubiquitination and proteasomal degradation in hepatocellular carcinoma, and is itself transcriptionally regulated by ZNF143, establishing a ZNF143-FBXO9-FBXW7 signaling axis.\",\n      \"method\": \"Loss- and gain-of-function experiments in vitro and in vivo, co-immunoprecipitation, ubiquitination assay, transcription factor binding assay\",\n      \"journal\": \"Frontiers in oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal CoIP and ubiquitination assay, single lab\",\n      \"pmids\": [\"35847937\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"FBXO9 ubiquitinates the V-ATPase catalytic subunit ATP6V1A, promoting its interaction with cytoplasmic chaperone HSPA8 and sequestration in the cytoplasm, thereby hindering V-ATPase assembly, reducing vesicular acidification, and suppressing pro-metastatic Wnt signaling in lung cancer.\",\n      \"method\": \"Immunoprecipitation, mass spectrometry, shRNA knockdown, CRISPR-KO, migration/clonogenic/sphere assays, ubiquitination assay, in vivo mouse model\",\n      \"journal\": \"Experimental hematology & oncology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (MS, CoIP, ubiquitination assay, KO, functional assays, in vivo), moderate evidence\",\n      \"pmids\": [\"38486234\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"FBXO9 targets DPPA5 for ubiquitylation and proteasomal degradation; silencing FBXO9 stabilizes DPPA5, facilitating induction of cellular pluripotency.\",\n      \"method\": \"RNAi screen during reprogramming, knockdown with pluripotency functional readouts, proteasomal degradation assay\",\n      \"journal\": \"Stem cells (Dayton, Ohio)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — RNAi screen identification with functional follow-up, single lab\",\n      \"pmids\": [\"38227647\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The Drosophila FBXO9 ortholog CG5961 is required for proper neuronal tissue formation; altered expression in dopaminergic neurons reduces lifespan, and the protein domains are highly conserved with human FBXO9.\",\n      \"method\": \"Directed expression and RNAi knockdown in Drosophila eye and dopaminergic neurons (UAS-GAL4 system), domain conservation analysis\",\n      \"journal\": \"Genetics and molecular research : GMR\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — Drosophila ortholog, single method per phenotype, weak mechanistic resolution\",\n      \"pmids\": [\"27173356\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"FBXO9, as part of the SCF-CRL1 complex, recognizes YAP through a conserved degron motif and facilitates K48-linked polyubiquitination at lysine K76, promoting proteasomal degradation; GSK-3β phosphorylation of YAP at Ser338 and Thr342 primes YAP for FBXO9 recognition, and Akt modulates this axis by regulating GSK-3β phosphorylation status.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ubiquitination assay, site-directed mutagenesis (K76 and phosphorylation sites), pharmacological Akt inhibition, proteasome inhibitor experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro ubiquitination assay with mutagenesis, CoIP, pharmacological validation, multiple orthogonal approaches in single study\",\n      \"pmids\": [\"40902979\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"FBXO9 directly binds PD-L1 protein and promotes its ubiquitination and degradation, impairing PD-L1 maturation and tumor immune evasion in pancreatic cancer; FBXO9 overexpression suppresses tumor growth and promotes cytotoxic T cell activation in vivo.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, in vivo tumor model with T cell functional readouts, gain-of-function experiments\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — CoIP and ubiquitination assay with in vivo immune functional readout, single lab\",\n      \"pmids\": [\"41646985\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"FBXO9 is the substrate-recognition subunit of the SCF (SKP1-Cullin1-RBX1) E3 ubiquitin ligase complex that promotes proteasomal degradation of diverse substrates—including Neurog2, FBXW7, ATP6V1A, YAP (via GSK-3β-primed phosphodegron), DPPA5, PD-L1, and p53—thereby controlling cell fate decisions (neural crest gliogenesis, pluripotency), proteostasis (proteasome and V-ATPase activity), and oncogenic signaling, with substrate recognition mediated through its F-box motif and, for YAP, through phosphorylation-dependent degron recognition.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"FBXO9 is the substrate-recognition subunit of an SCF (SKP1–CUL1–RBX1) E3 ubiquitin ligase that targets a diverse set of substrates for proteasomal degradation, thereby governing cell fate, proteostasis, and oncogenic signaling. Through its F-box motif, FBXO9 ubiquitinates Neurog2 downstream of Sox10 to direct neural crest progenitors toward gliogenesis [PMID:32029586], ubiquitinates ATP6V1A to impair V-ATPase assembly and suppress pro-metastatic Wnt signaling [PMID:38486234], and targets the tumor suppressor FBXW7 for degradation in hepatocellular carcinoma [PMID:35847937]. FBXO9 also recognizes a GSK-3β-phosphorylated degron on YAP, catalyzing K48-linked polyubiquitination at K76 to promote YAP turnover in an Akt-modulated manner [PMID:40902979], and degrades PD-L1 to enhance antitumor immunity [PMID:41646985], DPPA5 to restrain pluripotency [PMID:38227647], and p53 to regulate ferroptosis susceptibility in osteoclasts [PMID:35003915].\",\n  \"teleology\": [\n    {\n      \"year\": 2016,\n      \"claim\": \"Initial evidence that the FBXO9 ortholog CG5961 is required for neuronal tissue integrity in Drosophila established that the gene has a conserved role in nervous system development, motivating functional studies of the mammalian protein.\",\n      \"evidence\": \"UAS-GAL4 directed expression and RNAi in Drosophila eye and dopaminergic neurons\",\n      \"pmids\": [\"27173356\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Drosophila system only, no mammalian validation\", \"no substrate identified\", \"lifespan phenotype lacks mechanistic explanation\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Demonstration that FBXO9 functions as an SCF E3 ligase subunit whose loss increases proteasome activity in AML established its role in proteostasis regulation in hematopoietic malignancy.\",\n      \"evidence\": \"Conditional CRISPR/Cas9 knockout mouse model with quantitative mass spectrometry and in vitro proteasome activity assay\",\n      \"pmids\": [\"31684170\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"specific proteasomal substrates targeted by FBXO9 in AML not identified\", \"mechanism linking FBXO9 loss to increased proteasome activity unknown\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identification of Neurog2 as a direct FBXO9 substrate linked FBXO9-mediated ubiquitination to a developmental cell-fate switch, showing that Sox10 induces FBXO9 to degrade Neurog2 and promote gliogenesis over neurogenesis in neural crest progenitors.\",\n      \"evidence\": \"Gain/loss-of-function in avian neural crest progenitors, co-immunoprecipitation, ubiquitination assay, epistasis analysis\",\n      \"pmids\": [\"32029586\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"degron on Neurog2 not mapped\", \"whether phosphorylation primes Neurog2 for FBXO9 recognition not tested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Discovery that FBXO9 ubiquitinates p53 and promotes its degradation expanded the substrate repertoire to a major tumor suppressor, linking FBXO9 to ferroptosis regulation in osteoclasts.\",\n      \"evidence\": \"Co-immunoprecipitation, knockdown/overexpression with functional readouts of ferroptosis markers\",\n      \"pmids\": [\"35003915\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"single lab study without in vitro reconstitution\", \"ubiquitination site on p53 not mapped\", \"relationship to MDM2-mediated p53 degradation not addressed\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identification of FBXW7 as an FBXO9 substrate revealed an unusual E3-degrades-E3 regulatory circuit in hepatocellular carcinoma, with ZNF143 transcriptionally driving FBXO9 expression.\",\n      \"evidence\": \"Reciprocal co-immunoprecipitation, ubiquitination assay, gain/loss-of-function in vitro and in vivo, transcription factor binding assay\",\n      \"pmids\": [\"35847937\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"degron on FBXW7 not mapped\", \"functional consequences on FBXW7 substrate pool not fully characterized\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Two studies expanded FBXO9 substrate repertoire to ATP6V1A and DPPA5: ubiquitination of ATP6V1A sequesters it with HSPA8 in the cytoplasm to impair V-ATPase assembly and Wnt signaling, while degradation of DPPA5 restricts pluripotency induction.\",\n      \"evidence\": \"Mass spectrometry, CRISPR-KO, in vivo mouse tumor model, ubiquitination assay (ATP6V1A); RNAi screen with reprogramming functional readouts (DPPA5)\",\n      \"pmids\": [\"38486234\", \"38227647\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"ubiquitination sites on ATP6V1A not mapped\", \"DPPA5 interaction domain on FBXO9 not defined\", \"whether HSPA8 sequestration is a general feature of FBXO9 substrate handling is unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Mechanistic dissection of YAP as an FBXO9 substrate revealed the first phosphodegron-dependent recognition mode for FBXO9: GSK-3β phosphorylates YAP at Ser338/Thr342 to create a degron recognized by FBXO9, which then catalyzes K48-linked polyubiquitination at K76.\",\n      \"evidence\": \"In vitro ubiquitination assay, site-directed mutagenesis of K76 and phosphorylation sites, pharmacological Akt inhibition, co-immunoprecipitation\",\n      \"pmids\": [\"40902979\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"structural basis of FBXO9-phosphodegron interaction not determined\", \"whether other FBXO9 substrates use analogous phosphodegrons is untested\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Demonstration that FBXO9 ubiquitinates PD-L1 to block its maturation and promote antitumor T cell responses extended FBXO9 function to immune checkpoint regulation.\",\n      \"evidence\": \"Co-immunoprecipitation, ubiquitination assay, in vivo pancreatic tumor model with cytotoxic T cell functional readouts\",\n      \"pmids\": [\"41646985\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"PD-L1 ubiquitination sites not mapped\", \"whether FBXO9-mediated PD-L1 degradation is phosphorylation-dependent is unknown\", \"single cancer type tested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A unifying structural and biochemical framework for how FBXO9 discriminates among its many substrates — including whether phosphodegron recognition (established for YAP) is a general mechanism — remains to be defined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"no crystal or cryo-EM structure of FBXO9 or an FBXO9–substrate complex\", \"substrate degron motifs mapped only for YAP\", \"in vivo physiological relevance established mainly for Neurog2 and ATP6V1A; other substrates lack genetic models\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 2, 3, 4, 5, 7, 8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [2, 7]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 2, 3, 4, 5, 7, 8]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [4, 7]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"complexes\": [\"SCF (SKP1-CUL1-RBX1-FBXO9)\"],\n    \"partners\": [\"SKP1\", \"CUL1\", \"NEUROG2\", \"FBXW7\", \"ATP6V1A\", \"YAP1\", \"DPPA5\", \"CD274\"],\n    \"other_free_text\": []\n  }\n}\n```"}