{"gene":"FBXO9","run_date":"2026-06-09T23:54:43","timeline":{"discoveries":[{"year":2021,"finding":"FBXO9 interacts with p53 and promotes its ubiquitination and degradation, thereby decreasing p53 protein stability; zoledronic acid suppresses FBXO9 expression, stabilizing p53 and inducing osteoclast ferroptosis.","method":"Co-immunoprecipitation, knockdown/overexpression functional assays, CCK-8 viability assay, ferroptosis marker measurement (Fe2+, ROS, MDA, GPX4, GSH)","journal":"PeerJ","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — single lab, Co-IP interaction and functional rescue shown, but no in vitro ubiquitination reconstitution or mutagenesis","pmids":["35003915"],"is_preprint":false},{"year":2019,"finding":"FBXO9 is the substrate recognition component of an SCF E3 ubiquitin ligase; its loss in AML leads to increased proteasome activity and more aggressive leukemia in an inv(16) mouse model, indicating FBXO9 normally restrains proteasome activity.","method":"CRISPR/Cas9 conditional knockout mouse model, quantitative mass spectrometry of primary tumors, in vitro proteasome activity assay, bortezomib sensitivity assay","journal":"Cancers","confidence":"High","confidence_rationale":"Tier 2 / Moderate — in vivo genetic knockout with defined leukemia phenotype, quantitative proteomics, and functional proteasome activity assay in a single rigorous study","pmids":["31684170"],"is_preprint":false},{"year":2020,"finding":"Fbxo9, acting downstream of Sox10, functions as a substrate receptor of an SCF-type ubiquitin ligase that binds Neurog2 through its F-box motif and promotes Neurog2 ubiquitination and proteasomal destabilization, thereby directing neural crest progenitors toward glial rather than neuronal fate.","method":"Co-immunoprecipitation (Fbxo9–Neurog2 interaction), overexpression and knockdown in avian trunk neural crest, epistasis analysis with Sox10, transcriptional profiling, ubiquitination assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal interaction, ubiquitination assay, genetic epistasis with Sox10, gain- and loss-of-function with defined fate-choice phenotype, multiple orthogonal methods","pmids":["32029586"],"is_preprint":false},{"year":2024,"finding":"FBXO9, as part of the SKP1-cullin-1-RBX1 ubiquitin ligase complex, ubiquitinates the V-ATPase catalytic subunit ATP6V1A; this ubiquitination promotes ATP6V1A interaction with the cytoplasmic chaperone HSPA8 and its sequestration in the cytoplasm, thereby inhibiting functional V-ATPase assembly, reducing vesicular acidification, and suppressing pro-metastatic Wnt signaling in lung cancer cells.","method":"Mass spectrometry-based protein interaction studies, co-immunoprecipitation, immunoblot, CRISPR-Cas9 knockout, shRNA knockdown, migration/clonogenic/tumor sphere assays, in vivo mouse metastasis model, V-ATPase inhibitor treatment","journal":"Experimental hematology & oncology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — MS-identified interaction, reciprocal Co-IP, multiple functional assays, in vivo validation, multiple orthogonal methods in single study","pmids":["38486234"],"is_preprint":false},{"year":2022,"finding":"FBXO9 targets FBXW7 for ubiquitination and degradation; ZNF143 acts as a direct upstream transcription factor of FBXO9, forming a ZNF143-FBXO9-FBXW7 regulatory axis that promotes HCC cell proliferation and metastasis.","method":"Loss- and gain-of-function experiments in vitro and in vivo, co-immunoprecipitation, ubiquitination assay, ChIP or reporter assay for ZNF143 as transcriptional regulator, drug sensitivity assay","journal":"Frontiers in oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — single lab, ubiquitination and interaction assays shown, in vivo data included, but no structural validation or independent replication","pmids":["35847937"],"is_preprint":false},{"year":2025,"finding":"The SCF-FBXO9-CRL1 E3 ligase complex recognizes YAP through a conserved degron motif and facilitates K48-linked polyubiquitination at lysine K76, promoting proteasomal degradation; GSK-3β-mediated phosphorylation of YAP at Ser338 and Thr342 is required for FBXO9 recognition, and Akt kinase modulates this axis by controlling GSK-3β phosphorylation status.","method":"Co-immunoprecipitation, ubiquitination assay (K48-linkage specific), site-directed mutagenesis of YAP K76 and phosphosites, pharmacological inhibition of Akt/GSK-3β, chemosensitivity assays in cancer models","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis of substrate degron and ubiquitination site, K48-linkage specificity demonstrated, pharmacological and genetic epistasis of kinase cascade, multiple orthogonal methods in single study","pmids":["40902979"],"is_preprint":false},{"year":2024,"finding":"FBXO9 targets DPPA5 (a pluripotency-associated protein) for ubiquitylation and proteasomal degradation; FBXO9 silencing decreases DPPA5 degradation and facilitates induction of cellular reprogramming to pluripotency.","method":"RNAi screen during cellular reprogramming, FBXO9 silencing with functional pluripotency readouts, proteasomal degradation assay for DPPA5","journal":"Stem cells (Dayton, Ohio)","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — single lab, RNAi screen plus targeted validation of DPPA5 as substrate, but limited mechanistic detail in abstract regarding direct ubiquitination reconstitution","pmids":["38227647"],"is_preprint":false},{"year":2026,"finding":"FBXO9 directly binds PD-L1 protein and promotes its ubiquitination and degradation, thereby impeding PD-L1 maturation and impairing tumor immune evasion in pancreatic cancer; FBXO9 overexpression suppresses tumor growth and promotes cytotoxic T cell activation in vivo.","method":"Co-immunoprecipitation (FBXO9–PD-L1 interaction), ubiquitination assay, overexpression in vivo tumor model, cytotoxic T cell activation readout","journal":"Frontiers in immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — single lab, Co-IP interaction and ubiquitination assay with in vivo functional data, but no independent replication or structural validation","pmids":["41646985"],"is_preprint":false},{"year":2016,"finding":"The Drosophila FBXO9 homologue CG5961 is required for normal neuronal tissue formation; altered expression of CG5961 in dopaminergic neurons reduces lifespan, suggesting conserved neuronal function for this F-box protein family member.","method":"Targeted UAS-EP overexpression and RNAi knockdown in Drosophila dopaminergic neurons and eye, lifespan analysis, protein domain conservation analysis","journal":"Genetics and molecular research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Drosophila ortholog model, single lab, phenotypic readout only with no direct biochemical mechanism established","pmids":["27173356"],"is_preprint":false}],"current_model":"FBXO9 is the substrate-recognition subunit of an SCF (SKP1-Cullin1-RBX1) E3 ubiquitin ligase complex that ubiquitinates and promotes proteasomal degradation of multiple substrates—including Neurog2 (directing neural crest fate toward glia), YAP (via a GSK-3β/Akt-primed phosphodegron), ATP6V1A (inhibiting V-ATPase assembly and Wnt-driven metastasis), FBXW7, DPPA5, PD-L1, and p53—thereby functioning as a context-dependent regulator of differentiation, pluripotency, proteasome homeostasis, and tumor suppression."},"narrative":{"mechanistic_narrative":"FBXO9 is the substrate-recognition subunit of an SCF (SKP1–Cullin1–RBX1) E3 ubiquitin ligase that selects diverse phosphorylation- and degron-defined substrates for K48-linked polyubiquitination and proteasomal degradation, functioning as a context-dependent regulator of differentiation, proteasome homeostasis, and tumor biology [PMID:31684170, PMID:32029586, PMID:40902979]. In hematopoietic cells FBXO9 restrains proteasome activity, and its loss accelerates AML in vivo, establishing a tumor-suppressive role tied to proteostasis control [PMID:31684170]. Its substrate repertoire spans developmental and oncogenic programs: it binds Neurog2 through its F-box motif to drive neural crest progenitors toward glial fate downstream of Sox10 [PMID:32029586], targets the pluripotency factor DPPA5 to oppose reprogramming [PMID:38227647], and degrades the V-ATPase catalytic subunit ATP6V1A—promoting its HSPA8-dependent cytoplasmic sequestration to block V-ATPase assembly, vesicular acidification, and Wnt-driven metastasis [PMID:38486234]. FBXO9 recognizes YAP via a conserved phosphodegron that requires GSK-3β-mediated phosphorylation at Ser338/Thr342, with Akt modulating the cascade, and ubiquitinates YAP at K76 for degradation [PMID:40902979]. It additionally directs degradation of FBXW7 within a ZNF143-driven axis [PMID:35847937], PD-L1 to limit tumor immune evasion [PMID:41646985], and p53, where it lowers p53 stability [PMID:35003915]. A conserved neuronal requirement for the Drosophila homologue supports an ancestral role for this F-box protein in neural tissue [PMID:27173356].","teleology":[{"year":2016,"claim":"Before biochemical substrates were known, conservation analysis asked whether this F-box protein had an ancestral physiological role, establishing a conserved neuronal requirement.","evidence":"UAS-EP overexpression and RNAi knockdown of the Drosophila homologue CG5961 in dopaminergic neurons with lifespan analysis","pmids":["27173356"],"confidence":"Low","gaps":["Phenotypic readout only with no direct biochemical mechanism","No substrate identified in the ortholog","Relevance to mammalian FBXO9 function not established"]},{"year":2019,"claim":"Defined FBXO9 as the substrate-recognition subunit of an SCF E3 ligase and showed it normally restrains proteasome activity, linking its loss to aggressive leukemia.","evidence":"CRISPR/Cas9 conditional knockout in an inv(16) AML mouse model, quantitative mass spectrometry, in vitro proteasome activity and bortezomib sensitivity assays","pmids":["31684170"],"confidence":"High","gaps":["Substrate(s) responsible for proteasome restraint not fully resolved","Mechanism connecting FBXO9 loss to elevated proteasome activity not defined"]},{"year":2020,"claim":"Identified Neurog2 as a direct degradation substrate and placed FBXO9 downstream of Sox10 in a developmental fate decision, showing ubiquitin-mediated control of neural crest cell fate.","evidence":"Reciprocal Co-IP, ubiquitination assay, Sox10 epistasis, and gain/loss-of-function in avian trunk neural crest with fate-choice readout","pmids":["32029586"],"confidence":"High","gaps":["Degron on Neurog2 not mapped","No in vitro reconstitution of the ubiquitination reaction"]},{"year":2021,"claim":"Extended FBXO9 substrates to p53, proposing it lowers p53 stability and linking zoledronic acid to p53 stabilization and osteoclast ferroptosis.","evidence":"Co-IP, knockdown/overexpression rescue, CCK-8 viability and ferroptosis marker measurements","pmids":["35003915"],"confidence":"Medium","gaps":["No in vitro ubiquitination reconstitution or degron mutagenesis","Single lab without independent replication","Direct vs indirect effect on p53 not distinguished"]},{"year":2022,"claim":"Placed FBXO9 within a transcriptional regulatory axis by identifying ZNF143 as an upstream activator and FBXW7 as a degradation target, connecting FBXO9 to HCC proliferation and metastasis.","evidence":"In vitro/in vivo loss- and gain-of-function, Co-IP, ubiquitination assay, ChIP/reporter assay for ZNF143","pmids":["35847937"],"confidence":"Medium","gaps":["No structural validation of FBXW7 recognition","Not independently replicated","Degron on FBXW7 unmapped"]},{"year":2024,"claim":"Defined a non-canonical degradation outcome by showing FBXO9 ubiquitinates ATP6V1A to drive HSPA8-dependent cytoplasmic sequestration, inhibiting V-ATPase assembly and Wnt-driven metastasis.","evidence":"MS-based interactome, reciprocal Co-IP, CRISPR/shRNA, migration/sphere assays, in vivo metastasis model and V-ATPase inhibitor treatment","pmids":["38486234"],"confidence":"High","gaps":["Mechanism by which ubiquitination promotes HSPA8 binding not defined","Ubiquitin linkage type not specified"]},{"year":2024,"claim":"Identified DPPA5 as a substrate from a reprogramming screen, showing FBXO9 opposes pluripotency induction by degrading a pluripotency-associated protein.","evidence":"RNAi screen during cellular reprogramming with FBXO9 silencing and DPPA5 proteasomal degradation readout","pmids":["38227647"],"confidence":"Medium","gaps":["No reconstitution confirming direct ubiquitination of DPPA5","Degron not mapped","Single lab"]},{"year":2025,"claim":"Established the most detailed substrate-recognition mechanism: a GSK-3β/Akt-primed YAP phosphodegron and K48-linked ubiquitination at K76, defining kinase-gated degron logic for FBXO9.","evidence":"Co-IP, K48-linkage-specific ubiquitination assay, mutagenesis of YAP K76 and phosphosites, pharmacological Akt/GSK-3β inhibition, chemosensitivity assays","pmids":["40902979"],"confidence":"High","gaps":["Structural basis of degron engagement not resolved","In vivo relevance of the YAP axis not established in this study"]},{"year":2026,"claim":"Linked FBXO9 to anti-tumor immunity by showing it degrades PD-L1 and impedes its maturation, impairing immune evasion in pancreatic cancer.","evidence":"Co-IP, ubiquitination assay, in vivo overexpression tumor model, cytotoxic T cell activation readout","pmids":["41646985"],"confidence":"Medium","gaps":["No structural validation or degron mapping on PD-L1","Not independently replicated","Mechanism of maturation interference vs degradation not separated"]},{"year":null,"claim":"How FBXO9 selects among its many substrates in a given cell type, and whether common degron features or upstream signals coordinate this context-dependent specificity, remains unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No structural model of FBXO9 substrate engagement","Determinants of tissue-specific substrate choice unknown","No unified degron consensus across substrates established"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,2,3,5]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[1,2,5]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[1,3]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[3]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[1,2,5]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[1,3,4]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,5]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[2,6]}],"complexes":["SCF (SKP1-Cullin1-RBX1) E3 ubiquitin ligase"],"partners":["SKP1","CUL1","RBX1","NEUROG2","YAP","ATP6V1A","FBXW7","PD-L1"],"other_free_text":[]}},"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":37,"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":"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":12,"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":"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":2,"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":"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":6868,"output_tokens":2494,"usd":0.029007,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9640,"output_tokens":3384,"usd":0.0664,"stage2_stop_reason":"end_turn"},"total_usd":0.095407,"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\": 2021,\n      \"finding\": \"FBXO9 interacts with p53 and promotes its ubiquitination and degradation, thereby decreasing p53 protein stability; zoledronic acid suppresses FBXO9 expression, stabilizing p53 and inducing osteoclast ferroptosis.\",\n      \"method\": \"Co-immunoprecipitation, knockdown/overexpression functional assays, CCK-8 viability assay, ferroptosis marker measurement (Fe2+, ROS, MDA, GPX4, GSH)\",\n      \"journal\": \"PeerJ\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — single lab, Co-IP interaction and functional rescue shown, but no in vitro ubiquitination reconstitution or mutagenesis\",\n      \"pmids\": [\"35003915\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"FBXO9 is the substrate recognition component of an SCF E3 ubiquitin ligase; its loss in AML leads to increased proteasome activity and more aggressive leukemia in an inv(16) mouse model, indicating FBXO9 normally restrains proteasome activity.\",\n      \"method\": \"CRISPR/Cas9 conditional knockout mouse model, quantitative mass spectrometry of primary tumors, in vitro proteasome activity assay, bortezomib sensitivity assay\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo genetic knockout with defined leukemia phenotype, quantitative proteomics, and functional proteasome activity assay in a single rigorous study\",\n      \"pmids\": [\"31684170\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Fbxo9, acting downstream of Sox10, functions as a substrate receptor of an SCF-type ubiquitin ligase that binds Neurog2 through its F-box motif and promotes Neurog2 ubiquitination and proteasomal destabilization, thereby directing neural crest progenitors toward glial rather than neuronal fate.\",\n      \"method\": \"Co-immunoprecipitation (Fbxo9–Neurog2 interaction), overexpression and knockdown in avian trunk neural crest, epistasis analysis with Sox10, transcriptional profiling, ubiquitination assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal interaction, ubiquitination assay, genetic epistasis with Sox10, gain- and loss-of-function with defined fate-choice phenotype, multiple orthogonal methods\",\n      \"pmids\": [\"32029586\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"FBXO9, as part of the SKP1-cullin-1-RBX1 ubiquitin ligase complex, ubiquitinates the V-ATPase catalytic subunit ATP6V1A; this ubiquitination promotes ATP6V1A interaction with the cytoplasmic chaperone HSPA8 and its sequestration in the cytoplasm, thereby inhibiting functional V-ATPase assembly, reducing vesicular acidification, and suppressing pro-metastatic Wnt signaling in lung cancer cells.\",\n      \"method\": \"Mass spectrometry-based protein interaction studies, co-immunoprecipitation, immunoblot, CRISPR-Cas9 knockout, shRNA knockdown, migration/clonogenic/tumor sphere assays, in vivo mouse metastasis model, V-ATPase inhibitor treatment\",\n      \"journal\": \"Experimental hematology & oncology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS-identified interaction, reciprocal Co-IP, multiple functional assays, in vivo validation, multiple orthogonal methods in single study\",\n      \"pmids\": [\"38486234\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"FBXO9 targets FBXW7 for ubiquitination and degradation; ZNF143 acts as a direct upstream transcription factor of FBXO9, forming a ZNF143-FBXO9-FBXW7 regulatory axis that promotes HCC cell proliferation and metastasis.\",\n      \"method\": \"Loss- and gain-of-function experiments in vitro and in vivo, co-immunoprecipitation, ubiquitination assay, ChIP or reporter assay for ZNF143 as transcriptional regulator, drug sensitivity assay\",\n      \"journal\": \"Frontiers in oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — single lab, ubiquitination and interaction assays shown, in vivo data included, but no structural validation or independent replication\",\n      \"pmids\": [\"35847937\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The SCF-FBXO9-CRL1 E3 ligase complex recognizes YAP through a conserved degron motif and facilitates K48-linked polyubiquitination at lysine K76, promoting proteasomal degradation; GSK-3β-mediated phosphorylation of YAP at Ser338 and Thr342 is required for FBXO9 recognition, and Akt kinase modulates this axis by controlling GSK-3β phosphorylation status.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay (K48-linkage specific), site-directed mutagenesis of YAP K76 and phosphosites, pharmacological inhibition of Akt/GSK-3β, chemosensitivity assays in cancer models\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis of substrate degron and ubiquitination site, K48-linkage specificity demonstrated, pharmacological and genetic epistasis of kinase cascade, multiple orthogonal methods in single study\",\n      \"pmids\": [\"40902979\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"FBXO9 targets DPPA5 (a pluripotency-associated protein) for ubiquitylation and proteasomal degradation; FBXO9 silencing decreases DPPA5 degradation and facilitates induction of cellular reprogramming to pluripotency.\",\n      \"method\": \"RNAi screen during cellular reprogramming, FBXO9 silencing with functional pluripotency readouts, proteasomal degradation assay for DPPA5\",\n      \"journal\": \"Stem cells (Dayton, Ohio)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — single lab, RNAi screen plus targeted validation of DPPA5 as substrate, but limited mechanistic detail in abstract regarding direct ubiquitination reconstitution\",\n      \"pmids\": [\"38227647\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"FBXO9 directly binds PD-L1 protein and promotes its ubiquitination and degradation, thereby impeding PD-L1 maturation and impairing tumor immune evasion in pancreatic cancer; FBXO9 overexpression suppresses tumor growth and promotes cytotoxic T cell activation in vivo.\",\n      \"method\": \"Co-immunoprecipitation (FBXO9–PD-L1 interaction), ubiquitination assay, overexpression in vivo tumor model, cytotoxic T cell activation readout\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — single lab, Co-IP interaction and ubiquitination assay with in vivo functional data, but no independent replication or structural validation\",\n      \"pmids\": [\"41646985\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The Drosophila FBXO9 homologue CG5961 is required for normal neuronal tissue formation; altered expression of CG5961 in dopaminergic neurons reduces lifespan, suggesting conserved neuronal function for this F-box protein family member.\",\n      \"method\": \"Targeted UAS-EP overexpression and RNAi knockdown in Drosophila dopaminergic neurons and eye, lifespan analysis, protein domain conservation analysis\",\n      \"journal\": \"Genetics and molecular research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Drosophila ortholog model, single lab, phenotypic readout only with no direct biochemical mechanism established\",\n      \"pmids\": [\"27173356\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"FBXO9 is the substrate-recognition subunit of an SCF (SKP1-Cullin1-RBX1) E3 ubiquitin ligase complex that ubiquitinates and promotes proteasomal degradation of multiple substrates—including Neurog2 (directing neural crest fate toward glia), YAP (via a GSK-3β/Akt-primed phosphodegron), ATP6V1A (inhibiting V-ATPase assembly and Wnt-driven metastasis), FBXW7, DPPA5, PD-L1, and p53—thereby functioning as a context-dependent regulator of differentiation, pluripotency, proteasome homeostasis, and tumor suppression.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"FBXO9 is the substrate-recognition subunit of an SCF (SKP1–Cullin1–RBX1) E3 ubiquitin ligase that selects diverse phosphorylation- and degron-defined substrates for K48-linked polyubiquitination and proteasomal degradation, functioning as a context-dependent regulator of differentiation, proteasome homeostasis, and tumor biology [#1, #2, #5]. In hematopoietic cells FBXO9 restrains proteasome activity, and its loss accelerates AML in vivo, establishing a tumor-suppressive role tied to proteostasis control [#1]. Its substrate repertoire spans developmental and oncogenic programs: it binds Neurog2 through its F-box motif to drive neural crest progenitors toward glial fate downstream of Sox10 [#2], targets the pluripotency factor DPPA5 to oppose reprogramming [#6], and degrades the V-ATPase catalytic subunit ATP6V1A—promoting its HSPA8-dependent cytoplasmic sequestration to block V-ATPase assembly, vesicular acidification, and Wnt-driven metastasis [#3]. FBXO9 recognizes YAP via a conserved phosphodegron that requires GSK-3β-mediated phosphorylation at Ser338/Thr342, with Akt modulating the cascade, and ubiquitinates YAP at K76 for degradation [#5]. It additionally directs degradation of FBXW7 within a ZNF143-driven axis [#4], PD-L1 to limit tumor immune evasion [#7], and p53, where it lowers p53 stability [#0]. A conserved neuronal requirement for the Drosophila homologue supports an ancestral role for this F-box protein in neural tissue [#8].\",\n  \"teleology\": [\n    {\n      \"year\": 2016,\n      \"claim\": \"Before biochemical substrates were known, conservation analysis asked whether this F-box protein had an ancestral physiological role, establishing a conserved neuronal requirement.\",\n      \"evidence\": \"UAS-EP overexpression and RNAi knockdown of the Drosophila homologue CG5961 in dopaminergic neurons with lifespan analysis\",\n      \"pmids\": [\"27173356\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Phenotypic readout only with no direct biochemical mechanism\", \"No substrate identified in the ortholog\", \"Relevance to mammalian FBXO9 function not established\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defined FBXO9 as the substrate-recognition subunit of an SCF E3 ligase and showed it normally restrains proteasome activity, linking its loss to aggressive leukemia.\",\n      \"evidence\": \"CRISPR/Cas9 conditional knockout in an inv(16) AML mouse model, quantitative mass spectrometry, in vitro proteasome activity and bortezomib sensitivity assays\",\n      \"pmids\": [\"31684170\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Substrate(s) responsible for proteasome restraint not fully resolved\", \"Mechanism connecting FBXO9 loss to elevated proteasome activity not defined\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identified Neurog2 as a direct degradation substrate and placed FBXO9 downstream of Sox10 in a developmental fate decision, showing ubiquitin-mediated control of neural crest cell fate.\",\n      \"evidence\": \"Reciprocal Co-IP, ubiquitination assay, Sox10 epistasis, and gain/loss-of-function in avian trunk neural crest with fate-choice readout\",\n      \"pmids\": [\"32029586\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Degron on Neurog2 not mapped\", \"No in vitro reconstitution of the ubiquitination reaction\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Extended FBXO9 substrates to p53, proposing it lowers p53 stability and linking zoledronic acid to p53 stabilization and osteoclast ferroptosis.\",\n      \"evidence\": \"Co-IP, knockdown/overexpression rescue, CCK-8 viability and ferroptosis marker measurements\",\n      \"pmids\": [\"35003915\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in vitro ubiquitination reconstitution or degron mutagenesis\", \"Single lab without independent replication\", \"Direct vs indirect effect on p53 not distinguished\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Placed FBXO9 within a transcriptional regulatory axis by identifying ZNF143 as an upstream activator and FBXW7 as a degradation target, connecting FBXO9 to HCC proliferation and metastasis.\",\n      \"evidence\": \"In vitro/in vivo loss- and gain-of-function, Co-IP, ubiquitination assay, ChIP/reporter assay for ZNF143\",\n      \"pmids\": [\"35847937\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural validation of FBXW7 recognition\", \"Not independently replicated\", \"Degron on FBXW7 unmapped\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined a non-canonical degradation outcome by showing FBXO9 ubiquitinates ATP6V1A to drive HSPA8-dependent cytoplasmic sequestration, inhibiting V-ATPase assembly and Wnt-driven metastasis.\",\n      \"evidence\": \"MS-based interactome, reciprocal Co-IP, CRISPR/shRNA, migration/sphere assays, in vivo metastasis model and V-ATPase inhibitor treatment\",\n      \"pmids\": [\"38486234\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which ubiquitination promotes HSPA8 binding not defined\", \"Ubiquitin linkage type not specified\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified DPPA5 as a substrate from a reprogramming screen, showing FBXO9 opposes pluripotency induction by degrading a pluripotency-associated protein.\",\n      \"evidence\": \"RNAi screen during cellular reprogramming with FBXO9 silencing and DPPA5 proteasomal degradation readout\",\n      \"pmids\": [\"38227647\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No reconstitution confirming direct ubiquitination of DPPA5\", \"Degron not mapped\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Established the most detailed substrate-recognition mechanism: a GSK-3β/Akt-primed YAP phosphodegron and K48-linked ubiquitination at K76, defining kinase-gated degron logic for FBXO9.\",\n      \"evidence\": \"Co-IP, K48-linkage-specific ubiquitination assay, mutagenesis of YAP K76 and phosphosites, pharmacological Akt/GSK-3β inhibition, chemosensitivity assays\",\n      \"pmids\": [\"40902979\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of degron engagement not resolved\", \"In vivo relevance of the YAP axis not established in this study\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Linked FBXO9 to anti-tumor immunity by showing it degrades PD-L1 and impedes its maturation, impairing immune evasion in pancreatic cancer.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, in vivo overexpression tumor model, cytotoxic T cell activation readout\",\n      \"pmids\": [\"41646985\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural validation or degron mapping on PD-L1\", \"Not independently replicated\", \"Mechanism of maturation interference vs degradation not separated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How FBXO9 selects among its many substrates in a given cell type, and whether common degron features or upstream signals coordinate this context-dependent specificity, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structural model of FBXO9 substrate engagement\", \"Determinants of tissue-specific substrate choice unknown\", \"No unified degron consensus across substrates established\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 2, 3, 5]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [1, 2, 5]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 2, 5]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [1, 3, 4]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 5]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [2, 6]}\n    ],\n    \"complexes\": [\"SCF (SKP1-Cullin1-RBX1) E3 ubiquitin ligase\"],\n    \"partners\": [\"SKP1\", \"CUL1\", \"RBX1\", \"Neurog2\", \"YAP\", \"ATP6V1A\", \"FBXW7\", \"PD-L1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}