{"gene":"FBXL6","run_date":"2026-04-28T17:46:03","timeline":{"discoveries":[{"year":2020,"finding":"FBXL6, as a substrate recognition subunit of the SCF (Skp1-Cul1-F-box protein) E3 ubiquitin ligase complex, binds HSP90AA1 and promotes its K63-linked polyubiquitination, leading to HSP90AA1 stabilization (not degradation). Stabilized HSP90AA1 in turn prevents c-MYC degradation, activating c-MYC signaling. Activated c-MYC then directly binds the FBXL6 promoter to transcriptionally induce FBXL6 expression, forming a feed-forward loop.","method":"IP/Mass Spectrometry, Co-immunoprecipitation, in vivo ubiquitination assay, luciferase reporter assay, chromatin immunoprecipitation (ChIP), shRNA knockdown with proliferation/colony formation assays, xenograft tumor model","journal":"Cell communication and signaling : CCS","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (Co-IP, ubiquitination assay, ChIP, reporter assay) in a single study with in vivo validation","pmids":["32576198"],"is_preprint":false},{"year":2021,"finding":"FBXL6 physically interacts with phosphorylated p53 (phospho-Ser315) and mediates its K48-linked polyubiquitination and proteasomal degradation, thereby suppressing p53 signaling. Conversely, p53 transcriptionally represses FBXL6 expression by binding the FBXL6 core promoter, establishing a reciprocal feed-forward loop.","method":"Co-immunoprecipitation, in vivo ubiquitination assay, proteasome inhibitor rescue, ChIP, shRNA knockdown with cell cycle and apoptosis readouts (flow cytometry), colony formation assay","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods including Co-IP, ubiquitination assay, ChIP, and functional KD phenotypes","pmids":["33568778"],"is_preprint":false},{"year":2023,"finding":"FBXL6 promotes K63-linked polyubiquitination of both wild-type KRAS and mutant KRASG12D at lysine 128, leading to KRAS activation and enhanced binding to RAF, which activates MEK/ERK/mTOR signaling. This oncogenic axis depends on PRELID2-induced ROS generation. Validated in transgenic mouse models (LC, KC, KLC).","method":"Co-immunoprecipitation, Western blotting, in vivo ubiquitination assay, RAS activity detection assay, transgenic mouse models, multiomics, pharmacological inhibition of MEK/mTOR","journal":"Military Medical Research","confidence":"High","confidence_rationale":"Tier 1–2 — ubiquitination site mapping (K128), activity assay, Co-IP, and in vivo transgenic mouse validation with multiple orthogonal methods","pmids":["38124228"],"is_preprint":false},{"year":2023,"finding":"VRK2 kinase phosphorylates transketolase (TKT) at Thr287, which then recruits FBXL6 to ubiquitinate and activate TKT. Activated TKT drives ROS-mTOR signaling, upregulating PD-L1 and VRK2, leading to immune evasion and HCC metastasis. FBXL6 thus acts downstream of VRK2 phosphorylation to regulate TKT activity via ubiquitination.","method":"Co-immunoprecipitation, ubiquitination assay, phosphorylation assay, shRNA knockdown, transgenic mouse models (Alb-Cre driven), in vitro and in vivo functional assays","journal":"Experimental & molecular medicine","confidence":"High","confidence_rationale":"Tier 2 — Co-IP, ubiquitination assay, in vivo transgenic mouse validation, and functional rescue experiments with multiple orthogonal methods","pmids":["37653031"],"is_preprint":false},{"year":2023,"finding":"FBXL6 acts as an E3 ubiquitin ligase that ubiquitinates newly synthesized mitochondrial ribosomal proteins (MRPs) as part of a ribosome-associated quality control mechanism. FBXL6 physically binds chaperones involved in folding/trafficking of newly synthesized peptides and ribosomal-associated quality control (RQC) proteins. Deletion of these interacting partners abolishes FBXL6–substrate interactions. FBXL6 KO cells fail to degrade mistranslated MRPs, display MRP aggregation, altered mitochondrial metabolism, and inhibited cell cycle under oxidative conditions.","method":"Co-immunoprecipitation, ubiquitination assay, FBXL6 knockout cells, mitochondrial fractionation/metabolic assays, cell cycle analysis, protein aggregation assays","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 — KO model with multiple functional readouts, Co-IP with chaperones and RQC factors, ubiquitination assay, mechanistic rescue experiments","pmids":["37267103"],"is_preprint":false},{"year":2025,"finding":"FBXL6 physically interacts with ATAD3A and promotes its K63-linked polyubiquitination, stabilizing ATAD3A protein. Stabilized ATAD3A activates aerobic glycolysis (Warburg effect), thereby promoting TNBC tumor malignancy.","method":"Co-immunoprecipitation, ubiquitination assay, ATAD3A genetic depletion, Western blotting, cellular and xenograft tumor models","journal":"International journal of biological macromolecules","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP and ubiquitination assay with in vivo model, but single study with limited mechanistic detail on K63 linkage functional consequence","pmids":["40975350"],"is_preprint":false},{"year":2025,"finding":"FBXL6 physically interacts with CDKN1C/p57Kip2 and promotes its polyubiquitination and proteasomal degradation, thereby destabilizing this CDK inhibitor/tumor suppressor and driving cell cycle progression, proliferation, migration, and invasion in lung adenocarcinoma.","method":"Co-immunoprecipitation, ubiquitination assay, shRNA knockdown and overexpression rescue assays, in vivo xenograft and metastasis models","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP, ubiquitination assay, and rescue experiments in a single study","pmids":["41443404"],"is_preprint":false},{"year":2022,"finding":"FBXL6 promotes proliferation of keloid fibroblasts by inducing c-MYC expression, and the pro-proliferative effect of FBXL6 requires c-MYC as an epistatic downstream effector; FBXL6 knockdown reduces c-MYC, cyclin A1, cyclin D2, cyclin E1, and collagen I levels, while c-MYC overexpression rescues the proliferation defect caused by FBXL6 shRNA.","method":"shRNA knockdown, overexpression, c-MYC epistasis rescue, CCK-8 viability assay, Western blot, RT-PCR","journal":"International wound journal","confidence":"Medium","confidence_rationale":"Tier 3 — epistasis experiment (c-MYC rescue of FBXL6 KD) but no direct biochemical interaction or ubiquitination assay performed","pmids":["35606330"],"is_preprint":false}],"current_model":"FBXL6 is an SCF-type E3 ubiquitin ligase (F-box/LRR protein) that stabilizes or activates multiple oncogenic substrates via K63-linked polyubiquitination (HSP90AA1, KRAS, ATAD3A, TKT) and degrades tumor suppressors via K48-linked polyubiquitination (phospho-p53-S315, CDKN1C), while also acting in cytosolic ribosome-associated quality control to ubiquitinate mistranslated mitochondrial ribosomal proteins, collectively driving cell proliferation, metabolic reprogramming, and tumor progression."},"narrative":{"teleology":[{"year":2020,"claim":"The first mechanistic characterization established that FBXL6 functions as an SCF E3 ligase subunit that K63-ubiquitinates HSP90AA1, stabilizing it to sustain c-MYC signaling, with c-MYC reciprocally transactivating FBXL6—revealing a feed-forward oncogenic loop.","evidence":"IP/MS substrate identification, Co-IP, in vivo ubiquitination assay, ChIP, luciferase reporter, shRNA KD, and xenograft models in gastric cancer cells","pmids":["32576198"],"confidence":"High","gaps":["Whether the FBXL6–HSP90AA1–c-MYC loop operates in tissues beyond gastric cancer","Structural basis for FBXL6 recognition of HSP90AA1","Whether other SCF adaptors can substitute for FBXL6 in HSP90AA1 stabilization"]},{"year":2021,"claim":"Discovery that FBXL6 can also deploy K48-linked ubiquitination—targeting phospho-p53 (Ser315) for proteasomal degradation—demonstrated that FBXL6 is not limited to K63 stabilization but can switch ubiquitin linkage type to degrade tumor suppressors, with p53 reciprocally repressing FBXL6 transcription.","evidence":"Co-IP, in vivo ubiquitination assay, MG132 rescue, ChIP on FBXL6 promoter, cell-cycle and apoptosis flow cytometry in hepatocellular carcinoma cells","pmids":["33568778"],"confidence":"High","gaps":["Whether FBXL6 recognizes only Ser315-phosphorylated p53 or additional phospho-forms","How FBXL6 determines K48 versus K63 linkage on different substrates","Kinase(s) responsible for the Ser315 phosphorylation event that enables FBXL6 recruitment"]},{"year":2022,"claim":"Functional epistasis experiments in keloid fibroblasts confirmed that the pro-proliferative activity of FBXL6 is c-MYC-dependent in a non-cancer context, generalizing the FBXL6–c-MYC axis beyond carcinomas.","evidence":"shRNA KD with c-MYC overexpression rescue, CCK-8 viability, Western blot in keloid fibroblasts","pmids":["35606330"],"confidence":"Medium","gaps":["No direct ubiquitination or Co-IP data in this system—mechanism inferred from prior work","Whether fibroblast-specific cofactors modulate FBXL6 substrate selection"]},{"year":2023,"claim":"Three independent studies broadened FBXL6's substrate repertoire to KRAS, TKT, and mitochondrial ribosomal proteins, revealing that FBXL6 activates RAS–RAF–MEK/ERK signaling through K63 ubiquitination of KRAS-K128, links VRK2-mediated TKT phosphorylation to metabolic reprogramming and immune evasion, and participates in cytosolic ribosome-associated quality control of mistranslated MRPs.","evidence":"Co-IP, site-directed mutagenesis (K128), RAS-GTP pulldown, transgenic mouse models for KRAS; Co-IP, phospho-mapping, Alb-Cre mouse models for TKT; FBXL6 KO, chaperone/RQC-factor interaction mapping, MRP aggregation and metabolic assays for quality control","pmids":["38124228","37653031","37267103"],"confidence":"High","gaps":["Whether FBXL6-mediated K63 ubiquitination of KRAS and TKT uses the same E2 conjugating enzyme","Structural basis for how phospho-Thr287 on TKT promotes FBXL6 recruitment","Identity of the specific RQC factors required for FBXL6-dependent MRP ubiquitination","Whether the MRP quality-control function operates independently of the SCF complex"]},{"year":2025,"claim":"The substrate catalog was further extended to ATAD3A (K63 stabilization driving aerobic glycolysis in TNBC) and CDKN1C/p57 (degradative ubiquitination promoting lung adenocarcinoma), consolidating FBXL6 as a dual-linkage E3 ligase targeting both oncogenic effectors and tumor suppressors.","evidence":"Co-IP and ubiquitination assays with xenograft models for ATAD3A; Co-IP, ubiquitination assay, shRNA/overexpression rescue, xenograft and metastasis models for CDKN1C","pmids":["40975350","41443404"],"confidence":"Medium","gaps":["Ubiquitin-linkage specificity for CDKN1C not explicitly mapped (K48 inferred from degradation)","No structural or mutagenesis data identifying the FBXL6-binding degron on CDKN1C or ATAD3A","Whether ATAD3A stabilization and glycolysis induction are direct or partly mediated by downstream effectors"]},{"year":null,"claim":"The determinants of FBXL6 ubiquitin-linkage switching (K63 stabilization vs. K48 degradation), the identity of the E2 conjugating enzymes used for each substrate class, and the physiological role of FBXL6 in normal non-transformed tissues remain unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No E2 enzyme identified for either K63 or K48 FBXL6-dependent reactions","No structural model of FBXL6 LRR domain with any substrate","Phenotype of whole-organism FBXL6 knockout in mammals not reported","Regulatory post-translational modifications on FBXL6 itself are uncharacterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,2,3,4,5,6]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[4]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1,2,3,4,5,6]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,3]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[1,6]}],"complexes":["SCF (Skp1-Cul1-FBXL6)"],"partners":["HSP90AA1","TP53","KRAS","TKT","CDKN1C","ATAD3A","SKP1","CUL1"],"other_free_text":[]},"mechanistic_narrative":"FBXL6 is an F-box/leucine-rich-repeat protein that functions as the substrate-recognition subunit of an SCF (Skp1–Cul1–F-box) E3 ubiquitin ligase, coupling K63-linked polyubiquitination to the stabilization and activation of oncogenic substrates and K48-linked polyubiquitination to the degradation of tumor suppressors. FBXL6 stabilizes HSP90AA1 via K63-linked ubiquitination, thereby preventing c-MYC degradation and establishing a c-MYC–FBXL6 transcriptional feed-forward loop, and similarly activates KRAS (at K128) and TKT (downstream of VRK2-mediated phosphorylation) through K63-linked ubiquitination to promote MEK/ERK/mTOR and ROS-mTOR signaling, respectively [PMID:32576198, PMID:38124228, PMID:37653031]. Conversely, FBXL6 targets phospho-p53 (Ser315) and the CDK inhibitor CDKN1C/p57 for K48-linked polyubiquitination and proteasomal degradation, suppressing tumor-suppressive checkpoints and driving cell cycle progression [PMID:33568778, PMID:41443404]. Independent of its oncogenic substrates, FBXL6 participates in cytosolic ribosome-associated quality control by ubiquitinating mistranslated mitochondrial ribosomal proteins; its loss causes MRP aggregation, altered mitochondrial metabolism, and impaired cell-cycle progression under oxidative stress [PMID:37267103]."},"prefetch_data":{"uniprot":{"accession":"Q8N531","full_name":"F-box/LRR-repeat protein 6","aliases":["F-box and leucine-rich repeat protein 6","F-box protein FBL6","FBL6A"],"length_aa":539,"mass_kda":58.6,"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/Q8N531/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/FBXL6","classification":"Not Classified","n_dependent_lines":42,"n_total_lines":1208,"dependency_fraction":0.0347682119205298},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/FBXL6","total_profiled":1310},"omim":[{"mim_id":"609076","title":"F-BOX AND LEUCINE-RICH REPEAT PROTEIN 6; FBXL6","url":"https://www.omim.org/entry/609076"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Vesicles","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/FBXL6"},"hgnc":{"alias_symbol":["FBL6"],"prev_symbol":[]},"alphafold":{"accession":"Q8N531","domains":[{"cath_id":"-","chopping":"424-539","consensus_level":"medium","plddt":89.7234,"start":424,"end":539},{"cath_id":"1.10.10","chopping":"114-157","consensus_level":"medium","plddt":90.038,"start":114,"end":157}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8N531","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8N531-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8N531-F1-predicted_aligned_error_v6.png","plddt_mean":78.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=FBXL6","jax_strain_url":"https://www.jax.org/strain/search?query=FBXL6"},"sequence":{"accession":"Q8N531","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8N531.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8N531/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8N531"}},"corpus_meta":[{"pmid":"32576198","id":"PMC_32576198","title":"FBXL6 governs c-MYC to promote hepatocellular carcinoma through ubiquitination and stabilization of HSP90AA1.","date":"2020","source":"Cell communication and signaling : CCS","url":"https://pubmed.ncbi.nlm.nih.gov/32576198","citation_count":69,"is_preprint":false},{"pmid":"38124228","id":"PMC_38124228","title":"Elevated FBXL6 activates both wild-type KRAS and mutant KRASG12D and drives HCC tumorigenesis via the ERK/mTOR/PRELID2/ROS axis in mice.","date":"2023","source":"Military Medical Research","url":"https://pubmed.ncbi.nlm.nih.gov/38124228","citation_count":43,"is_preprint":false},{"pmid":"33568778","id":"PMC_33568778","title":"FBXL6 degrades phosphorylated p53 to promote tumor growth.","date":"2021","source":"Cell death and differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/33568778","citation_count":30,"is_preprint":false},{"pmid":"37653031","id":"PMC_37653031","title":"Elevated FBXL6 expression in hepatocytes activates VRK2-transketolase-ROS-mTOR-mediated immune evasion and liver cancer metastasis in mice.","date":"2023","source":"Experimental & molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/37653031","citation_count":28,"is_preprint":false},{"pmid":"35069490","id":"PMC_35069490","title":"Complete Genome Sequencing and Comparative Genomics of Three Potential Probiotic Strains, Lacticaseibacillus casei FBL6, Lacticaseibacillus chiayiensis FBL7, and Lacticaseibacillus zeae FBL8.","date":"2022","source":"Frontiers in microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/35069490","citation_count":26,"is_preprint":false},{"pmid":"36183674","id":"PMC_36183674","title":"FBXL6 depletion restrains clear cell renal cell carcinoma progression.","date":"2022","source":"Translational oncology","url":"https://pubmed.ncbi.nlm.nih.gov/36183674","citation_count":9,"is_preprint":false},{"pmid":"35606330","id":"PMC_35606330","title":"FBXL6 is dysregulated in keloids and promotes keloid fibroblast growth by inducing c-Myc expression.","date":"2022","source":"International wound journal","url":"https://pubmed.ncbi.nlm.nih.gov/35606330","citation_count":8,"is_preprint":false},{"pmid":"37267103","id":"PMC_37267103","title":"The E3 ubiquitin ligase FBXL6 controls the quality of newly synthesized mitochondrial ribosomal proteins.","date":"2023","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/37267103","citation_count":4,"is_preprint":false},{"pmid":"40975350","id":"PMC_40975350","title":"Elevated FBXL6 activates ATAD3A through K63-linked polyubiquitination and promotes the malignant progression of TNBC via metabolic reprogramming.","date":"2025","source":"International journal of biological macromolecules","url":"https://pubmed.ncbi.nlm.nih.gov/40975350","citation_count":1,"is_preprint":false},{"pmid":"41443404","id":"PMC_41443404","title":"FBXL6 drives tumorigenesis in lung adenocarcinoma through ubiquitination and proteasomal degradation of CDKN1C.","date":"2025","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/41443404","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7047,"output_tokens":2342,"usd":0.028136},"stage2":{"model":"claude-opus-4-6","input_tokens":5619,"output_tokens":2413,"usd":0.13263},"total_usd":0.160766,"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\": 2020,\n      \"finding\": \"FBXL6, as a substrate recognition subunit of the SCF (Skp1-Cul1-F-box protein) E3 ubiquitin ligase complex, binds HSP90AA1 and promotes its K63-linked polyubiquitination, leading to HSP90AA1 stabilization (not degradation). Stabilized HSP90AA1 in turn prevents c-MYC degradation, activating c-MYC signaling. Activated c-MYC then directly binds the FBXL6 promoter to transcriptionally induce FBXL6 expression, forming a feed-forward loop.\",\n      \"method\": \"IP/Mass Spectrometry, Co-immunoprecipitation, in vivo ubiquitination assay, luciferase reporter assay, chromatin immunoprecipitation (ChIP), shRNA knockdown with proliferation/colony formation assays, xenograft tumor model\",\n      \"journal\": \"Cell communication and signaling : CCS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (Co-IP, ubiquitination assay, ChIP, reporter assay) in a single study with in vivo validation\",\n      \"pmids\": [\"32576198\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"FBXL6 physically interacts with phosphorylated p53 (phospho-Ser315) and mediates its K48-linked polyubiquitination and proteasomal degradation, thereby suppressing p53 signaling. Conversely, p53 transcriptionally represses FBXL6 expression by binding the FBXL6 core promoter, establishing a reciprocal feed-forward loop.\",\n      \"method\": \"Co-immunoprecipitation, in vivo ubiquitination assay, proteasome inhibitor rescue, ChIP, shRNA knockdown with cell cycle and apoptosis readouts (flow cytometry), colony formation assay\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including Co-IP, ubiquitination assay, ChIP, and functional KD phenotypes\",\n      \"pmids\": [\"33568778\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"FBXL6 promotes K63-linked polyubiquitination of both wild-type KRAS and mutant KRASG12D at lysine 128, leading to KRAS activation and enhanced binding to RAF, which activates MEK/ERK/mTOR signaling. This oncogenic axis depends on PRELID2-induced ROS generation. Validated in transgenic mouse models (LC, KC, KLC).\",\n      \"method\": \"Co-immunoprecipitation, Western blotting, in vivo ubiquitination assay, RAS activity detection assay, transgenic mouse models, multiomics, pharmacological inhibition of MEK/mTOR\",\n      \"journal\": \"Military Medical Research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — ubiquitination site mapping (K128), activity assay, Co-IP, and in vivo transgenic mouse validation with multiple orthogonal methods\",\n      \"pmids\": [\"38124228\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"VRK2 kinase phosphorylates transketolase (TKT) at Thr287, which then recruits FBXL6 to ubiquitinate and activate TKT. Activated TKT drives ROS-mTOR signaling, upregulating PD-L1 and VRK2, leading to immune evasion and HCC metastasis. FBXL6 thus acts downstream of VRK2 phosphorylation to regulate TKT activity via ubiquitination.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, phosphorylation assay, shRNA knockdown, transgenic mouse models (Alb-Cre driven), in vitro and in vivo functional assays\",\n      \"journal\": \"Experimental & molecular medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP, ubiquitination assay, in vivo transgenic mouse validation, and functional rescue experiments with multiple orthogonal methods\",\n      \"pmids\": [\"37653031\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"FBXL6 acts as an E3 ubiquitin ligase that ubiquitinates newly synthesized mitochondrial ribosomal proteins (MRPs) as part of a ribosome-associated quality control mechanism. FBXL6 physically binds chaperones involved in folding/trafficking of newly synthesized peptides and ribosomal-associated quality control (RQC) proteins. Deletion of these interacting partners abolishes FBXL6–substrate interactions. FBXL6 KO cells fail to degrade mistranslated MRPs, display MRP aggregation, altered mitochondrial metabolism, and inhibited cell cycle under oxidative conditions.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, FBXL6 knockout cells, mitochondrial fractionation/metabolic assays, cell cycle analysis, protein aggregation assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO model with multiple functional readouts, Co-IP with chaperones and RQC factors, ubiquitination assay, mechanistic rescue experiments\",\n      \"pmids\": [\"37267103\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"FBXL6 physically interacts with ATAD3A and promotes its K63-linked polyubiquitination, stabilizing ATAD3A protein. Stabilized ATAD3A activates aerobic glycolysis (Warburg effect), thereby promoting TNBC tumor malignancy.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, ATAD3A genetic depletion, Western blotting, cellular and xenograft tumor models\",\n      \"journal\": \"International journal of biological macromolecules\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP and ubiquitination assay with in vivo model, but single study with limited mechanistic detail on K63 linkage functional consequence\",\n      \"pmids\": [\"40975350\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"FBXL6 physically interacts with CDKN1C/p57Kip2 and promotes its polyubiquitination and proteasomal degradation, thereby destabilizing this CDK inhibitor/tumor suppressor and driving cell cycle progression, proliferation, migration, and invasion in lung adenocarcinoma.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, shRNA knockdown and overexpression rescue assays, in vivo xenograft and metastasis models\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP, ubiquitination assay, and rescue experiments in a single study\",\n      \"pmids\": [\"41443404\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"FBXL6 promotes proliferation of keloid fibroblasts by inducing c-MYC expression, and the pro-proliferative effect of FBXL6 requires c-MYC as an epistatic downstream effector; FBXL6 knockdown reduces c-MYC, cyclin A1, cyclin D2, cyclin E1, and collagen I levels, while c-MYC overexpression rescues the proliferation defect caused by FBXL6 shRNA.\",\n      \"method\": \"shRNA knockdown, overexpression, c-MYC epistasis rescue, CCK-8 viability assay, Western blot, RT-PCR\",\n      \"journal\": \"International wound journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — epistasis experiment (c-MYC rescue of FBXL6 KD) but no direct biochemical interaction or ubiquitination assay performed\",\n      \"pmids\": [\"35606330\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"FBXL6 is an SCF-type E3 ubiquitin ligase (F-box/LRR protein) that stabilizes or activates multiple oncogenic substrates via K63-linked polyubiquitination (HSP90AA1, KRAS, ATAD3A, TKT) and degrades tumor suppressors via K48-linked polyubiquitination (phospho-p53-S315, CDKN1C), while also acting in cytosolic ribosome-associated quality control to ubiquitinate mistranslated mitochondrial ribosomal proteins, collectively driving cell proliferation, metabolic reprogramming, and tumor progression.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"FBXL6 is an F-box/leucine-rich-repeat protein that functions as the substrate-recognition subunit of an SCF (Skp1–Cul1–F-box) E3 ubiquitin ligase, coupling K63-linked polyubiquitination to the stabilization and activation of oncogenic substrates and K48-linked polyubiquitination to the degradation of tumor suppressors. FBXL6 stabilizes HSP90AA1 via K63-linked ubiquitination, thereby preventing c-MYC degradation and establishing a c-MYC–FBXL6 transcriptional feed-forward loop, and similarly activates KRAS (at K128) and TKT (downstream of VRK2-mediated phosphorylation) through K63-linked ubiquitination to promote MEK/ERK/mTOR and ROS-mTOR signaling, respectively [PMID:32576198, PMID:38124228, PMID:37653031]. Conversely, FBXL6 targets phospho-p53 (Ser315) and the CDK inhibitor CDKN1C/p57 for K48-linked polyubiquitination and proteasomal degradation, suppressing tumor-suppressive checkpoints and driving cell cycle progression [PMID:33568778, PMID:41443404]. Independent of its oncogenic substrates, FBXL6 participates in cytosolic ribosome-associated quality control by ubiquitinating mistranslated mitochondrial ribosomal proteins; its loss causes MRP aggregation, altered mitochondrial metabolism, and impaired cell-cycle progression under oxidative stress [PMID:37267103].\",\n  \"teleology\": [\n    {\n      \"year\": 2020,\n      \"claim\": \"The first mechanistic characterization established that FBXL6 functions as an SCF E3 ligase subunit that K63-ubiquitinates HSP90AA1, stabilizing it to sustain c-MYC signaling, with c-MYC reciprocally transactivating FBXL6—revealing a feed-forward oncogenic loop.\",\n      \"evidence\": \"IP/MS substrate identification, Co-IP, in vivo ubiquitination assay, ChIP, luciferase reporter, shRNA KD, and xenograft models in gastric cancer cells\",\n      \"pmids\": [\"32576198\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether the FBXL6–HSP90AA1–c-MYC loop operates in tissues beyond gastric cancer\",\n        \"Structural basis for FBXL6 recognition of HSP90AA1\",\n        \"Whether other SCF adaptors can substitute for FBXL6 in HSP90AA1 stabilization\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Discovery that FBXL6 can also deploy K48-linked ubiquitination—targeting phospho-p53 (Ser315) for proteasomal degradation—demonstrated that FBXL6 is not limited to K63 stabilization but can switch ubiquitin linkage type to degrade tumor suppressors, with p53 reciprocally repressing FBXL6 transcription.\",\n      \"evidence\": \"Co-IP, in vivo ubiquitination assay, MG132 rescue, ChIP on FBXL6 promoter, cell-cycle and apoptosis flow cytometry in hepatocellular carcinoma cells\",\n      \"pmids\": [\"33568778\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether FBXL6 recognizes only Ser315-phosphorylated p53 or additional phospho-forms\",\n        \"How FBXL6 determines K48 versus K63 linkage on different substrates\",\n        \"Kinase(s) responsible for the Ser315 phosphorylation event that enables FBXL6 recruitment\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Functional epistasis experiments in keloid fibroblasts confirmed that the pro-proliferative activity of FBXL6 is c-MYC-dependent in a non-cancer context, generalizing the FBXL6–c-MYC axis beyond carcinomas.\",\n      \"evidence\": \"shRNA KD with c-MYC overexpression rescue, CCK-8 viability, Western blot in keloid fibroblasts\",\n      \"pmids\": [\"35606330\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No direct ubiquitination or Co-IP data in this system—mechanism inferred from prior work\",\n        \"Whether fibroblast-specific cofactors modulate FBXL6 substrate selection\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Three independent studies broadened FBXL6's substrate repertoire to KRAS, TKT, and mitochondrial ribosomal proteins, revealing that FBXL6 activates RAS–RAF–MEK/ERK signaling through K63 ubiquitination of KRAS-K128, links VRK2-mediated TKT phosphorylation to metabolic reprogramming and immune evasion, and participates in cytosolic ribosome-associated quality control of mistranslated MRPs.\",\n      \"evidence\": \"Co-IP, site-directed mutagenesis (K128), RAS-GTP pulldown, transgenic mouse models for KRAS; Co-IP, phospho-mapping, Alb-Cre mouse models for TKT; FBXL6 KO, chaperone/RQC-factor interaction mapping, MRP aggregation and metabolic assays for quality control\",\n      \"pmids\": [\"38124228\", \"37653031\", \"37267103\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether FBXL6-mediated K63 ubiquitination of KRAS and TKT uses the same E2 conjugating enzyme\",\n        \"Structural basis for how phospho-Thr287 on TKT promotes FBXL6 recruitment\",\n        \"Identity of the specific RQC factors required for FBXL6-dependent MRP ubiquitination\",\n        \"Whether the MRP quality-control function operates independently of the SCF complex\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"The substrate catalog was further extended to ATAD3A (K63 stabilization driving aerobic glycolysis in TNBC) and CDKN1C/p57 (degradative ubiquitination promoting lung adenocarcinoma), consolidating FBXL6 as a dual-linkage E3 ligase targeting both oncogenic effectors and tumor suppressors.\",\n      \"evidence\": \"Co-IP and ubiquitination assays with xenograft models for ATAD3A; Co-IP, ubiquitination assay, shRNA/overexpression rescue, xenograft and metastasis models for CDKN1C\",\n      \"pmids\": [\"40975350\", \"41443404\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Ubiquitin-linkage specificity for CDKN1C not explicitly mapped (K48 inferred from degradation)\",\n        \"No structural or mutagenesis data identifying the FBXL6-binding degron on CDKN1C or ATAD3A\",\n        \"Whether ATAD3A stabilization and glycolysis induction are direct or partly mediated by downstream effectors\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The determinants of FBXL6 ubiquitin-linkage switching (K63 stabilization vs. K48 degradation), the identity of the E2 conjugating enzymes used for each substrate class, and the physiological role of FBXL6 in normal non-transformed tissues remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No E2 enzyme identified for either K63 or K48 FBXL6-dependent reactions\",\n        \"No structural model of FBXL6 LRR domain with any substrate\",\n        \"Phenotype of whole-organism FBXL6 knockout in mammals not reported\",\n        \"Regulatory post-translational modifications on FBXL6 itself are uncharacterized\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 2, 3, 4, 5, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 2, 3, 4, 5, 6]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [1, 6]}\n    ],\n    \"complexes\": [\n      \"SCF (Skp1-Cul1-FBXL6)\"\n    ],\n    \"partners\": [\n      \"HSP90AA1\",\n      \"TP53\",\n      \"KRAS\",\n      \"TKT\",\n      \"CDKN1C\",\n      \"ATAD3A\",\n      \"SKP1\",\n      \"CUL1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}