{"gene":"FBXL8","run_date":"2026-06-09T23:54:43","timeline":{"discoveries":[{"year":2020,"finding":"SCF-Fbxl8 is identified as the E3 ubiquitin ligase for phospho-Thr-283 cyclin D3; it poly-ubiquitylates p-Thr-283 cyclin D3 targeting it for proteasomal degradation, thereby antagonizing cell cycle progression, hematopoietic cell proliferation, and oncogene-induced transformation.","method":"Co-immunoprecipitation, ubiquitylation assays, cyclin D3T283A phosphorylation-deficient mutant rescue experiments, cell proliferation and transformation assays","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, ubiquitylation assay, mutagenesis (T283A non-phosphorylatable mutant), functional rescue, single lab with multiple orthogonal methods","pmids":["33122824"],"is_preprint":false},{"year":2022,"finding":"SCF-Fbxl8 binds and ubiquitylates c-myc independent of phosphorylation (i.e., independent of the Fbxw7 phospho-degron), targeting a distinct pool of c-myc for degradation; loss of Fbxl8 increases c-myc protein levels and stability and increases cell division, while concurrent loss of Fbxl8 and Fbxw7 causes a synergistic increase in c-myc protein.","method":"Co-immunoprecipitation, ubiquitylation assay, loss-of-function (Fbxl8 knockdown/loss), gain-of-function (Fbxl8 overexpression), protein stability assay, double-loss epistasis (Fbxl8 + Fbxw7)","journal":"Cancer biology & therapy","confidence":"High","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitylation assay, gain- and loss-of-function, epistasis with Fbxw7, multiple orthogonal methods in single lab","pmids":["35438057"],"is_preprint":false},{"year":2020,"finding":"FBXL8, as an E3 ubiquitin ligase, interacts with (by Co-IP) and promotes degradation of CCND2 (cyclin D2) and IRF5 in breast cancer cells; knockdown of FBXL8 causes accumulation of CCND2 and IRF5, induces apoptosis, and inhibits cell migration and invasion.","method":"Co-immunoprecipitation, FBXL8 knockdown with protein accumulation readout, cell migration/invasion assays, apoptosis assays","journal":"Cancers","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — single Co-IP with correlated protein levels, functional knockdown phenotype, single lab, no direct ubiquitylation assay reported in abstract","pmids":["32784654"],"is_preprint":false},{"year":2021,"finding":"FBXL8 and FZR1 each independently pull down CCNF (cyclin F); double knockdown of both FBXL8 and FZR1 causes CCNF protein accumulation, indicating both E3 ligases contribute to CCNF degradation in breast cancer cells.","method":"Co-immunoprecipitation (pulldown), double knockdown with protein accumulation readout","journal":"Cancers","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP pulldown and double-KD epistasis, single lab, no direct ubiquitylation assay described in abstract","pmids":["34201347"],"is_preprint":false},{"year":2023,"finding":"SCF-FBXL8 directly interacts with and ubiquitinates TP53 (p53), promoting its degradation via the proteasome in colorectal cancer cells; FBXL8 knockout stabilizes p53 and inhibits tumor proliferation, invasion, migration, stem-cell-like properties, and liver metastasis in vivo.","method":"Co-immunoprecipitation, ubiquitination assay, FBXL8 knockout, xenograft mouse model, western blotting","journal":"Clinical and translational medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, in vivo knockout model, single lab, multiple orthogonal methods","pmids":["36855778"],"is_preprint":false},{"year":2024,"finding":"FBXL8 interacts with Snail1 via its C3-binding domain and promotes Snail1 degradation through the ubiquitin-proteasome system, leading to decreased RhoA activation; FBXL8 overexpression in cardiac fibroblasts suppresses TGFβ-induced fibroblast activation (proliferation, migration, contraction, collagen secretion), and AAV9-mediated FBXL8 overexpression ameliorated post-myocardial infarction cardiac fibrosis in vivo.","method":"Co-immunoprecipitation, proteasome inhibition assay, FBXL8ΔC3 deletion mutant, FBXL8 overexpression/knockdown in cardiac fibroblasts, Snail1 rescue experiments, AAV9-mediated overexpression in MI mouse model, echocardiography and histology","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 2 / Moderate — Co-IP, domain-deletion mutagenesis, rescue with ectopic Snail1, in vivo AAV model with functional readouts, multiple orthogonal methods in single lab","pmids":["38615011"],"is_preprint":false},{"year":2026,"finding":"FBXL8 binds dephosphorylated IκBα (at S32/S36) and mediates K63-linked polyubiquitination at the K38 site of IκBα, thereby stabilizing IκBα (non-degradative ubiquitination) and inhibiting NF-κB p65 nuclear translocation; additionally, NF-κB p65 transcriptionally upregulates YY1, which in turn transcriptionally represses FBXL8, forming a feedforward loop.","method":"Co-immunoprecipitation, ubiquitination assay (K63-linkage-specific), site-directed mutagenesis (K38), dephosphorylation experiments, NF-κB p65 nuclear translocation assay, transcription factor binding (YY1-ChIP implied), in vitro and in vivo functional assays","journal":"International journal of biological sciences","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — Co-IP, K63-ubiquitination assay with lysine-site mutagenesis, functional nuclear translocation readout, in vivo data, multiple orthogonal methods in single lab","pmids":["41608629"],"is_preprint":false}],"current_model":"FBXL8 is an F-box protein that functions as the substrate-recognition subunit of SCF (Skp1-Cullin1-F-box) E3 ubiquitin ligase complexes, with multiple established substrates: it promotes K48-linked proteasomal degradation of phospho-Thr-283 cyclin D3, c-myc (phosphorylation-independent), CCND2, IRF5, CCNF, TP53, and Snail1, while conversely stabilizing IκBα through K63-linked non-degradative ubiquitination to suppress NF-κB signaling; collectively, its substrate repertoire places FBXL8 as a context-dependent regulator of cell cycle, apoptosis, EMT, and inflammation in multiple cancer and fibrotic settings."},"narrative":{"mechanistic_narrative":"FBXL8 is an F-box protein that operates as the substrate-recognition subunit of SCF (Skp1-Cullin1-F-box) E3 ubiquitin ligase complexes, coupling target recognition to ubiquitination across cell-cycle, oncogenic, and inflammatory programs [PMID:33122824, PMID:36855778]. Its canonical mode is K48-linked polyubiquitination driving proteasomal degradation: it targets phospho-Thr-283 cyclin D3 to antagonize cell-cycle progression and oncogene-induced transformation [PMID:33122824], and it degrades c-myc through a phosphorylation-independent route distinct from the Fbxw7 phospho-degron, such that combined loss of FBXL8 and Fbxw7 synergistically elevates c-myc [PMID:35438057]. In cancer settings FBXL8 likewise promotes turnover of TP53, with FBXL8 knockout stabilizing p53 and suppressing colorectal tumor proliferation, invasion, and liver metastasis in vivo [PMID:36855778], and it engages Snail1 via its C3-binding domain to limit RhoA activation and TGFβ-induced cardiac fibroblast activation, with AAV9-delivered FBXL8 ameliorating post-myocardial-infarction fibrosis [PMID:38615011]. Beyond degradative activity, FBXL8 binds dephosphorylated IκBα and assembles K63-linked, non-degradative polyubiquitin at K38, stabilizing IκBα to block NF-κB p65 nuclear translocation within a YY1-mediated feedforward loop [PMID:41608629]. This dual degradative/stabilizing repertoire positions FBXL8 as a context-dependent regulator of proliferation, apoptosis, EMT, and inflammation.","teleology":[{"year":2020,"claim":"Established the first defined SCF-FBXL8 substrate, answering whether FBXL8 acts as a functional E3 recognition subunit and linking it to cell-cycle control via phospho-degron recognition.","evidence":"Co-IP, ubiquitylation assays, and cyclin D3 T283A non-phosphorylatable mutant rescue with proliferation/transformation readouts","pmids":["33122824"],"confidence":"High","gaps":["Did not define the kinase generating the phospho-Thr-283 degron in this context","Structural basis of FBXL8 substrate binding not resolved"]},{"year":2020,"claim":"Extended the FBXL8 substrate set to CCND2 and IRF5 in breast cancer, addressing whether FBXL8 loss has tumor-relevant functional consequences.","evidence":"Co-IP with protein accumulation on FBXL8 knockdown plus migration, invasion, and apoptosis assays in breast cancer cells","pmids":["32784654"],"confidence":"Medium","gaps":["No direct ubiquitylation assay reported for CCND2 or IRF5","Linkage type and degradation route not directly demonstrated"]},{"year":2021,"claim":"Tested whether FBXL8 contributes to CCNF turnover, clarifying redundancy with another E3 pathway.","evidence":"Co-IP pulldown and double knockdown of FBXL8 and FZR1 with CCNF accumulation readout in breast cancer cells","pmids":["34201347"],"confidence":"Medium","gaps":["No direct ubiquitylation assay for CCNF","Relative contribution of FBXL8 versus FZR1 not quantified"]},{"year":2022,"claim":"Resolved whether FBXL8 targets c-myc independently of the canonical Fbxw7 phospho-degron, defining a parallel route of c-myc control.","evidence":"Co-IP, ubiquitylation assay, gain/loss-of-function, protein stability assay, and Fbxl8+Fbxw7 double-loss epistasis","pmids":["35438057"],"confidence":"High","gaps":["Degron or interaction surface on c-myc recognized by FBXL8 not mapped","Physiological setting where this pool dominates not defined"]},{"year":2023,"claim":"Demonstrated FBXL8-mediated degradation of TP53 in vivo, connecting FBXL8 to tumor suppressor turnover and metastatic phenotypes.","evidence":"Co-IP, ubiquitination assay, FBXL8 knockout, and xenograft mouse model in colorectal cancer","pmids":["36855778"],"confidence":"Medium","gaps":["Ubiquitin linkage type on p53 not specified","Relationship to MDM2-dependent p53 control not addressed"]},{"year":2024,"claim":"Identified a domain-specific Snail1 interaction and degradation by FBXL8, establishing an anti-fibrotic role via RhoA signaling.","evidence":"Co-IP, FBXL8ΔC3 deletion mutant, proteasome inhibition, Snail1 rescue, and AAV9 overexpression in a myocardial infarction mouse model","pmids":["38615011"],"confidence":"High","gaps":["Direct ubiquitin linkage on Snail1 not characterized","Mechanism linking Snail1 loss to reduced RhoA activation not fully resolved"]},{"year":2026,"claim":"Revealed a non-degradative FBXL8 function, answering how FBXL8 can suppress rather than promote substrate function through K63-linked ubiquitination.","evidence":"Co-IP, K63-linkage-specific ubiquitination assay with K38 site mutagenesis, dephosphorylation experiments, p65 nuclear translocation readout, and YY1 transcriptional feedback analysis","pmids":["41608629"],"confidence":"High","gaps":["Whether the same SCF complex configuration mediates both degradative and stabilizing activities is unresolved","Determinants selecting K63 versus K48 output by FBXL8 not defined"]},{"year":null,"claim":"How FBXL8 selects between degradative (K48) and stabilizing (K63) ubiquitin outputs across its diverse substrates, and the structural basis of its phosphorylation-dependent and -independent substrate recognition, remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of FBXL8-substrate complexes","Mechanism determining linkage-type choice unknown","No unified accounting of which substrates dominate in which tissue contexts"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,1,4,6]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,4,5,6]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[6]}],"localization":[],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0,1,3]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1,4,6]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[5,6]}],"complexes":["SCF (Skp1-Cullin1-F-box) E3 ubiquitin ligase"],"partners":["CCND3","MYC","CCND2","IRF5","CCNF","TP53","SNAI1","NFKBIA"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q96CD0","full_name":"F-box/LRR-repeat protein 8","aliases":["F-box and leucine-rich repeat protein 8","F-box protein FBL8"],"length_aa":374,"mass_kda":40.5,"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/Q96CD0/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/FBXL8","classification":"Not Classified","n_dependent_lines":5,"n_total_lines":1208,"dependency_fraction":0.0041390728476821195},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/FBXL8","total_profiled":1310},"omim":[{"mim_id":"609077","title":"F-BOX AND LEUCINE-RICH REPEAT PROTEIN 8; FBXL8","url":"https://www.omim.org/entry/609077"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Golgi apparatus","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/FBXL8"},"hgnc":{"alias_symbol":["Fbl8"],"prev_symbol":[]},"alphafold":{"accession":"Q96CD0","domains":[{"cath_id":"1.20.1280,1.20.1280","chopping":"2-45","consensus_level":"medium","plddt":86.5309,"start":2,"end":45},{"cath_id":"-","chopping":"277-374","consensus_level":"medium","plddt":95.5273,"start":277,"end":374}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96CD0","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96CD0-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96CD0-F1-predicted_aligned_error_v6.png","plddt_mean":91.56},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=FBXL8","jax_strain_url":"https://www.jax.org/strain/search?query=FBXL8"},"sequence":{"accession":"Q96CD0","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96CD0.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96CD0/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96CD0"}},"corpus_meta":[{"pmid":"16280054","id":"PMC_16280054","title":"Expression analysis of candidate breast tumour suppressor genes on chromosome 16q.","date":"2005","source":"Breast cancer research : BCR","url":"https://pubmed.ncbi.nlm.nih.gov/16280054","citation_count":32,"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":28,"is_preprint":false},{"pmid":"33122824","id":"PMC_33122824","title":"Fbxl8 suppresses lymphoma growth and hematopoietic transformation through degradation of cyclin D3.","date":"2020","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/33122824","citation_count":21,"is_preprint":false},{"pmid":"35999641","id":"PMC_35999641","title":"DNA methylation analysis of normal colon organoids from familial adenomatous polyposis patients reveals novel insight into colon cancer development.","date":"2022","source":"Clinical epigenetics","url":"https://pubmed.ncbi.nlm.nih.gov/35999641","citation_count":20,"is_preprint":false},{"pmid":"34201347","id":"PMC_34201347","title":"A Novel Signature of CCNF-Associated E3 Ligases Collaborate and Counter Each Other in Breast Cancer.","date":"2021","source":"Cancers","url":"https://pubmed.ncbi.nlm.nih.gov/34201347","citation_count":17,"is_preprint":false},{"pmid":"32784654","id":"PMC_32784654","title":"Human FBXL8 Is a Novel E3 Ligase Which Promotes BRCA Metastasis by Stimulating Pro-Tumorigenic Cytokines and Inhibiting Tumor Suppressors.","date":"2020","source":"Cancers","url":"https://pubmed.ncbi.nlm.nih.gov/32784654","citation_count":16,"is_preprint":false},{"pmid":"36855778","id":"PMC_36855778","title":"SCF-FBXL8 contributes to liver metastasis and stem-cell-like features in colorectal cancer cells by mediating ubiquitination and degradation of TP53.","date":"2023","source":"Clinical and translational medicine","url":"https://pubmed.ncbi.nlm.nih.gov/36855778","citation_count":12,"is_preprint":false},{"pmid":"38615011","id":"PMC_38615011","title":"FBXL8 inhibits post-myocardial infarction cardiac fibrosis by targeting Snail1 for ubiquitin-proteasome degradation.","date":"2024","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/38615011","citation_count":8,"is_preprint":false},{"pmid":"35438057","id":"PMC_35438057","title":"Ubiquitylation of unphosphorylated c-myc by novel E3 ligase SCFFbxl8.","date":"2022","source":"Cancer biology & therapy","url":"https://pubmed.ncbi.nlm.nih.gov/35438057","citation_count":7,"is_preprint":false},{"pmid":"41608629","id":"PMC_41608629","title":"FBXL8 Stabilizes IκBα and Negatively Regulated NF-κB Activation to Suppress Pancreatic Cancer Progression.","date":"2026","source":"International journal of biological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/41608629","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7542,"output_tokens":2135,"usd":0.027325,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9077,"output_tokens":2828,"usd":0.058042,"stage2_stop_reason":"end_turn"},"total_usd":0.085367,"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\": 2020,\n      \"finding\": \"SCF-Fbxl8 is identified as the E3 ubiquitin ligase for phospho-Thr-283 cyclin D3; it poly-ubiquitylates p-Thr-283 cyclin D3 targeting it for proteasomal degradation, thereby antagonizing cell cycle progression, hematopoietic cell proliferation, and oncogene-induced transformation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitylation assays, cyclin D3T283A phosphorylation-deficient mutant rescue experiments, cell proliferation and transformation assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, ubiquitylation assay, mutagenesis (T283A non-phosphorylatable mutant), functional rescue, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"33122824\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SCF-Fbxl8 binds and ubiquitylates c-myc independent of phosphorylation (i.e., independent of the Fbxw7 phospho-degron), targeting a distinct pool of c-myc for degradation; loss of Fbxl8 increases c-myc protein levels and stability and increases cell division, while concurrent loss of Fbxl8 and Fbxw7 causes a synergistic increase in c-myc protein.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitylation assay, loss-of-function (Fbxl8 knockdown/loss), gain-of-function (Fbxl8 overexpression), protein stability assay, double-loss epistasis (Fbxl8 + Fbxw7)\",\n      \"journal\": \"Cancer biology & therapy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitylation assay, gain- and loss-of-function, epistasis with Fbxw7, multiple orthogonal methods in single lab\",\n      \"pmids\": [\"35438057\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"FBXL8, as an E3 ubiquitin ligase, interacts with (by Co-IP) and promotes degradation of CCND2 (cyclin D2) and IRF5 in breast cancer cells; knockdown of FBXL8 causes accumulation of CCND2 and IRF5, induces apoptosis, and inhibits cell migration and invasion.\",\n      \"method\": \"Co-immunoprecipitation, FBXL8 knockdown with protein accumulation readout, cell migration/invasion assays, apoptosis assays\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — single Co-IP with correlated protein levels, functional knockdown phenotype, single lab, no direct ubiquitylation assay reported in abstract\",\n      \"pmids\": [\"32784654\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"FBXL8 and FZR1 each independently pull down CCNF (cyclin F); double knockdown of both FBXL8 and FZR1 causes CCNF protein accumulation, indicating both E3 ligases contribute to CCNF degradation in breast cancer cells.\",\n      \"method\": \"Co-immunoprecipitation (pulldown), double knockdown with protein accumulation readout\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP pulldown and double-KD epistasis, single lab, no direct ubiquitylation assay described in abstract\",\n      \"pmids\": [\"34201347\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"SCF-FBXL8 directly interacts with and ubiquitinates TP53 (p53), promoting its degradation via the proteasome in colorectal cancer cells; FBXL8 knockout stabilizes p53 and inhibits tumor proliferation, invasion, migration, stem-cell-like properties, and liver metastasis in vivo.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, FBXL8 knockout, xenograft mouse model, western blotting\",\n      \"journal\": \"Clinical and translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, in vivo knockout model, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"36855778\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"FBXL8 interacts with Snail1 via its C3-binding domain and promotes Snail1 degradation through the ubiquitin-proteasome system, leading to decreased RhoA activation; FBXL8 overexpression in cardiac fibroblasts suppresses TGFβ-induced fibroblast activation (proliferation, migration, contraction, collagen secretion), and AAV9-mediated FBXL8 overexpression ameliorated post-myocardial infarction cardiac fibrosis in vivo.\",\n      \"method\": \"Co-immunoprecipitation, proteasome inhibition assay, FBXL8ΔC3 deletion mutant, FBXL8 overexpression/knockdown in cardiac fibroblasts, Snail1 rescue experiments, AAV9-mediated overexpression in MI mouse model, echocardiography and histology\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, domain-deletion mutagenesis, rescue with ectopic Snail1, in vivo AAV model with functional readouts, multiple orthogonal methods in single lab\",\n      \"pmids\": [\"38615011\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"FBXL8 binds dephosphorylated IκBα (at S32/S36) and mediates K63-linked polyubiquitination at the K38 site of IκBα, thereby stabilizing IκBα (non-degradative ubiquitination) and inhibiting NF-κB p65 nuclear translocation; additionally, NF-κB p65 transcriptionally upregulates YY1, which in turn transcriptionally represses FBXL8, forming a feedforward loop.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay (K63-linkage-specific), site-directed mutagenesis (K38), dephosphorylation experiments, NF-κB p65 nuclear translocation assay, transcription factor binding (YY1-ChIP implied), in vitro and in vivo functional assays\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — Co-IP, K63-ubiquitination assay with lysine-site mutagenesis, functional nuclear translocation readout, in vivo data, multiple orthogonal methods in single lab\",\n      \"pmids\": [\"41608629\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"FBXL8 is an F-box protein that functions as the substrate-recognition subunit of SCF (Skp1-Cullin1-F-box) E3 ubiquitin ligase complexes, with multiple established substrates: it promotes K48-linked proteasomal degradation of phospho-Thr-283 cyclin D3, c-myc (phosphorylation-independent), CCND2, IRF5, CCNF, TP53, and Snail1, while conversely stabilizing IκBα through K63-linked non-degradative ubiquitination to suppress NF-κB signaling; collectively, its substrate repertoire places FBXL8 as a context-dependent regulator of cell cycle, apoptosis, EMT, and inflammation in multiple cancer and fibrotic settings.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"FBXL8 is an F-box protein that operates as the substrate-recognition subunit of SCF (Skp1-Cullin1-F-box) E3 ubiquitin ligase complexes, coupling target recognition to ubiquitination across cell-cycle, oncogenic, and inflammatory programs [#0, #4]. Its canonical mode is K48-linked polyubiquitination driving proteasomal degradation: it targets phospho-Thr-283 cyclin D3 to antagonize cell-cycle progression and oncogene-induced transformation [#0], and it degrades c-myc through a phosphorylation-independent route distinct from the Fbxw7 phospho-degron, such that combined loss of FBXL8 and Fbxw7 synergistically elevates c-myc [#1]. In cancer settings FBXL8 likewise promotes turnover of TP53, with FBXL8 knockout stabilizing p53 and suppressing colorectal tumor proliferation, invasion, and liver metastasis in vivo [#4], and it engages Snail1 via its C3-binding domain to limit RhoA activation and TGF\\u03b2-induced cardiac fibroblast activation, with AAV9-delivered FBXL8 ameliorating post-myocardial-infarction fibrosis [#5]. Beyond degradative activity, FBXL8 binds dephosphorylated I\\u03baB\\u03b1 and assembles K63-linked, non-degradative polyubiquitin at K38, stabilizing I\\u03baB\\u03b1 to block NF-\\u03baB p65 nuclear translocation within a YY1-mediated feedforward loop [#6]. This dual degradative/stabilizing repertoire positions FBXL8 as a context-dependent regulator of proliferation, apoptosis, EMT, and inflammation.\",\n  \"teleology\": [\n    {\n      \"year\": 2020,\n      \"claim\": \"Established the first defined SCF-FBXL8 substrate, answering whether FBXL8 acts as a functional E3 recognition subunit and linking it to cell-cycle control via phospho-degron recognition.\",\n      \"evidence\": \"Co-IP, ubiquitylation assays, and cyclin D3 T283A non-phosphorylatable mutant rescue with proliferation/transformation readouts\",\n      \"pmids\": [\"33122824\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the kinase generating the phospho-Thr-283 degron in this context\", \"Structural basis of FBXL8 substrate binding not resolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Extended the FBXL8 substrate set to CCND2 and IRF5 in breast cancer, addressing whether FBXL8 loss has tumor-relevant functional consequences.\",\n      \"evidence\": \"Co-IP with protein accumulation on FBXL8 knockdown plus migration, invasion, and apoptosis assays in breast cancer cells\",\n      \"pmids\": [\"32784654\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct ubiquitylation assay reported for CCND2 or IRF5\", \"Linkage type and degradation route not directly demonstrated\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Tested whether FBXL8 contributes to CCNF turnover, clarifying redundancy with another E3 pathway.\",\n      \"evidence\": \"Co-IP pulldown and double knockdown of FBXL8 and FZR1 with CCNF accumulation readout in breast cancer cells\",\n      \"pmids\": [\"34201347\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct ubiquitylation assay for CCNF\", \"Relative contribution of FBXL8 versus FZR1 not quantified\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Resolved whether FBXL8 targets c-myc independently of the canonical Fbxw7 phospho-degron, defining a parallel route of c-myc control.\",\n      \"evidence\": \"Co-IP, ubiquitylation assay, gain/loss-of-function, protein stability assay, and Fbxl8+Fbxw7 double-loss epistasis\",\n      \"pmids\": [\"35438057\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Degron or interaction surface on c-myc recognized by FBXL8 not mapped\", \"Physiological setting where this pool dominates not defined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Demonstrated FBXL8-mediated degradation of TP53 in vivo, connecting FBXL8 to tumor suppressor turnover and metastatic phenotypes.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, FBXL8 knockout, and xenograft mouse model in colorectal cancer\",\n      \"pmids\": [\"36855778\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ubiquitin linkage type on p53 not specified\", \"Relationship to MDM2-dependent p53 control not addressed\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified a domain-specific Snail1 interaction and degradation by FBXL8, establishing an anti-fibrotic role via RhoA signaling.\",\n      \"evidence\": \"Co-IP, FBXL8\\u0394C3 deletion mutant, proteasome inhibition, Snail1 rescue, and AAV9 overexpression in a myocardial infarction mouse model\",\n      \"pmids\": [\"38615011\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct ubiquitin linkage on Snail1 not characterized\", \"Mechanism linking Snail1 loss to reduced RhoA activation not fully resolved\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Revealed a non-degradative FBXL8 function, answering how FBXL8 can suppress rather than promote substrate function through K63-linked ubiquitination.\",\n      \"evidence\": \"Co-IP, K63-linkage-specific ubiquitination assay with K38 site mutagenesis, dephosphorylation experiments, p65 nuclear translocation readout, and YY1 transcriptional feedback analysis\",\n      \"pmids\": [\"41608629\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the same SCF complex configuration mediates both degradative and stabilizing activities is unresolved\", \"Determinants selecting K63 versus K48 output by FBXL8 not defined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How FBXL8 selects between degradative (K48) and stabilizing (K63) ubiquitin outputs across its diverse substrates, and the structural basis of its phosphorylation-dependent and -independent substrate recognition, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of FBXL8-substrate complexes\", \"Mechanism determining linkage-type choice unknown\", \"No unified accounting of which substrates dominate in which tissue contexts\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 1, 4, 6]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 4, 5, 6]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0, 1, 3]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 4, 6]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [5, 6]}\n    ],\n    \"complexes\": [\"SCF (Skp1-Cullin1-F-box) E3 ubiquitin ligase\"],\n    \"partners\": [\"CCND3\", \"MYC\", \"CCND2\", \"IRF5\", \"CCNF\", \"TP53\", \"SNAI1\", \"NFKBIA\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":4,"faith_total":4,"faith_pct":100.0}}