{"gene":"FBXO25","run_date":"2026-04-28T17:46:03","timeline":{"discoveries":[{"year":2005,"finding":"FBXO25 (hFBX25) was established as an F-box protein that interacts with Skp1 and Cul1, forming a functional SCF-type ubiquitin ligase complex. An atypical serine residue in the F-box domain was identified as crucial for Skp1 binding. FBXO25 localizes predominantly to the nucleus and is strongly expressed in brain, with neuronal expression in mouse embryos.","method":"Yeast two-hybrid and co-immunoprecipitation to verify Skp1/Cul1 interaction; site-directed mutagenesis of the F-box serine; immunolocalization","journal":"Biochimica et biophysica acta","confidence":"High","confidence_rationale":"Tier 1-2 — interaction verified by Co-IP/pulldown with mutagenesis identifying key residue, plus subcellular localization","pmids":["16278047"],"is_preprint":false},{"year":2006,"finding":"FBXO25 contains a functional F-box domain that binds Skp1, Roc1, and Cul1 to form a productive SCF-type E3 ubiquitin ligase complex with ubiquitin ligase activity in vitro. FBXO25 is highly expressed in brain, kidney, and intestine but not striated muscle, and is not induced during muscle atrophy.","method":"Co-immunoprecipitation, in vitro ubiquitin ligase activity assay, RT-PCR across tissues, atrophy models (fasting, dexamethasone, streptozotocin-diabetes)","journal":"Biochimica et biophysica acta","confidence":"High","confidence_rationale":"Tier 1 — in vitro E3 ligase reconstitution with Co-IP confirming complex assembly","pmids":["16714087"],"is_preprint":false},{"year":2008,"finding":"FBXO25, Skp1, Cul1, and Rbx1 form a functional ubiquitin ligase complex that localizes to novel dot-like nuclear structures (FBXO25-associated nuclear domains, FANDs) distinct from clastosomes, which are enriched in ubiquitin conjugates and 20S proteasome. Transcription inhibition (actinomycin D) or heat shock disrupts FANDs. FBXO25-dependent ubiquitin ligase activity prevents aggregation of polyglutamine-containing huntingtin in the nucleus.","method":"Confocal immunofluorescence with affinity-purified antibodies, biochemical fractionation, immunoblot, functional ubiquitination assay with polyglutamine-huntingtin aggregation readout","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 — direct localization with functional consequence, plus functional ubiquitination assay with multiple orthogonal methods","pmids":["18287534"],"is_preprint":false},{"year":2010,"finding":"FBXO25 physically interacts with beta-actin through its N-terminus; beta-actin is enriched in FBXO25-associated nuclear domains (FANDs). Inhibitors of actin polymerization disrupt FANDs, indicating nuclear actin organization influences FBXO25 compartments. FBXO25 antibodies interfered with RNA polymerase II transcription in vitro, linking the FAND compartment to transcriptional regulation.","method":"Two-step affinity purification followed by mass spectrometry, yeast two-hybrid screen, Co-IP, confocal imaging, in vitro transcription assay with FBXO25 antibody interference","journal":"Proteomics","confidence":"Medium","confidence_rationale":"Tier 2 — MS interactome plus Co-IP for beta-actin; in vitro transcription interference is indirect","pmids":["20473970"],"is_preprint":false},{"year":2011,"finding":"FBXO25 functions as a nuclear E3 ubiquitin ligase in cardiomyocytes that ubiquitinates and promotes proteasomal degradation of cardiac transcription factors Nkx2-5, Isl1, and Hand1. FBXO25 expression is higher in fetal heart than adult heart.","method":"Ubiquitination assay (co-expression with ubiquitin constructs), proteasome inhibitor (MG132) rescue of substrate levels, immunofluorescence showing nuclear localization in cardiomyocytes","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 — ubiquitination assays with proteasome inhibitor confirmation, but single lab","pmids":["21596019"],"is_preprint":false},{"year":2013,"finding":"FBXO25 interacts with the transcription factor ELK-1 and mediates its ubiquitination and proteasomal degradation via the SCF(FBXO25) complex, suppressing ELK-1 target genes c-fos and egr-1 in response to PMA. ELK-1 was identified as an FBXO25 substrate using an in-chip ubiquitination screen on a human protein microarray.","method":"Human protein microarray ubiquitination screen, co-immunoprecipitation, ubiquitination assay in HEK293T cells, protein turnover assay, gene expression analysis (c-fos, egr-1)","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal methods (microarray screen, Co-IP, ubiquitination assay, functional gene expression); however, this substrate relationship was later contested","pmids":["23940030"],"is_preprint":false},{"year":2014,"finding":"FBXO25 is the substrate-specifying subunit of SCF(FBXO25) E3 ubiquitin ligase that targets the prosurvival protein HAX-1 for ubiquitination and degradation after apoptotic stress. Protein kinase Cδ (PRKCD) phosphorylates both FBXO25 and HAX-1, directing nuclear FBXO25 to mitochondrial HAX-1 to enable substrate recognition. FBXO25 acts as a haploinsufficient tumor suppressor in mantle cell lymphoma (MCL), where monoallelic deletion of FBXO25 and stabilizing HAX1 phosphodegron mutations were identified in primary human MCL.","method":"Unbiased substrate screen, Co-immunoprecipitation, ubiquitination assay, genetic epistasis (Eμ-Myc lymphoma mouse model, MCL xenotransplant), phosphorylation assays with PRKCD, phosphodegron mutant analysis, patient sample genomics","journal":"Nature medicine","confidence":"High","confidence_rationale":"Tier 1-2 — substrate identified by unbiased screen, confirmed by Co-IP and ubiquitination assay, mechanistic kinase regulation validated in vivo mouse models and human patient samples","pmids":["25419709"],"is_preprint":false},{"year":2015,"finding":"FBXO25 directly interacts with cardiac transcription factors Tbx5 and Nkx2-5 in vitro and in vivo, and mediates their ubiquitination and proteasomal degradation via the SCF complex. A dominant-negative Fbxo25 mutant (residues 1-236) prevents Tbx5 degradation and increases Tbx5 transcriptional activity. Silencing endogenous Fbxo25 suppresses cardiomyocyte differentiation from mESCs.","method":"Co-immunoprecipitation (in vitro and in vivo), proteasome inhibitor MG132 rescue, temperature-sensitive ubiquitin system (ts20 cells), dominant-negative mutant expression, luciferase reporter assay, siRNA knockdown with differentiation readout","journal":"Biochimica et biophysica acta","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal methods including dominant-negative mutagenesis, ts20 ubiquitin system, and Co-IP","pmids":["25725482"],"is_preprint":false},{"year":2017,"finding":"FBXO25 negatively regulates MAPK/ERK signaling by decreasing ERK1/2 phosphorylation independently of MEK1/2. In FBXO25 knockout HAP1 cells, ERK1/2 is more active upon PMA treatment and cell proliferation is increased under receptor-mediated ERK pathway activation.","method":"FBXO25 overexpression and CRISPR knockout (FBXO25KO HAP1 cells), phospho-ERK1/2 immunoblot, MEK inhibitor epistasis, cell proliferation assay","journal":"Archives of biochemistry and biophysics","confidence":"Medium","confidence_rationale":"Tier 2 — genetic KO with defined phosphorylation phenotype and epistasis with MEK inhibitor, single lab","pmids":["28389297"],"is_preprint":false},{"year":2019,"finding":"FBXO25 is targeted for proteasome-dependent degradation by the lncRNA ODIR1, which recruits Cullin 3 (CUL3) to facilitate FBXO25 degradation. FBXO25 increases mono-ubiquitination of H2BK120 (H2BK120ub), which subsequently promotes H3K4 trimethylation (H3K4me3), inducing a loose chromatin structure and transcription of the osteoblast transcription factor OSX.","method":"RNA pulldown/co-immunoprecipitation (ODIR1-FBXO25 interaction), ChIP assay (H2BK120ub, H3K4me3 at OSX locus), shRNA knockdown and overexpression of ODIR1 in hUC-MSCs, in vitro and in vivo osteogenesis assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 — RNA-protein interaction with ChIP for histone marks, functional in vitro/in vivo osteogenesis, single lab","pmids":["31827076"],"is_preprint":false},{"year":2020,"finding":"FBXO25 and ELK-1 interact in HEK293T cells by co-immunoprecipitation, and FBXO25 is active toward Hand1 and HAX1 substrates; however, FBXO25 does not promote ubiquitination or proteasomal degradation of ELK-1 nor affect ELK-1 transcriptional activity, contradicting an earlier report. This indicates another E3 ligase regulates ELK-1 ubiquitination.","method":"Co-immunoprecipitation, protein turnover assay (cycloheximide chase), ubiquitination assay, RNA interference, gene expression analysis in HEK293T cells","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal assays refuting prior claim while confirming FBXO25 activity toward Hand1 and HAX1; single lab but rigorous","pmids":["33428929"],"is_preprint":false},{"year":2020,"finding":"In cutaneous squamous cell carcinoma (cSCC), FBXO25 promotes tumor growth and metastasis through cyclin D1 stabilization. FBXO25 interacts with Oct-1, leading to Oct-1 downregulation and consequent cyclin D1 stabilization. Stable silencing of FBXO25 reduces cSCC tumor growth and cyclin D1 levels, while cyclin D1 overexpression rescues growth in FBXO25-deficient tumors.","method":"Stable shRNA knockdown, stable cyclin D1 overexpression rescue experiment, co-immunoprecipitation (FBXO25-Oct-1 interaction), in vivo tumor growth assay (SCC13 cells)","journal":"The Journal of investigative dermatology","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP with functional rescue experiment in vivo, single lab","pmids":["32335130"],"is_preprint":false}],"current_model":"FBXO25 is the substrate-recognition subunit of an SCF(FBXO25) E3 ubiquitin ligase complex (with SKP1, CUL1, RBX1) that localizes to novel nuclear compartments (FANDs) and targets multiple substrates for ubiquitination and proteasomal degradation, including the prosurvival protein HAX-1 (directed by PRKCD-mediated phosphorylation of both FBXO25 and HAX-1), cardiac transcription factors (Nkx2-5, Isl1, Hand1, Tbx5), and huntingtin with polyglutamine expansions; it also negatively regulates MAPK/ERK signaling independently of MEK, promotes H2BK120 monoubiquitination to drive H3K4me3 and chromatin opening at osteogenic loci, and functions as a haploinsufficient tumor suppressor in mantle cell lymphoma."},"narrative":{"teleology":[{"year":2005,"claim":"Establishing that FBXO25 is a bona fide F-box protein that assembles into an SCF complex resolved whether this orphan F-box protein could function as an E3 ligase subunit.","evidence":"Yeast two-hybrid, Co-IP with Skp1/Cul1, and site-directed mutagenesis of a critical F-box serine in HEK293 cells","pmids":["16278047"],"confidence":"High","gaps":["No substrate identified","Functional consequence of nuclear localization unknown","Mechanism of nuclear targeting undetermined"]},{"year":2006,"claim":"Demonstrating reconstituted ubiquitin ligase activity in vitro confirmed that the SCF(FBXO25) complex is catalytically competent, not merely a structural assembly.","evidence":"In vitro ubiquitin ligase assay with Co-IP-purified complex, tissue expression profiling by RT-PCR","pmids":["16714087"],"confidence":"High","gaps":["Endogenous substrates still unknown","Physiological role in brain (highest expression tissue) uncharacterized"]},{"year":2008,"claim":"Discovery of FANDs — novel nuclear domains containing FBXO25, ubiquitin conjugates, and 20S proteasome — established a dedicated subnuclear compartment for FBXO25-mediated proteolysis and showed the complex prevents polyglutamine-huntingtin aggregation.","evidence":"Confocal immunofluorescence with affinity-purified antibodies, actinomycin D/heat-shock perturbation, polyglutamine aggregation assay","pmids":["18287534"],"confidence":"High","gaps":["How FANDs are nucleated is unknown","Whether huntingtin is a direct ubiquitination substrate or an indirect target is unresolved"]},{"year":2010,"claim":"Identifying beta-actin as a FAND component and showing that actin-polymerization inhibitors disrupt FANDs linked nuclear actin organization to FBXO25 compartment integrity and suggested a connection to RNA Pol II transcription.","evidence":"TAP-MS interactome, Co-IP for beta-actin, latrunculin treatment, in vitro transcription assay with FBXO25 antibody interference","pmids":["20473970"],"confidence":"Medium","gaps":["Transcription link is indirect (antibody interference)","Whether beta-actin is a substrate or structural partner is unclear","FAND biogenesis mechanism still unknown"]},{"year":2011,"claim":"Identifying cardiac transcription factors Nkx2-5, Isl1, and Hand1 as FBXO25 substrates provided the first physiological substrates and implicated FBXO25 in cardiac development.","evidence":"Ubiquitination assays with ubiquitin co-expression, MG132 proteasome inhibitor rescue in cardiomyocytes","pmids":["21596019"],"confidence":"Medium","gaps":["Degron motifs on cardiac substrates not mapped","In vivo cardiac phenotype of FBXO25 loss not tested","Phosphorylation-dependent recognition not explored"]},{"year":2014,"claim":"Identification of HAX-1 as a phosphodegron-dependent substrate and PRKCD as the upstream kinase provided the first complete substrate-recognition mechanism for FBXO25, while genetic evidence in mouse lymphoma models and human MCL established FBXO25 as a haploinsufficient tumor suppressor.","evidence":"Unbiased substrate screen, Co-IP, ubiquitination assay, PRKCD phosphorylation assays, phosphodegron mutant analysis, Eμ-Myc mouse model, MCL xenotransplant, patient genomics","pmids":["25419709"],"confidence":"High","gaps":["Whether PRKCD-dependent mechanism applies to other substrates is unknown","Full spectrum of FBXO25 tumor-suppressive targets beyond HAX-1 not defined"]},{"year":2015,"claim":"Extending cardiac substrate repertoire to Tbx5 and demonstrating that FBXO25 silencing impairs cardiomyocyte differentiation established a functional role for FBXO25-mediated proteolysis in cardiac lineage specification.","evidence":"Co-IP (in vitro and in vivo), ts20 temperature-sensitive ubiquitin system, dominant-negative FBXO25 mutant, siRNA knockdown with mESC differentiation readout","pmids":["25725482"],"confidence":"High","gaps":["No Fbxo25 knockout mouse cardiac phenotype reported","Whether FBXO25 degrades cardiac factors during normal development or only in overexpression models is unclear"]},{"year":2017,"claim":"Showing that FBXO25 knockout increases ERK1/2 phosphorylation independently of MEK revealed a non-canonical role in restraining MAPK signaling, though the direct mechanism remains undefined.","evidence":"CRISPR FBXO25 KO in HAP1 cells, phospho-ERK1/2 immunoblot, MEK inhibitor epistasis, cell proliferation assay","pmids":["28389297"],"confidence":"Medium","gaps":["Direct MAPK pathway substrate of FBXO25 not identified","Whether ERK regulation is ubiquitination-dependent or indirect is unresolved","Single cell line used"]},{"year":2019,"claim":"Discovery that FBXO25 promotes H2BK120 monoubiquitination and consequent H3K4me3 at the OSX locus, and that the lncRNA ODIR1 recruits CUL3 to degrade FBXO25, revealed a chromatin-remodeling function and an upstream mechanism controlling FBXO25 abundance during osteogenic differentiation.","evidence":"RNA pulldown, Co-IP (ODIR1–FBXO25, CUL3–FBXO25), ChIP for H2BK120ub and H3K4me3 at OSX, shRNA/overexpression in hUC-MSCs, in vivo osteogenesis","pmids":["31827076"],"confidence":"Medium","gaps":["Whether FBXO25 directly ubiquitinates H2B or acts through an intermediary is unknown","CUL3 adaptor bridging ODIR1 to FBXO25 not identified","Single differentiation system tested"]},{"year":2020,"claim":"Rigorous re-examination disproved ELK-1 as an FBXO25 substrate while reconfirming Hand1 and HAX-1, refining the validated substrate list and highlighting the need for independent replication of F-box protein substrate assignments.","evidence":"Co-IP, cycloheximide chase, ubiquitination assay, RNAi, gene expression analysis in HEK293T cells","pmids":["33428929"],"confidence":"Medium","gaps":["Identity of the true ELK-1 E3 ligase remains unknown","Whether FBXO25–ELK-1 physical interaction serves a non-degradative function is unexplored"]},{"year":2020,"claim":"In cutaneous SCC, FBXO25 was found to promote tumor growth through Oct-1 downregulation and consequent cyclin D1 stabilization, revealing a context-dependent oncogenic role contrasting with its tumor-suppressive function in MCL.","evidence":"Stable shRNA knockdown, cyclin D1 overexpression rescue, Co-IP (FBXO25–Oct-1), in vivo SCC13 xenograft","pmids":["32335130"],"confidence":"Medium","gaps":["Whether Oct-1 is a direct ubiquitination substrate or indirectly regulated is not demonstrated","Reconciliation of oncogenic (cSCC) versus tumor-suppressive (MCL) roles not mechanistically resolved","Single cancer cell line"]},{"year":null,"claim":"Key unresolved questions include the structural basis of FBXO25 substrate recognition, the complete inventory of direct substrates and their degron motifs, the biogenesis and regulation of FANDs, whether FBXO25 directly ubiquitinates H2B, and the in vivo developmental consequences of FBXO25 loss in mammals.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural or cryo-EM model of SCF(FBXO25) exists","No Fbxo25 knockout mouse phenotype reported","Degron motifs beyond the HAX-1 phosphodegron are unmapped"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,2,4,6,7]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,1,2,6]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,2,3,4,7]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[1,2,6,7]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[8]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[4,7]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[6]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[9]}],"complexes":["SCF(FBXO25)"],"partners":["SKP1","CUL1","RBX1","HAX1","NKX2-5","TBX5","HAND1","PRKCD"],"other_free_text":[]},"mechanistic_narrative":"FBXO25 is the substrate-recognition subunit of an SCF-type E3 ubiquitin ligase complex (with SKP1, CUL1, and RBX1) that ubiquitinates diverse nuclear and cytoplasmic substrates to regulate transcription, apoptosis, signaling, and chromatin remodeling. Within the nucleus, FBXO25 localizes to novel proteasome-enriched compartments (FANDs) where it targets cardiac transcription factors (Nkx2-5, Isl1, Hand1, Tbx5) for proteasomal degradation, promotes H2BK120 monoubiquitination to drive H3K4me3 and osteogenic gene expression, prevents polyglutamine-huntingtin aggregation, and negatively regulates MAPK/ERK signaling independently of MEK [PMID:18287534, PMID:25725482, PMID:31827076, PMID:28389297]. PRKCD-mediated phosphorylation directs FBXO25 from the nucleus to mitochondrial HAX-1 for ubiquitination-dependent degradation during apoptotic stress, and monoallelic loss of FBXO25 or stabilizing HAX-1 phosphodegron mutations occur in mantle cell lymphoma, establishing FBXO25 as a haploinsufficient tumor suppressor [PMID:25419709]. FBXO25 itself is subject to CUL3-dependent proteasomal turnover recruited by the lncRNA ODIR1, linking its abundance to differentiation-coupled chromatin remodeling [PMID:31827076]."},"prefetch_data":{"uniprot":{"accession":"Q8TCJ0","full_name":"F-box only protein 25","aliases":[],"length_aa":367,"mass_kda":43.3,"function":"Substrate-recognition component of the SCF (SKP1-CUL1-F-box protein)-type E3 ubiquitin ligase complex. May play a role in accumulation of expanded polyglutamine (polyQ) protein huntingtin (HTT) (By similarity)","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/Q8TCJ0/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/FBXO25","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/FBXO25","total_profiled":1310},"omim":[{"mim_id":"609098","title":"F-BOX ONLY PROTEIN 25; FBXO25","url":"https://www.omim.org/entry/609098"},{"mim_id":"606604","title":"F-BOX ONLY PROTEIN 32; FBXO32","url":"https://www.omim.org/entry/606604"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"testis","ntpm":91.6}],"url":"https://www.proteinatlas.org/search/FBXO25"},"hgnc":{"alias_symbol":["FBX25"],"prev_symbol":[]},"alphafold":{"accession":"Q8TCJ0","domains":[{"cath_id":"-","chopping":"93-216","consensus_level":"high","plddt":87.2114,"start":93,"end":216},{"cath_id":"1.20.1280.50","chopping":"229-307","consensus_level":"medium","plddt":90.1115,"start":229,"end":307},{"cath_id":"-","chopping":"318-358","consensus_level":"medium","plddt":81.6022,"start":318,"end":358}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8TCJ0","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8TCJ0-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8TCJ0-F1-predicted_aligned_error_v6.png","plddt_mean":76.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=FBXO25","jax_strain_url":"https://www.jax.org/strain/search?query=FBXO25"},"sequence":{"accession":"Q8TCJ0","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8TCJ0.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8TCJ0/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8TCJ0"}},"corpus_meta":[{"pmid":"31827076","id":"PMC_31827076","title":"LncRNA ODIR1 inhibits osteogenic differentiation of hUC-MSCs through the FBXO25/H2BK120ub/H3K4me3/OSX axis.","date":"2019","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/31827076","citation_count":70,"is_preprint":false},{"pmid":"25419709","id":"PMC_25419709","title":"Disruption of the PRKCD-FBXO25-HAX-1 axis attenuates the apoptotic response and drives lymphomagenesis.","date":"2014","source":"Nature medicine","url":"https://pubmed.ncbi.nlm.nih.gov/25419709","citation_count":53,"is_preprint":false},{"pmid":"20601059","id":"PMC_20601059","title":"Molecular characterization of atrogin-1/F-box protein-32 (FBXO32) and F-box protein-25 (FBXO25) in rainbow trout (Oncorhynchus mykiss): Expression across tissues in response to feed deprivation.","date":"2010","source":"Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/20601059","citation_count":43,"is_preprint":false},{"pmid":"21596019","id":"PMC_21596019","title":"A novel Fbxo25 acts as an E3 ligase for destructing cardiac specific transcription factors.","date":"2011","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/21596019","citation_count":26,"is_preprint":false},{"pmid":"16278047","id":"PMC_16278047","title":"Characterization of FBX25, encoding a novel brain-expressed F-box protein.","date":"2005","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/16278047","citation_count":20,"is_preprint":false},{"pmid":"23940030","id":"PMC_23940030","title":"The F-box protein FBXO25 promotes the proteasome-dependent degradation of ELK-1 protein.","date":"2013","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/23940030","citation_count":16,"is_preprint":false},{"pmid":"18287534","id":"PMC_18287534","title":"FBXO25-associated nuclear domains: a novel subnuclear structure.","date":"2008","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/18287534","citation_count":16,"is_preprint":false},{"pmid":"25725482","id":"PMC_25725482","title":"Fbxo25 controls Tbx5 and Nkx2-5 transcriptional activity to regulate cardiomyocyte development.","date":"2015","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/25725482","citation_count":15,"is_preprint":false},{"pmid":"16714087","id":"PMC_16714087","title":"FBXO25, an F-box protein homologue of atrogin-1, is not induced in atrophying muscle.","date":"2006","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/16714087","citation_count":13,"is_preprint":false},{"pmid":"32335130","id":"PMC_32335130","title":"FBXO25 Promotes Cutaneous Squamous Cell Carcinoma Growth and Metastasis through Cyclin D1.","date":"2020","source":"The Journal of investigative dermatology","url":"https://pubmed.ncbi.nlm.nih.gov/32335130","citation_count":10,"is_preprint":false},{"pmid":"27596142","id":"PMC_27596142","title":"FBXO25 promotes cell proliferation, invasion, and migration of NSCLC.","date":"2016","source":"Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine","url":"https://pubmed.ncbi.nlm.nih.gov/27596142","citation_count":9,"is_preprint":false},{"pmid":"28389297","id":"PMC_28389297","title":"FBXO25 regulates MAPK signaling pathway through inhibition of ERK1/2 phosphorylation.","date":"2017","source":"Archives of biochemistry and biophysics","url":"https://pubmed.ncbi.nlm.nih.gov/28389297","citation_count":8,"is_preprint":false},{"pmid":"20473970","id":"PMC_20473970","title":"Identification of FBXO25-interacting proteins using an integrated proteomics approach.","date":"2010","source":"Proteomics","url":"https://pubmed.ncbi.nlm.nih.gov/20473970","citation_count":8,"is_preprint":false},{"pmid":"31849056","id":"PMC_31849056","title":"From man to fly - convergent evidence links FBXO25 to ADHD and comorbid psychiatric phenotypes.","date":"2019","source":"Journal of child psychology and psychiatry, and allied disciplines","url":"https://pubmed.ncbi.nlm.nih.gov/31849056","citation_count":7,"is_preprint":false},{"pmid":"33428929","id":"PMC_33428929","title":"ELK-1 ubiquitination status and transcriptional activity are modulated independently of F-Box protein FBXO25.","date":"2020","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/33428929","citation_count":4,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8505,"output_tokens":3620,"usd":0.039907},"stage2":{"model":"claude-opus-4-6","input_tokens":6968,"output_tokens":3186,"usd":0.171735},"total_usd":0.211642,"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\": 2005,\n      \"finding\": \"FBXO25 (hFBX25) was established as an F-box protein that interacts with Skp1 and Cul1, forming a functional SCF-type ubiquitin ligase complex. An atypical serine residue in the F-box domain was identified as crucial for Skp1 binding. FBXO25 localizes predominantly to the nucleus and is strongly expressed in brain, with neuronal expression in mouse embryos.\",\n      \"method\": \"Yeast two-hybrid and co-immunoprecipitation to verify Skp1/Cul1 interaction; site-directed mutagenesis of the F-box serine; immunolocalization\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — interaction verified by Co-IP/pulldown with mutagenesis identifying key residue, plus subcellular localization\",\n      \"pmids\": [\"16278047\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"FBXO25 contains a functional F-box domain that binds Skp1, Roc1, and Cul1 to form a productive SCF-type E3 ubiquitin ligase complex with ubiquitin ligase activity in vitro. FBXO25 is highly expressed in brain, kidney, and intestine but not striated muscle, and is not induced during muscle atrophy.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ubiquitin ligase activity assay, RT-PCR across tissues, atrophy models (fasting, dexamethasone, streptozotocin-diabetes)\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro E3 ligase reconstitution with Co-IP confirming complex assembly\",\n      \"pmids\": [\"16714087\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"FBXO25, Skp1, Cul1, and Rbx1 form a functional ubiquitin ligase complex that localizes to novel dot-like nuclear structures (FBXO25-associated nuclear domains, FANDs) distinct from clastosomes, which are enriched in ubiquitin conjugates and 20S proteasome. Transcription inhibition (actinomycin D) or heat shock disrupts FANDs. FBXO25-dependent ubiquitin ligase activity prevents aggregation of polyglutamine-containing huntingtin in the nucleus.\",\n      \"method\": \"Confocal immunofluorescence with affinity-purified antibodies, biochemical fractionation, immunoblot, functional ubiquitination assay with polyglutamine-huntingtin aggregation readout\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct localization with functional consequence, plus functional ubiquitination assay with multiple orthogonal methods\",\n      \"pmids\": [\"18287534\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"FBXO25 physically interacts with beta-actin through its N-terminus; beta-actin is enriched in FBXO25-associated nuclear domains (FANDs). Inhibitors of actin polymerization disrupt FANDs, indicating nuclear actin organization influences FBXO25 compartments. FBXO25 antibodies interfered with RNA polymerase II transcription in vitro, linking the FAND compartment to transcriptional regulation.\",\n      \"method\": \"Two-step affinity purification followed by mass spectrometry, yeast two-hybrid screen, Co-IP, confocal imaging, in vitro transcription assay with FBXO25 antibody interference\",\n      \"journal\": \"Proteomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — MS interactome plus Co-IP for beta-actin; in vitro transcription interference is indirect\",\n      \"pmids\": [\"20473970\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"FBXO25 functions as a nuclear E3 ubiquitin ligase in cardiomyocytes that ubiquitinates and promotes proteasomal degradation of cardiac transcription factors Nkx2-5, Isl1, and Hand1. FBXO25 expression is higher in fetal heart than adult heart.\",\n      \"method\": \"Ubiquitination assay (co-expression with ubiquitin constructs), proteasome inhibitor (MG132) rescue of substrate levels, immunofluorescence showing nuclear localization in cardiomyocytes\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ubiquitination assays with proteasome inhibitor confirmation, but single lab\",\n      \"pmids\": [\"21596019\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"FBXO25 interacts with the transcription factor ELK-1 and mediates its ubiquitination and proteasomal degradation via the SCF(FBXO25) complex, suppressing ELK-1 target genes c-fos and egr-1 in response to PMA. ELK-1 was identified as an FBXO25 substrate using an in-chip ubiquitination screen on a human protein microarray.\",\n      \"method\": \"Human protein microarray ubiquitination screen, co-immunoprecipitation, ubiquitination assay in HEK293T cells, protein turnover assay, gene expression analysis (c-fos, egr-1)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (microarray screen, Co-IP, ubiquitination assay, functional gene expression); however, this substrate relationship was later contested\",\n      \"pmids\": [\"23940030\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"FBXO25 is the substrate-specifying subunit of SCF(FBXO25) E3 ubiquitin ligase that targets the prosurvival protein HAX-1 for ubiquitination and degradation after apoptotic stress. Protein kinase Cδ (PRKCD) phosphorylates both FBXO25 and HAX-1, directing nuclear FBXO25 to mitochondrial HAX-1 to enable substrate recognition. FBXO25 acts as a haploinsufficient tumor suppressor in mantle cell lymphoma (MCL), where monoallelic deletion of FBXO25 and stabilizing HAX1 phosphodegron mutations were identified in primary human MCL.\",\n      \"method\": \"Unbiased substrate screen, Co-immunoprecipitation, ubiquitination assay, genetic epistasis (Eμ-Myc lymphoma mouse model, MCL xenotransplant), phosphorylation assays with PRKCD, phosphodegron mutant analysis, patient sample genomics\",\n      \"journal\": \"Nature medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — substrate identified by unbiased screen, confirmed by Co-IP and ubiquitination assay, mechanistic kinase regulation validated in vivo mouse models and human patient samples\",\n      \"pmids\": [\"25419709\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"FBXO25 directly interacts with cardiac transcription factors Tbx5 and Nkx2-5 in vitro and in vivo, and mediates their ubiquitination and proteasomal degradation via the SCF complex. A dominant-negative Fbxo25 mutant (residues 1-236) prevents Tbx5 degradation and increases Tbx5 transcriptional activity. Silencing endogenous Fbxo25 suppresses cardiomyocyte differentiation from mESCs.\",\n      \"method\": \"Co-immunoprecipitation (in vitro and in vivo), proteasome inhibitor MG132 rescue, temperature-sensitive ubiquitin system (ts20 cells), dominant-negative mutant expression, luciferase reporter assay, siRNA knockdown with differentiation readout\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods including dominant-negative mutagenesis, ts20 ubiquitin system, and Co-IP\",\n      \"pmids\": [\"25725482\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"FBXO25 negatively regulates MAPK/ERK signaling by decreasing ERK1/2 phosphorylation independently of MEK1/2. In FBXO25 knockout HAP1 cells, ERK1/2 is more active upon PMA treatment and cell proliferation is increased under receptor-mediated ERK pathway activation.\",\n      \"method\": \"FBXO25 overexpression and CRISPR knockout (FBXO25KO HAP1 cells), phospho-ERK1/2 immunoblot, MEK inhibitor epistasis, cell proliferation assay\",\n      \"journal\": \"Archives of biochemistry and biophysics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with defined phosphorylation phenotype and epistasis with MEK inhibitor, single lab\",\n      \"pmids\": [\"28389297\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"FBXO25 is targeted for proteasome-dependent degradation by the lncRNA ODIR1, which recruits Cullin 3 (CUL3) to facilitate FBXO25 degradation. FBXO25 increases mono-ubiquitination of H2BK120 (H2BK120ub), which subsequently promotes H3K4 trimethylation (H3K4me3), inducing a loose chromatin structure and transcription of the osteoblast transcription factor OSX.\",\n      \"method\": \"RNA pulldown/co-immunoprecipitation (ODIR1-FBXO25 interaction), ChIP assay (H2BK120ub, H3K4me3 at OSX locus), shRNA knockdown and overexpression of ODIR1 in hUC-MSCs, in vitro and in vivo osteogenesis assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — RNA-protein interaction with ChIP for histone marks, functional in vitro/in vivo osteogenesis, single lab\",\n      \"pmids\": [\"31827076\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"FBXO25 and ELK-1 interact in HEK293T cells by co-immunoprecipitation, and FBXO25 is active toward Hand1 and HAX1 substrates; however, FBXO25 does not promote ubiquitination or proteasomal degradation of ELK-1 nor affect ELK-1 transcriptional activity, contradicting an earlier report. This indicates another E3 ligase regulates ELK-1 ubiquitination.\",\n      \"method\": \"Co-immunoprecipitation, protein turnover assay (cycloheximide chase), ubiquitination assay, RNA interference, gene expression analysis in HEK293T cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal assays refuting prior claim while confirming FBXO25 activity toward Hand1 and HAX1; single lab but rigorous\",\n      \"pmids\": [\"33428929\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"In cutaneous squamous cell carcinoma (cSCC), FBXO25 promotes tumor growth and metastasis through cyclin D1 stabilization. FBXO25 interacts with Oct-1, leading to Oct-1 downregulation and consequent cyclin D1 stabilization. Stable silencing of FBXO25 reduces cSCC tumor growth and cyclin D1 levels, while cyclin D1 overexpression rescues growth in FBXO25-deficient tumors.\",\n      \"method\": \"Stable shRNA knockdown, stable cyclin D1 overexpression rescue experiment, co-immunoprecipitation (FBXO25-Oct-1 interaction), in vivo tumor growth assay (SCC13 cells)\",\n      \"journal\": \"The Journal of investigative dermatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP with functional rescue experiment in vivo, single lab\",\n      \"pmids\": [\"32335130\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"FBXO25 is the substrate-recognition subunit of an SCF(FBXO25) E3 ubiquitin ligase complex (with SKP1, CUL1, RBX1) that localizes to novel nuclear compartments (FANDs) and targets multiple substrates for ubiquitination and proteasomal degradation, including the prosurvival protein HAX-1 (directed by PRKCD-mediated phosphorylation of both FBXO25 and HAX-1), cardiac transcription factors (Nkx2-5, Isl1, Hand1, Tbx5), and huntingtin with polyglutamine expansions; it also negatively regulates MAPK/ERK signaling independently of MEK, promotes H2BK120 monoubiquitination to drive H3K4me3 and chromatin opening at osteogenic loci, and functions as a haploinsufficient tumor suppressor in mantle cell lymphoma.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"FBXO25 is the substrate-recognition subunit of an SCF-type E3 ubiquitin ligase complex (with SKP1, CUL1, and RBX1) that ubiquitinates diverse nuclear and cytoplasmic substrates to regulate transcription, apoptosis, signaling, and chromatin remodeling. Within the nucleus, FBXO25 localizes to novel proteasome-enriched compartments (FANDs) where it targets cardiac transcription factors (Nkx2-5, Isl1, Hand1, Tbx5) for proteasomal degradation, promotes H2BK120 monoubiquitination to drive H3K4me3 and osteogenic gene expression, prevents polyglutamine-huntingtin aggregation, and negatively regulates MAPK/ERK signaling independently of MEK [PMID:18287534, PMID:25725482, PMID:31827076, PMID:28389297]. PRKCD-mediated phosphorylation directs FBXO25 from the nucleus to mitochondrial HAX-1 for ubiquitination-dependent degradation during apoptotic stress, and monoallelic loss of FBXO25 or stabilizing HAX-1 phosphodegron mutations occur in mantle cell lymphoma, establishing FBXO25 as a haploinsufficient tumor suppressor [PMID:25419709]. FBXO25 itself is subject to CUL3-dependent proteasomal turnover recruited by the lncRNA ODIR1, linking its abundance to differentiation-coupled chromatin remodeling [PMID:31827076].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Establishing that FBXO25 is a bona fide F-box protein that assembles into an SCF complex resolved whether this orphan F-box protein could function as an E3 ligase subunit.\",\n      \"evidence\": \"Yeast two-hybrid, Co-IP with Skp1/Cul1, and site-directed mutagenesis of a critical F-box serine in HEK293 cells\",\n      \"pmids\": [\"16278047\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No substrate identified\", \"Functional consequence of nuclear localization unknown\", \"Mechanism of nuclear targeting undetermined\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Demonstrating reconstituted ubiquitin ligase activity in vitro confirmed that the SCF(FBXO25) complex is catalytically competent, not merely a structural assembly.\",\n      \"evidence\": \"In vitro ubiquitin ligase assay with Co-IP-purified complex, tissue expression profiling by RT-PCR\",\n      \"pmids\": [\"16714087\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous substrates still unknown\", \"Physiological role in brain (highest expression tissue) uncharacterized\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Discovery of FANDs — novel nuclear domains containing FBXO25, ubiquitin conjugates, and 20S proteasome — established a dedicated subnuclear compartment for FBXO25-mediated proteolysis and showed the complex prevents polyglutamine-huntingtin aggregation.\",\n      \"evidence\": \"Confocal immunofluorescence with affinity-purified antibodies, actinomycin D/heat-shock perturbation, polyglutamine aggregation assay\",\n      \"pmids\": [\"18287534\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How FANDs are nucleated is unknown\", \"Whether huntingtin is a direct ubiquitination substrate or an indirect target is unresolved\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Identifying beta-actin as a FAND component and showing that actin-polymerization inhibitors disrupt FANDs linked nuclear actin organization to FBXO25 compartment integrity and suggested a connection to RNA Pol II transcription.\",\n      \"evidence\": \"TAP-MS interactome, Co-IP for beta-actin, latrunculin treatment, in vitro transcription assay with FBXO25 antibody interference\",\n      \"pmids\": [\"20473970\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Transcription link is indirect (antibody interference)\", \"Whether beta-actin is a substrate or structural partner is unclear\", \"FAND biogenesis mechanism still unknown\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Identifying cardiac transcription factors Nkx2-5, Isl1, and Hand1 as FBXO25 substrates provided the first physiological substrates and implicated FBXO25 in cardiac development.\",\n      \"evidence\": \"Ubiquitination assays with ubiquitin co-expression, MG132 proteasome inhibitor rescue in cardiomyocytes\",\n      \"pmids\": [\"21596019\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Degron motifs on cardiac substrates not mapped\", \"In vivo cardiac phenotype of FBXO25 loss not tested\", \"Phosphorylation-dependent recognition not explored\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identification of HAX-1 as a phosphodegron-dependent substrate and PRKCD as the upstream kinase provided the first complete substrate-recognition mechanism for FBXO25, while genetic evidence in mouse lymphoma models and human MCL established FBXO25 as a haploinsufficient tumor suppressor.\",\n      \"evidence\": \"Unbiased substrate screen, Co-IP, ubiquitination assay, PRKCD phosphorylation assays, phosphodegron mutant analysis, Eμ-Myc mouse model, MCL xenotransplant, patient genomics\",\n      \"pmids\": [\"25419709\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PRKCD-dependent mechanism applies to other substrates is unknown\", \"Full spectrum of FBXO25 tumor-suppressive targets beyond HAX-1 not defined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Extending cardiac substrate repertoire to Tbx5 and demonstrating that FBXO25 silencing impairs cardiomyocyte differentiation established a functional role for FBXO25-mediated proteolysis in cardiac lineage specification.\",\n      \"evidence\": \"Co-IP (in vitro and in vivo), ts20 temperature-sensitive ubiquitin system, dominant-negative FBXO25 mutant, siRNA knockdown with mESC differentiation readout\",\n      \"pmids\": [\"25725482\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No Fbxo25 knockout mouse cardiac phenotype reported\", \"Whether FBXO25 degrades cardiac factors during normal development or only in overexpression models is unclear\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Showing that FBXO25 knockout increases ERK1/2 phosphorylation independently of MEK revealed a non-canonical role in restraining MAPK signaling, though the direct mechanism remains undefined.\",\n      \"evidence\": \"CRISPR FBXO25 KO in HAP1 cells, phospho-ERK1/2 immunoblot, MEK inhibitor epistasis, cell proliferation assay\",\n      \"pmids\": [\"28389297\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct MAPK pathway substrate of FBXO25 not identified\", \"Whether ERK regulation is ubiquitination-dependent or indirect is unresolved\", \"Single cell line used\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Discovery that FBXO25 promotes H2BK120 monoubiquitination and consequent H3K4me3 at the OSX locus, and that the lncRNA ODIR1 recruits CUL3 to degrade FBXO25, revealed a chromatin-remodeling function and an upstream mechanism controlling FBXO25 abundance during osteogenic differentiation.\",\n      \"evidence\": \"RNA pulldown, Co-IP (ODIR1–FBXO25, CUL3–FBXO25), ChIP for H2BK120ub and H3K4me3 at OSX, shRNA/overexpression in hUC-MSCs, in vivo osteogenesis\",\n      \"pmids\": [\"31827076\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether FBXO25 directly ubiquitinates H2B or acts through an intermediary is unknown\", \"CUL3 adaptor bridging ODIR1 to FBXO25 not identified\", \"Single differentiation system tested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Rigorous re-examination disproved ELK-1 as an FBXO25 substrate while reconfirming Hand1 and HAX-1, refining the validated substrate list and highlighting the need for independent replication of F-box protein substrate assignments.\",\n      \"evidence\": \"Co-IP, cycloheximide chase, ubiquitination assay, RNAi, gene expression analysis in HEK293T cells\",\n      \"pmids\": [\"33428929\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Identity of the true ELK-1 E3 ligase remains unknown\", \"Whether FBXO25–ELK-1 physical interaction serves a non-degradative function is unexplored\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"In cutaneous SCC, FBXO25 was found to promote tumor growth through Oct-1 downregulation and consequent cyclin D1 stabilization, revealing a context-dependent oncogenic role contrasting with its tumor-suppressive function in MCL.\",\n      \"evidence\": \"Stable shRNA knockdown, cyclin D1 overexpression rescue, Co-IP (FBXO25–Oct-1), in vivo SCC13 xenograft\",\n      \"pmids\": [\"32335130\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether Oct-1 is a direct ubiquitination substrate or indirectly regulated is not demonstrated\", \"Reconciliation of oncogenic (cSCC) versus tumor-suppressive (MCL) roles not mechanistically resolved\", \"Single cancer cell line\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the structural basis of FBXO25 substrate recognition, the complete inventory of direct substrates and their degron motifs, the biogenesis and regulation of FANDs, whether FBXO25 directly ubiquitinates H2B, and the in vivo developmental consequences of FBXO25 loss in mammals.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural or cryo-EM model of SCF(FBXO25) exists\", \"No Fbxo25 knockout mouse phenotype reported\", \"Degron motifs beyond the HAX-1 phosphodegron are unmapped\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 2, 4, 6, 7]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 1, 2, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 2, 3, 4, 7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": []},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 2, 6, 7]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [4, 7]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [9]}\n    ],\n    \"complexes\": [\n      \"SCF(FBXO25)\"\n    ],\n    \"partners\": [\n      \"SKP1\",\n      \"CUL1\",\n      \"RBX1\",\n      \"HAX1\",\n      \"NKX2-5\",\n      \"TBX5\",\n      \"HAND1\",\n      \"PRKCD\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}