{"gene":"FBXO3","run_date":"2026-06-09T23:54:43","timeline":{"discoveries":[{"year":2013,"finding":"FBXO3 functions as an E3 ubiquitin ligase F-box component that mediates the polyubiquitination and proteasomal degradation of FBXL2, thereby preventing FBXL2 from inhibiting TRAF proteins. This leads to elevated TRAF protein levels and potent stimulation of proinflammatory cytokine secretion from human inflammatory cells. The C-terminal ApaG domain of FBXO3 was found to be indispensable for mediating FBXL2 disposal and cytokine secretion.","method":"Co-immunoprecipitation, siRNA knockdown, cytokine secretion assays, murine models of viral pneumonia/septic shock/colitis, small-molecule inhibitor targeting ApaG domain","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, loss-of-function with defined cellular phenotype, in vivo disease models, replicated across multiple disease contexts in one rigorous study","pmids":["24123678"],"is_preprint":false},{"year":2014,"finding":"RVFV NSs virulence factor recruits host cell F-box protein FBXO3 to degrade TFIIH subunit p62 via the ubiquitin/proteasome pathway, thereby suppressing type I interferon transcription. siRNA depletion of FBXO3 rescued p62 levels and elevated IFN transcription ~10-fold. FBXO3 forms SCF complexes with Skp1; knockdown of Skp1 also protected p62, but knockdown of cullin 1, cullin 7, or Rbx1 did not rescue p62 degradation.","method":"siRNA knockdown, co-immunoprecipitation, protein level rescue assays, IFN transcription reporter assays","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, siRNA depletion with defined molecular phenotype (p62 rescue), IFN transcription measurement, multiple genetic perturbations tested","pmids":["24403578"],"is_preprint":false},{"year":2015,"finding":"FBXO3-dependent ubiquitination and degradation of FBXL2 leads to de-ubiquitination (stabilization) of TRAF2 in spinal dorsal horn neurons. This TRAF2 stabilization drives TNIK/GluR1 phosphorylation and GluR1 trafficking to the plasma membrane, mediating neuropathic allodynia after spinal nerve ligation. Intrathecal BC-1215 (FBXO3 inhibitor) prevented FBXL2 ubiquitination and ameliorated allodynia.","method":"Spinal nerve ligation rat model, siRNA knockdown, intrathecal pharmacological inhibition (BC-1215), behavioral allodynia assays, Western blot for ubiquitination and phosphorylation","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic loss-of-function and pharmacological inhibition with defined molecular pathway readouts and behavioral phenotype","pmids":["26674878"],"is_preprint":false},{"year":2015,"finding":"FBXO3 targets Smurf1 (an HECT-type E3 ubiquitin ligase) for polyubiquitination and proteasomal degradation. Unlike Fbxl15, FBXO3 also targets all Nedd4 family members for degradation, implicating FBXO3 in BMP signaling through control of Nedd4-family ligase stability.","method":"Co-immunoprecipitation, ubiquitination assays, proteasomal degradation assays","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP and ubiquitination assay, single lab, single study without full mechanistic follow-up","pmids":["25721664"],"is_preprint":false},{"year":2016,"finding":"FBXO3 promotes ubiquitylation and increases transcriptional activity of AIRE (Autoimmune Regulator). AIRE, phosphorylated on two specific N-terminal residues, binds the SCF(FBXO3) (SKP1-CUL1-FBXO3) complex, which ubiquitylates AIRE and enhances its binding to P-TEFb (positive transcription elongation factor b), thereby potentiating AIRE-driven transcription of tissue-specific antigens in the thymus.","method":"Co-immunoprecipitation, ubiquitylation assays, transcriptional reporter assays, phosphorylation mapping, mutagenesis of phosphorylation sites","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, ubiquitylation assay, phosphorylation site mutagenesis, transcriptional readout; multiple orthogonal methods in one study","pmids":["27365398"],"is_preprint":false},{"year":2016,"finding":"The X-ray crystal structure of the human FBXO3 ApaG domain (residues 278–407) was solved at 2.0 Å resolution, revealing a classic Immunoglobulin/Fibronectin III-type fold with a seven-stranded β-sheet core and four extended loops. Loop 1 (residues 294–303) is critical for interaction with FBXL2 in the context of the full-length protein. The isolated ApaG domain alone is necessary but not sufficient for binding full-length FBXL2, as shown by coimmunoprecipitation. No binding to Mg²⁺, Co²⁺, or dinucleotide polyphosphates was detected.","method":"X-ray crystallography (2.0 Å), NMR titration, coimmunoprecipitation, loop mutagenesis","journal":"The FEBS journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with functional validation by mutagenesis and Co-IP, single lab","pmids":["27010866"],"is_preprint":false},{"year":2016,"finding":"Spinal nerve ligation induces Fbxo3-dependent Fbxl2 ubiquitination, reducing Fbxl2-mediated ubiquitination of the presynaptic active zone protein RIM1α. De-ubiquitinated (stabilized) RIM1α accumulates in synaptic plasma membranes and directly binds CaV2.2, increasing CaV2.2 expression and synaptic vesicle exocytosis in dorsal horn, thus driving neuropathic allodynia.","method":"Spinal nerve ligation rat model, siRNA knockdown, intrathecal BC-1215 administration, co-immunoprecipitation (RIM1α–CaV2.2 interaction), electrophysiology (sEPSC), Western blot for synaptic plasma membrane fractions","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Moderate — in vivo loss-of-function, Co-IP demonstrating RIM1α–CaV2.2 interaction, pharmacological inhibition, multiple orthogonal phenotypic readouts","pmids":["27629721"],"is_preprint":false},{"year":2020,"finding":"Zebrafish fbxo3 interacts with transcription factors irf3 and irf7 and specifically catalyzes K27-linked polyubiquitination of irf3 and irf7, targeting them for proteasomal degradation, thereby suppressing antiviral IFN-I responses. Notably, the F-box domain of fbxo3 is not required for the interaction with irf3/irf7 or for inhibiting their transactivity.","method":"Co-immunoprecipitation, ubiquitination assays specifying K27-linkage, zebrafish knockout (fbxo3 disruption), survival assay upon viral challenge, overexpression studies","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — Co-IP, linkage-specific ubiquitination assay, in vivo fbxo3 knockout in zebrafish with defined antiviral phenotype; multiple orthogonal methods","pmids":["32859728"],"is_preprint":false},{"year":2020,"finding":"FBXO3 (as an SCF E3 ubiquitin ligase substrate receptor) binds and ubiquitylates C21ORF2, targeting it for proteasomal degradation. C21ORF2 stabilizes the kinase NEK1; consequently, loss of FBXO3 stabilizes both C21ORF2 and NEK1. Conversely, NEK1-mediated phosphorylation of C21ORF2 attenuates its interaction with FBXO3, stabilizing C21ORF2. The ALS-associated V58L mutant of C21ORF2 is hyperphosphorylated by NEK1 and therefore escapes FBXO3-mediated ubiquitylation, leading to its accumulation along with NEK1.","method":"Co-immunoprecipitation, ubiquitylation assays, proteasomal degradation assays, phosphorylation analysis, ALS mutant characterization, motor neuron differentiation from mouse ESCs with neurite outgrowth readout","journal":"iScience","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, ubiquitylation assay, genetic epistasis (NEK1 phosphorylation controls FBXO3 interaction), disease mutant validation; multiple orthogonal methods in one study","pmids":["32891887"],"is_preprint":false},{"year":2021,"finding":"Spinal Fbxo3 mediates ubiquitination and degradation of CARM1 (coactivator-associated arginine methyltransferase 1) after nerve injury. Reduced CARM1 decreases H3R17me2 at K+ channel promoters, causing epigenetic silencing of K+ channel genes and contributing to neuropathic allodynia. Intrathecal BC-1215 (Fbxo3 inhibitor) prevented CARM1 ubiquitination and blocked K+ channel gene silencing.","method":"Spinal nerve ligation rat model, siRNA knockdown, CARM1 inhibitor, intrathecal BC-1215, ChIP assay for H3R17me2 at K+ channel promoters, behavioral allodynia assays","journal":"Neurotherapeutics","confidence":"High","confidence_rationale":"Tier 2 / Moderate — in vivo loss-of-function, ChIP epigenetic readout, pharmacological inhibition, multiple orthogonal methods linking FBXO3 to substrate ubiquitination and gene silencing","pmids":["33415686"],"is_preprint":false},{"year":2022,"finding":"FBXO3 promotes ubiquitination and proteasomal degradation of HIPK2 (Homeodomain-Interacting Protein Kinase 2) in the context of cerebral ischemia/reperfusion injury, contributing to neuroinflammation. siRNA knockdown of FBXO3 and the FBXO3 inhibitor BC-1215 both preserved HIPK2 protein levels and reduced neuronal damage and inflammatory cytokine production in vivo and in vitro.","method":"MCAO/R rat model, OGD/R neuronal cell model, siRNA knockdown, BC-1215 pharmacological inhibition, Western blot for HIPK2, inflammatory cytokine measurement","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — loss-of-function with defined molecular and phenotypic readout, but interaction between FBXO3 and HIPK2 was inferred rather than directly demonstrated by Co-IP in this abstract; single lab","pmids":["36362432"],"is_preprint":false},{"year":2023,"finding":"FBXO3 stabilizes USP4 by disrupting the interaction between USP4 and DNPEP (aspartyl aminopeptidase), protecting USP4 from DNPEP-mediated degradation. Stabilized USP4 in turn stabilizes Twist1, promoting breast cancer cell migration and tumor metastasis. This function is independent of FBXO3's E3 ligase activity. Additionally, PI3K (p110αH1047R) facilitates FBXO3 phosphorylation and stabilization in an ERK1-dependent manner.","method":"Co-immunoprecipitation (FBXO3-USP4, USP4-DNPEP interactions), siRNA knockdown, mouse metastasis models, Western blot for protein stability, phosphorylation analysis","journal":"PLoS biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP demonstrating FBXO3-USP4 and USP4-DNPEP interactions, loss-of-function with in vivo metastasis phenotype, ERK1 phosphorylation mechanistic link; multiple orthogonal methods","pmids":["38134227"],"is_preprint":false},{"year":2024,"finding":"Ginsenoside Rg1 reduces FBXO3 protein stability in an m6A-YTHDF1-dependent manner. FBXO3 interacts with PGC-1α and promotes its ubiquitination, targeting PGC-1α for degradation. Reduction of FBXO3 by Rg1 activates the PGC-1α/Nrf2 signaling pathway, improving mitochondrial function in sepsis-induced acute lung injury. Co-IP confirmed FBXO3–YTHDF1 and FBXO3–PGC-1α interactions.","method":"Co-immunoprecipitation (FBXO3-YTHDF1 and FBXO3-PGC-1α), MeRIP assay (m6A modification of FBXO3 mRNA), RIP assay, ubiquitination assay, CLP rat model, LPS cell model","journal":"The AAPS journal","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP for binding partners, ubiquitination assay, in vivo model; single lab, moderate mechanistic depth","pmids":["38622374"],"is_preprint":false},{"year":2025,"finding":"The cryo-EM structure of the human SCF(FBXO3) complex (CUL1-RBX1-SKP1-FBXO3) was solved at 3.70 Å nominal resolution. The F-box domain of FBXO3 associates with SKP1 via extensive hydrophobic interactions and interacts with the N-terminal region of CUL1 via hydrophobic interactions. The weak density for the RBX1 globular region near the FBXO3 ApaG domain suggests an unmodified closed conformation, and CUL1 neddylation is likely required for high E3 activity.","method":"Cryo-EM structure determination at 3.70 Å","journal":"Proteins","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — cryo-EM structure of the full SCF complex, but single study without mutagenesis validation or activity assays to confirm functional implications","pmids":["39921442"],"is_preprint":false},{"year":2025,"finding":"TBK1-dependent phosphorylation of FBXO3 facilitates its interaction with TMEM192, promoting TMEM192 ubiquitination and subsequent recognition by the autophagy receptor TAX1BP1, orchestrating lysophagic flux following lysosomal damage. Perturbing this TBK1-SCFFBXO3-TMEM192-TAX1BP1 axis significantly reduces lysophagic flux and causes accumulation of damaged lysosomes.","method":"Genetic perturbation of pathway components, phosphorylation assays, ubiquitination assays, lysophagy flux assays, co-immunoprecipitation","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — defined phosphorylation-dependent interaction and ubiquitination cascade with functional lysophagy readout; single lab, abstract-level detail","pmids":["40083080"],"is_preprint":false},{"year":2026,"finding":"FBXO3 interacts with DUSP9 and promotes its ubiquitination, activating the MAPK pathway and supporting leukemia stem cell (LSC) maintenance and TKI resistance in CML. FBXO3 deficiency induced apoptosis and reduced proliferation of CML LSCs in vitro and in vivo, with minimal effects on normal hematopoietic stem cells. DUSP9 knockdown partially reversed the effects of FBXO3 deficiency.","method":"Co-immunoprecipitation (FBXO3-DUSP9), ubiquitination assays, siRNA/genetic knockdown, in vitro and in vivo CML LSC models, scRNA-seq for expression context, pharmacological FBXO3 inhibition","journal":"Cell reports. Medicine","confidence":"High","confidence_rationale":"Tier 2 / Moderate — Co-IP for substrate identification, ubiquitination assay, in vivo loss-of-function with defined LSC phenotype, epistasis via DUSP9 knockdown rescue experiment","pmids":["41850237"],"is_preprint":false},{"year":2024,"finding":"A molecular glue degrader (dHTC3) was found to selectively dimerize the first bromodomain of BRD4 to SCF(FBXO3), identifying FBXO3 as an E3 ligase accessible for chemical rewiring via proximity pharmacology.","method":"High-throughput SuFEx chemistry screening, degrader activity assays, biochemical characterization of BRD4-dHTC3-SCFFBXO3 ternary complex","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — preprint, functional degrader assay implicating FBXO3 but limited structural/mechanistic detail on FBXO3 itself in the abstract","pmids":["bio_10.1101_2024.09.30.615685"],"is_preprint":true}],"current_model":"FBXO3 is an SCF E3 ubiquitin ligase substrate receptor that assembles into SKP1-CUL1-FBXO3-RBX1 complexes (cryo-EM structure resolved at 3.70 Å) and ubiquitylates a diverse set of substrates—including FBXL2 (promoting TRAF-driven cytokine production), AIRE (enhancing P-TEFb binding and transcription), C21ORF2 (regulated by NEK1 phosphorylation), Smurf1/Nedd4-family ligases, CARM1, HIPK2, TMEM192 (in a TBK1-phosphorylation-dependent lysophagy axis), DUSP9 (maintaining CML leukemia stem cells via MAPK activation), and viral/zebrafish substrates irf3/irf7; its C-terminal ApaG domain (Ig/FNIII fold, crystal structure at 2.0 Å) is required but not sufficient alone for substrate binding, and pharmacological inhibition of FBXO3 reduces inflammatory cytokine production, neuropathic pain, and CML stem cell maintenance."},"narrative":{"mechanistic_narrative":"FBXO3 is the substrate-recognition subunit of an SCF (SKP1–CUL1–RBX1) E3 ubiquitin ligase that controls inflammatory, neuronal, and oncogenic signaling by directing target proteins for polyubiquitination and proteasomal degradation [PMID:24123678, PMID:27365398, PMID:39921442]. Its C-terminal ApaG domain adopts an immunoglobulin/fibronectin-III fold whose loop 1 mediates substrate engagement; this domain is necessary but not sufficient for binding, and it is essential for productive substrate disposal [PMID:27010866, PMID:24123678]. A central recurring axis is FBXO3-driven degradation of the competing F-box protein FBXL2, which de-represses TRAF proteins to amplify proinflammatory cytokine production [PMID:24123678] and, in spinal dorsal horn neurons, stabilizes the FBXL2 substrates TRAF2 and RIM1α to drive GluR1 and CaV2.2-dependent synaptic potentiation underlying neuropathic allodynia [PMID:26674878, PMID:27629721]. FBXO3 acts on a broad substrate range with diverse outcomes: it degrades Smurf1 and other Nedd4-family ligases [PMID:25721664], the disease-relevant C21ORF2 in a manner reversed by NEK1 phosphorylation [PMID:32891887], CARM1 to epigenetically silence K+ channel genes [PMID:33415686], HIPK2 during cerebral ischemia [PMID:36362432], and DUSP9 to sustain MAPK signaling and leukemia stem cell maintenance in CML [PMID:41850237]. It also ubiquitylates AIRE to potentiate P-TEFb-dependent transcription [PMID:27365398] and operates in a TBK1-phosphorylation-dependent lysophagy axis through TMEM192 [PMID:40083080]. Not all FBXO3 functions require its catalytic ligase activity: it stabilizes USP4 by displacing the aminopeptidase DNPEP to promote breast cancer metastasis [PMID:38134227]. FBXO3 activity is itself regulated by signaling and RNA-modification inputs, including ERK1-dependent phosphorylation and m6A/YTHDF1-controlled protein stability [PMID:38134227, PMID:38622374]. Pharmacological inhibition of FBXO3 (BC-1215) blunts cytokine production, neuropathic pain, and CML stem cell maintenance, establishing it as a tractable drug target [PMID:24123678, PMID:26674878, PMID:41850237].","teleology":[{"year":2013,"claim":"Established FBXO3 as an SCF F-box protein with a defined immunological output by showing it degrades the competing F-box protein FBXL2 to release TRAF-driven cytokine production.","evidence":"Co-IP, siRNA knockdown, cytokine assays, and murine inflammation models with an ApaG-targeting inhibitor","pmids":["24123678"],"confidence":"High","gaps":["Linkage type and direct ubiquitin-transfer reconstitution not defined","ApaG residues mediating FBXL2 recognition not yet mapped"]},{"year":2014,"claim":"Showed FBXO3 can be hijacked by a viral virulence factor to degrade a TFIIH subunit, revealing it as a host substrate receptor for transcriptional suppression and SCF assembly via Skp1.","evidence":"siRNA depletion with p62 rescue and IFN reporter assays plus Co-IP in RVFV-infected cells","pmids":["24403578"],"confidence":"High","gaps":["Cullin requirement was paradoxical (cullin1/7 and Rbx1 knockdown did not rescue p62)","Direct ubiquitination of p62 not reconstituted"]},{"year":2015,"claim":"Extended the FBXO3–FBXL2 axis into neuropathic pain and broadened the substrate range to Nedd4-family HECT ligases, indicating FBXO3 regulates other ubiquitin enzymes.","evidence":"Spinal nerve ligation rat model with BC-1215 inhibition (TRAF2/GluR1 axis) and Co-IP/ubiquitination assays for Smurf1/Nedd4 degradation","pmids":["26674878","25721664"],"confidence":"High","gaps":["Smurf1/Nedd4 substrate selectivity over Fbxl15 not structurally explained","In vivo relevance of Nedd4-family degradation to BMP signaling untested"]},{"year":2016,"claim":"Defined the structural basis of FBXO3 substrate engagement and showed it can act as a non-degradative activator of transcription, indicating ubiquitylation outcomes are context-dependent.","evidence":"X-ray crystallography of the ApaG domain with loop mutagenesis/NMR/Co-IP, plus AIRE ubiquitylation, phospho-site mutagenesis, and transcription reporters","pmids":["27010866","27365398","27629721"],"confidence":"High","gaps":["Why isolated ApaG is insufficient for substrate binding not resolved","Mechanism by which AIRE ubiquitylation enhances P-TEFb binding not detailed"]},{"year":2020,"claim":"Demonstrated phosphorylation-gated substrate choice and linkage-specific ubiquitination, connecting FBXO3 to disease-relevant proteostasis (C21ORF2/NEK1) and conserved antiviral IRF control.","evidence":"Co-IP, ubiquitylation assays, NEK1 phospho-epistasis and ALS mutant analysis; zebrafish fbxo3 knockout with K27-linkage-specific irf3/irf7 ubiquitination","pmids":["32891887","32859728"],"confidence":"High","gaps":["F-box-independent IRF degradation mechanism in zebrafish unexplained","Conservation of K27-linkage activity to human FBXO3 not tested"]},{"year":2021,"claim":"Connected FBXO3 to epigenetic gene regulation by showing it degrades the methyltransferase CARM1 to silence K+ channel genes in neuropathic pain.","evidence":"Spinal nerve ligation model, ChIP for H3R17me2 at K+ channel promoters, and BC-1215 inhibition","pmids":["33415686"],"confidence":"High","gaps":["Direct FBXO3–CARM1 interaction interface not mapped","Substrate-recognition determinants for CARM1 unknown"]},{"year":2022,"claim":"Implicated FBXO3 in ischemic neuroinflammation through HIPK2 turnover, expanding its substrate set.","evidence":"MCAO/R and OGD/R models with siRNA and BC-1215, monitoring HIPK2 levels and cytokines","pmids":["36362432"],"confidence":"Medium","gaps":["FBXO3–HIPK2 interaction inferred rather than shown by Co-IP","Single lab without reciprocal validation"]},{"year":2023,"claim":"Revealed a catalysis-independent function in which FBXO3 stabilizes USP4 by displacing DNPEP, showing FBXO3 can act as a scaffold to promote, not destroy, partner stability and drive metastasis.","evidence":"Reciprocal Co-IP of FBXO3–USP4 and USP4–DNPEP, knockdown, mouse metastasis models, and ERK1 phosphorylation analysis","pmids":["38134227"],"confidence":"High","gaps":["Structural basis for DNPEP displacement unknown","How E3-independent and E3-dependent roles are partitioned in cells unclear"]},{"year":2024,"claim":"Identified upstream control of FBXO3 abundance via m6A/YTHDF1 and a new mitochondrial substrate (PGC-1α), linking FBXO3 to metabolic regulation in acute lung injury.","evidence":"MeRIP/RIP assays, Co-IP of FBXO3–YTHDF1 and FBXO3–PGC-1α, ubiquitination assays, and CLP/LPS sepsis models","pmids":["38622374"],"confidence":"Medium","gaps":["Direct ubiquitin transfer to PGC-1α not reconstituted","Single-lab abstract-level mechanistic depth"]},{"year":2025,"claim":"Resolved the full SCF(FBXO3) architecture and defined a phosphorylation-gated lysophagy role, clarifying both how FBXO3 assembles into the ligase and how it is signal-activated.","evidence":"Cryo-EM of CUL1-RBX1-SKP1-FBXO3 at 3.70 Å; TBK1-dependent phospho-FBXO3 promoting TMEM192 ubiquitination and TAX1BP1-mediated lysophagy","pmids":["39921442","40083080"],"confidence":"Medium","gaps":["Structure lacks mutagenesis/activity validation and neddylation-state confirmation","How TBK1 phosphorylation alters FBXO3 substrate selectivity not defined"]},{"year":2026,"claim":"Established a selective oncogenic dependency by showing FBXO3 degrades DUSP9 to sustain MAPK signaling and CML leukemia stem cell maintenance with minimal effect on normal HSCs.","evidence":"Co-IP, ubiquitination assays, in vitro/in vivo CML LSC knockdown with DUSP9 rescue epistasis and pharmacological inhibition","pmids":["41850237"],"confidence":"High","gaps":["DUSP9-binding determinants on FBXO3 unmapped","Basis for selectivity toward LSCs over normal HSCs unexplained"]},{"year":null,"claim":"How FBXO3 achieves its strikingly broad and partly non-catalytic substrate repertoire, and how upstream phosphorylation and m6A inputs are integrated to select among them, remains unresolved.","evidence":"No single study reconciles substrate breadth with the ApaG recognition mechanism or defines a unifying recruitment logic","pmids":[],"confidence":"Low","gaps":["No common substrate-recognition motif identified across the diverse targets","Linkage specificity and neddylation dependence not systematically compared across substrates","Catalytic versus scaffolding functions not delineated genome-wide"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,4,8,9,15]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,13]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,11]}],"localization":[],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,4,8,13]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,1,7]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,6,15]}],"complexes":["SCF(SKP1-CUL1-RBX1-FBXO3) E3 ubiquitin ligase"],"partners":["FBXL2","SKP1","CUL1","AIRE","C21ORF2","USP4","DUSP9","TMEM192"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9UK99","full_name":"F-box only protein 3","aliases":[],"length_aa":471,"mass_kda":54.6,"function":"Substrate recognition component of the SCF (SKP1-CUL1-F-box protein)-type E3 ubiquitin ligase complex, SCF(FBXO3), which mediates the ubiquitination and subsequent proteasomal degradation of target proteins (PubMed:18809579, PubMed:26037928). Mediates the ubiquitination of HIPK2 and probably that of EP300, leading to rapid degradation by the proteasome (PubMed:18809579). In the presence of PML, HIPK2 ubiquitination still occurs, but degradation is prevented (PubMed:18809579). PML, HIPK2 and FBXO3 may act synergically to activate p53/TP53-dependent transactivation (PubMed:18809579). The SCF(FBXO3) also acts as a regulator of inflammation by mediating ubiquitination and degradation of FBXL2 in response to lipopolysaccharide (LPS) (PubMed:26037928, PubMed:27010866). The SCF(FBXO3) complex specifically recognizes FBXL2 phosphorylated at 'Thr-404' and promotes its ubiquitination (PubMed:27010866) (Microbial infection) Associates with the Rift valley fever virus NSs to form a remodeled E3 ligase that triggers efficient proteasomal degradation of targeted proteins. The filamentous E3 ligase targets the TFIIH complex leading to robust inhibition of antiviral immunity and enhances viral pathogenesis","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9UK99/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/FBXO3","classification":"Not Classified","n_dependent_lines":13,"n_total_lines":1208,"dependency_fraction":0.01076158940397351},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/FBXO3","total_profiled":1310},"omim":[{"mim_id":"611519","title":"POLYMERASE DELTA-INTERACTING PROTEIN 2; POLDIP2","url":"https://www.omim.org/entry/611519"},{"mim_id":"609089","title":"F-BOX ONLY PROTEIN 3; FBXO3","url":"https://www.omim.org/entry/609089"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in 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This leads to elevated TRAF protein levels and potent stimulation of proinflammatory cytokine secretion from human inflammatory cells. The C-terminal ApaG domain of FBXO3 was found to be indispensable for mediating FBXL2 disposal and cytokine secretion.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, cytokine secretion assays, murine models of viral pneumonia/septic shock/colitis, small-molecule inhibitor targeting ApaG domain\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, loss-of-function with defined cellular phenotype, in vivo disease models, replicated across multiple disease contexts in one rigorous study\",\n      \"pmids\": [\"24123678\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"RVFV NSs virulence factor recruits host cell F-box protein FBXO3 to degrade TFIIH subunit p62 via the ubiquitin/proteasome pathway, thereby suppressing type I interferon transcription. siRNA depletion of FBXO3 rescued p62 levels and elevated IFN transcription ~10-fold. FBXO3 forms SCF complexes with Skp1; knockdown of Skp1 also protected p62, but knockdown of cullin 1, cullin 7, or Rbx1 did not rescue p62 degradation.\",\n      \"method\": \"siRNA knockdown, co-immunoprecipitation, protein level rescue assays, IFN transcription reporter assays\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, siRNA depletion with defined molecular phenotype (p62 rescue), IFN transcription measurement, multiple genetic perturbations tested\",\n      \"pmids\": [\"24403578\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"FBXO3-dependent ubiquitination and degradation of FBXL2 leads to de-ubiquitination (stabilization) of TRAF2 in spinal dorsal horn neurons. This TRAF2 stabilization drives TNIK/GluR1 phosphorylation and GluR1 trafficking to the plasma membrane, mediating neuropathic allodynia after spinal nerve ligation. Intrathecal BC-1215 (FBXO3 inhibitor) prevented FBXL2 ubiquitination and ameliorated allodynia.\",\n      \"method\": \"Spinal nerve ligation rat model, siRNA knockdown, intrathecal pharmacological inhibition (BC-1215), behavioral allodynia assays, Western blot for ubiquitination and phosphorylation\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic loss-of-function and pharmacological inhibition with defined molecular pathway readouts and behavioral phenotype\",\n      \"pmids\": [\"26674878\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"FBXO3 targets Smurf1 (an HECT-type E3 ubiquitin ligase) for polyubiquitination and proteasomal degradation. Unlike Fbxl15, FBXO3 also targets all Nedd4 family members for degradation, implicating FBXO3 in BMP signaling through control of Nedd4-family ligase stability.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, proteasomal degradation assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP and ubiquitination assay, single lab, single study without full mechanistic follow-up\",\n      \"pmids\": [\"25721664\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"FBXO3 promotes ubiquitylation and increases transcriptional activity of AIRE (Autoimmune Regulator). AIRE, phosphorylated on two specific N-terminal residues, binds the SCF(FBXO3) (SKP1-CUL1-FBXO3) complex, which ubiquitylates AIRE and enhances its binding to P-TEFb (positive transcription elongation factor b), thereby potentiating AIRE-driven transcription of tissue-specific antigens in the thymus.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitylation assays, transcriptional reporter assays, phosphorylation mapping, mutagenesis of phosphorylation sites\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, ubiquitylation assay, phosphorylation site mutagenesis, transcriptional readout; multiple orthogonal methods in one study\",\n      \"pmids\": [\"27365398\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The X-ray crystal structure of the human FBXO3 ApaG domain (residues 278–407) was solved at 2.0 Å resolution, revealing a classic Immunoglobulin/Fibronectin III-type fold with a seven-stranded β-sheet core and four extended loops. Loop 1 (residues 294–303) is critical for interaction with FBXL2 in the context of the full-length protein. The isolated ApaG domain alone is necessary but not sufficient for binding full-length FBXL2, as shown by coimmunoprecipitation. No binding to Mg²⁺, Co²⁺, or dinucleotide polyphosphates was detected.\",\n      \"method\": \"X-ray crystallography (2.0 Å), NMR titration, coimmunoprecipitation, loop mutagenesis\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with functional validation by mutagenesis and Co-IP, single lab\",\n      \"pmids\": [\"27010866\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Spinal nerve ligation induces Fbxo3-dependent Fbxl2 ubiquitination, reducing Fbxl2-mediated ubiquitination of the presynaptic active zone protein RIM1α. De-ubiquitinated (stabilized) RIM1α accumulates in synaptic plasma membranes and directly binds CaV2.2, increasing CaV2.2 expression and synaptic vesicle exocytosis in dorsal horn, thus driving neuropathic allodynia.\",\n      \"method\": \"Spinal nerve ligation rat model, siRNA knockdown, intrathecal BC-1215 administration, co-immunoprecipitation (RIM1α–CaV2.2 interaction), electrophysiology (sEPSC), Western blot for synaptic plasma membrane fractions\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo loss-of-function, Co-IP demonstrating RIM1α–CaV2.2 interaction, pharmacological inhibition, multiple orthogonal phenotypic readouts\",\n      \"pmids\": [\"27629721\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Zebrafish fbxo3 interacts with transcription factors irf3 and irf7 and specifically catalyzes K27-linked polyubiquitination of irf3 and irf7, targeting them for proteasomal degradation, thereby suppressing antiviral IFN-I responses. Notably, the F-box domain of fbxo3 is not required for the interaction with irf3/irf7 or for inhibiting their transactivity.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays specifying K27-linkage, zebrafish knockout (fbxo3 disruption), survival assay upon viral challenge, overexpression studies\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, linkage-specific ubiquitination assay, in vivo fbxo3 knockout in zebrafish with defined antiviral phenotype; multiple orthogonal methods\",\n      \"pmids\": [\"32859728\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"FBXO3 (as an SCF E3 ubiquitin ligase substrate receptor) binds and ubiquitylates C21ORF2, targeting it for proteasomal degradation. C21ORF2 stabilizes the kinase NEK1; consequently, loss of FBXO3 stabilizes both C21ORF2 and NEK1. Conversely, NEK1-mediated phosphorylation of C21ORF2 attenuates its interaction with FBXO3, stabilizing C21ORF2. The ALS-associated V58L mutant of C21ORF2 is hyperphosphorylated by NEK1 and therefore escapes FBXO3-mediated ubiquitylation, leading to its accumulation along with NEK1.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitylation assays, proteasomal degradation assays, phosphorylation analysis, ALS mutant characterization, motor neuron differentiation from mouse ESCs with neurite outgrowth readout\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, ubiquitylation assay, genetic epistasis (NEK1 phosphorylation controls FBXO3 interaction), disease mutant validation; multiple orthogonal methods in one study\",\n      \"pmids\": [\"32891887\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Spinal Fbxo3 mediates ubiquitination and degradation of CARM1 (coactivator-associated arginine methyltransferase 1) after nerve injury. Reduced CARM1 decreases H3R17me2 at K+ channel promoters, causing epigenetic silencing of K+ channel genes and contributing to neuropathic allodynia. Intrathecal BC-1215 (Fbxo3 inhibitor) prevented CARM1 ubiquitination and blocked K+ channel gene silencing.\",\n      \"method\": \"Spinal nerve ligation rat model, siRNA knockdown, CARM1 inhibitor, intrathecal BC-1215, ChIP assay for H3R17me2 at K+ channel promoters, behavioral allodynia assays\",\n      \"journal\": \"Neurotherapeutics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo loss-of-function, ChIP epigenetic readout, pharmacological inhibition, multiple orthogonal methods linking FBXO3 to substrate ubiquitination and gene silencing\",\n      \"pmids\": [\"33415686\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"FBXO3 promotes ubiquitination and proteasomal degradation of HIPK2 (Homeodomain-Interacting Protein Kinase 2) in the context of cerebral ischemia/reperfusion injury, contributing to neuroinflammation. siRNA knockdown of FBXO3 and the FBXO3 inhibitor BC-1215 both preserved HIPK2 protein levels and reduced neuronal damage and inflammatory cytokine production in vivo and in vitro.\",\n      \"method\": \"MCAO/R rat model, OGD/R neuronal cell model, siRNA knockdown, BC-1215 pharmacological inhibition, Western blot for HIPK2, inflammatory cytokine measurement\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — loss-of-function with defined molecular and phenotypic readout, but interaction between FBXO3 and HIPK2 was inferred rather than directly demonstrated by Co-IP in this abstract; single lab\",\n      \"pmids\": [\"36362432\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"FBXO3 stabilizes USP4 by disrupting the interaction between USP4 and DNPEP (aspartyl aminopeptidase), protecting USP4 from DNPEP-mediated degradation. Stabilized USP4 in turn stabilizes Twist1, promoting breast cancer cell migration and tumor metastasis. This function is independent of FBXO3's E3 ligase activity. Additionally, PI3K (p110αH1047R) facilitates FBXO3 phosphorylation and stabilization in an ERK1-dependent manner.\",\n      \"method\": \"Co-immunoprecipitation (FBXO3-USP4, USP4-DNPEP interactions), siRNA knockdown, mouse metastasis models, Western blot for protein stability, phosphorylation analysis\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP demonstrating FBXO3-USP4 and USP4-DNPEP interactions, loss-of-function with in vivo metastasis phenotype, ERK1 phosphorylation mechanistic link; multiple orthogonal methods\",\n      \"pmids\": [\"38134227\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Ginsenoside Rg1 reduces FBXO3 protein stability in an m6A-YTHDF1-dependent manner. FBXO3 interacts with PGC-1α and promotes its ubiquitination, targeting PGC-1α for degradation. Reduction of FBXO3 by Rg1 activates the PGC-1α/Nrf2 signaling pathway, improving mitochondrial function in sepsis-induced acute lung injury. Co-IP confirmed FBXO3–YTHDF1 and FBXO3–PGC-1α interactions.\",\n      \"method\": \"Co-immunoprecipitation (FBXO3-YTHDF1 and FBXO3-PGC-1α), MeRIP assay (m6A modification of FBXO3 mRNA), RIP assay, ubiquitination assay, CLP rat model, LPS cell model\",\n      \"journal\": \"The AAPS journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP for binding partners, ubiquitination assay, in vivo model; single lab, moderate mechanistic depth\",\n      \"pmids\": [\"38622374\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The cryo-EM structure of the human SCF(FBXO3) complex (CUL1-RBX1-SKP1-FBXO3) was solved at 3.70 Å nominal resolution. The F-box domain of FBXO3 associates with SKP1 via extensive hydrophobic interactions and interacts with the N-terminal region of CUL1 via hydrophobic interactions. The weak density for the RBX1 globular region near the FBXO3 ApaG domain suggests an unmodified closed conformation, and CUL1 neddylation is likely required for high E3 activity.\",\n      \"method\": \"Cryo-EM structure determination at 3.70 Å\",\n      \"journal\": \"Proteins\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — cryo-EM structure of the full SCF complex, but single study without mutagenesis validation or activity assays to confirm functional implications\",\n      \"pmids\": [\"39921442\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TBK1-dependent phosphorylation of FBXO3 facilitates its interaction with TMEM192, promoting TMEM192 ubiquitination and subsequent recognition by the autophagy receptor TAX1BP1, orchestrating lysophagic flux following lysosomal damage. Perturbing this TBK1-SCFFBXO3-TMEM192-TAX1BP1 axis significantly reduces lysophagic flux and causes accumulation of damaged lysosomes.\",\n      \"method\": \"Genetic perturbation of pathway components, phosphorylation assays, ubiquitination assays, lysophagy flux assays, co-immunoprecipitation\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — defined phosphorylation-dependent interaction and ubiquitination cascade with functional lysophagy readout; single lab, abstract-level detail\",\n      \"pmids\": [\"40083080\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"FBXO3 interacts with DUSP9 and promotes its ubiquitination, activating the MAPK pathway and supporting leukemia stem cell (LSC) maintenance and TKI resistance in CML. FBXO3 deficiency induced apoptosis and reduced proliferation of CML LSCs in vitro and in vivo, with minimal effects on normal hematopoietic stem cells. DUSP9 knockdown partially reversed the effects of FBXO3 deficiency.\",\n      \"method\": \"Co-immunoprecipitation (FBXO3-DUSP9), ubiquitination assays, siRNA/genetic knockdown, in vitro and in vivo CML LSC models, scRNA-seq for expression context, pharmacological FBXO3 inhibition\",\n      \"journal\": \"Cell reports. Medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP for substrate identification, ubiquitination assay, in vivo loss-of-function with defined LSC phenotype, epistasis via DUSP9 knockdown rescue experiment\",\n      \"pmids\": [\"41850237\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"A molecular glue degrader (dHTC3) was found to selectively dimerize the first bromodomain of BRD4 to SCF(FBXO3), identifying FBXO3 as an E3 ligase accessible for chemical rewiring via proximity pharmacology.\",\n      \"method\": \"High-throughput SuFEx chemistry screening, degrader activity assays, biochemical characterization of BRD4-dHTC3-SCFFBXO3 ternary complex\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — preprint, functional degrader assay implicating FBXO3 but limited structural/mechanistic detail on FBXO3 itself in the abstract\",\n      \"pmids\": [\"bio_10.1101_2024.09.30.615685\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"FBXO3 is an SCF E3 ubiquitin ligase substrate receptor that assembles into SKP1-CUL1-FBXO3-RBX1 complexes (cryo-EM structure resolved at 3.70 Å) and ubiquitylates a diverse set of substrates—including FBXL2 (promoting TRAF-driven cytokine production), AIRE (enhancing P-TEFb binding and transcription), C21ORF2 (regulated by NEK1 phosphorylation), Smurf1/Nedd4-family ligases, CARM1, HIPK2, TMEM192 (in a TBK1-phosphorylation-dependent lysophagy axis), DUSP9 (maintaining CML leukemia stem cells via MAPK activation), and viral/zebrafish substrates irf3/irf7; its C-terminal ApaG domain (Ig/FNIII fold, crystal structure at 2.0 Å) is required but not sufficient alone for substrate binding, and pharmacological inhibition of FBXO3 reduces inflammatory cytokine production, neuropathic pain, and CML stem cell maintenance.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"FBXO3 is the substrate-recognition subunit of an SCF (SKP1–CUL1–RBX1) E3 ubiquitin ligase that controls inflammatory, neuronal, and oncogenic signaling by directing target proteins for polyubiquitination and proteasomal degradation [#0, #4, #13]. Its C-terminal ApaG domain adopts an immunoglobulin/fibronectin-III fold whose loop 1 mediates substrate engagement; this domain is necessary but not sufficient for binding, and it is essential for productive substrate disposal [#5, #0]. A central recurring axis is FBXO3-driven degradation of the competing F-box protein FBXL2, which de-represses TRAF proteins to amplify proinflammatory cytokine production [#0] and, in spinal dorsal horn neurons, stabilizes the FBXL2 substrates TRAF2 and RIM1α to drive GluR1 and CaV2.2-dependent synaptic potentiation underlying neuropathic allodynia [#2, #6]. FBXO3 acts on a broad substrate range with diverse outcomes: it degrades Smurf1 and other Nedd4-family ligases [#3], the disease-relevant C21ORF2 in a manner reversed by NEK1 phosphorylation [#8], CARM1 to epigenetically silence K+ channel genes [#9], HIPK2 during cerebral ischemia [#10], and DUSP9 to sustain MAPK signaling and leukemia stem cell maintenance in CML [#15]. It also ubiquitylates AIRE to potentiate P-TEFb-dependent transcription [#4] and operates in a TBK1-phosphorylation-dependent lysophagy axis through TMEM192 [#14]. Not all FBXO3 functions require its catalytic ligase activity: it stabilizes USP4 by displacing the aminopeptidase DNPEP to promote breast cancer metastasis [#11]. FBXO3 activity is itself regulated by signaling and RNA-modification inputs, including ERK1-dependent phosphorylation and m6A/YTHDF1-controlled protein stability [#11, #12]. Pharmacological inhibition of FBXO3 (BC-1215) blunts cytokine production, neuropathic pain, and CML stem cell maintenance, establishing it as a tractable drug target [#0, #2, #15].\",\n  \"teleology\": [\n    {\n      \"year\": 2013,\n      \"claim\": \"Established FBXO3 as an SCF F-box protein with a defined immunological output by showing it degrades the competing F-box protein FBXL2 to release TRAF-driven cytokine production.\",\n      \"evidence\": \"Co-IP, siRNA knockdown, cytokine assays, and murine inflammation models with an ApaG-targeting inhibitor\",\n      \"pmids\": [\"24123678\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Linkage type and direct ubiquitin-transfer reconstitution not defined\", \"ApaG residues mediating FBXL2 recognition not yet mapped\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Showed FBXO3 can be hijacked by a viral virulence factor to degrade a TFIIH subunit, revealing it as a host substrate receptor for transcriptional suppression and SCF assembly via Skp1.\",\n      \"evidence\": \"siRNA depletion with p62 rescue and IFN reporter assays plus Co-IP in RVFV-infected cells\",\n      \"pmids\": [\"24403578\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cullin requirement was paradoxical (cullin1/7 and Rbx1 knockdown did not rescue p62)\", \"Direct ubiquitination of p62 not reconstituted\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Extended the FBXO3–FBXL2 axis into neuropathic pain and broadened the substrate range to Nedd4-family HECT ligases, indicating FBXO3 regulates other ubiquitin enzymes.\",\n      \"evidence\": \"Spinal nerve ligation rat model with BC-1215 inhibition (TRAF2/GluR1 axis) and Co-IP/ubiquitination assays for Smurf1/Nedd4 degradation\",\n      \"pmids\": [\"26674878\", \"25721664\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Smurf1/Nedd4 substrate selectivity over Fbxl15 not structurally explained\", \"In vivo relevance of Nedd4-family degradation to BMP signaling untested\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Defined the structural basis of FBXO3 substrate engagement and showed it can act as a non-degradative activator of transcription, indicating ubiquitylation outcomes are context-dependent.\",\n      \"evidence\": \"X-ray crystallography of the ApaG domain with loop mutagenesis/NMR/Co-IP, plus AIRE ubiquitylation, phospho-site mutagenesis, and transcription reporters\",\n      \"pmids\": [\"27010866\", \"27365398\", \"27629721\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why isolated ApaG is insufficient for substrate binding not resolved\", \"Mechanism by which AIRE ubiquitylation enhances P-TEFb binding not detailed\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Demonstrated phosphorylation-gated substrate choice and linkage-specific ubiquitination, connecting FBXO3 to disease-relevant proteostasis (C21ORF2/NEK1) and conserved antiviral IRF control.\",\n      \"evidence\": \"Co-IP, ubiquitylation assays, NEK1 phospho-epistasis and ALS mutant analysis; zebrafish fbxo3 knockout with K27-linkage-specific irf3/irf7 ubiquitination\",\n      \"pmids\": [\"32891887\", \"32859728\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"F-box-independent IRF degradation mechanism in zebrafish unexplained\", \"Conservation of K27-linkage activity to human FBXO3 not tested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Connected FBXO3 to epigenetic gene regulation by showing it degrades the methyltransferase CARM1 to silence K+ channel genes in neuropathic pain.\",\n      \"evidence\": \"Spinal nerve ligation model, ChIP for H3R17me2 at K+ channel promoters, and BC-1215 inhibition\",\n      \"pmids\": [\"33415686\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct FBXO3–CARM1 interaction interface not mapped\", \"Substrate-recognition determinants for CARM1 unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Implicated FBXO3 in ischemic neuroinflammation through HIPK2 turnover, expanding its substrate set.\",\n      \"evidence\": \"MCAO/R and OGD/R models with siRNA and BC-1215, monitoring HIPK2 levels and cytokines\",\n      \"pmids\": [\"36362432\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"FBXO3–HIPK2 interaction inferred rather than shown by Co-IP\", \"Single lab without reciprocal validation\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Revealed a catalysis-independent function in which FBXO3 stabilizes USP4 by displacing DNPEP, showing FBXO3 can act as a scaffold to promote, not destroy, partner stability and drive metastasis.\",\n      \"evidence\": \"Reciprocal Co-IP of FBXO3–USP4 and USP4–DNPEP, knockdown, mouse metastasis models, and ERK1 phosphorylation analysis\",\n      \"pmids\": [\"38134227\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for DNPEP displacement unknown\", \"How E3-independent and E3-dependent roles are partitioned in cells unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified upstream control of FBXO3 abundance via m6A/YTHDF1 and a new mitochondrial substrate (PGC-1α), linking FBXO3 to metabolic regulation in acute lung injury.\",\n      \"evidence\": \"MeRIP/RIP assays, Co-IP of FBXO3–YTHDF1 and FBXO3–PGC-1α, ubiquitination assays, and CLP/LPS sepsis models\",\n      \"pmids\": [\"38622374\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ubiquitin transfer to PGC-1α not reconstituted\", \"Single-lab abstract-level mechanistic depth\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Resolved the full SCF(FBXO3) architecture and defined a phosphorylation-gated lysophagy role, clarifying both how FBXO3 assembles into the ligase and how it is signal-activated.\",\n      \"evidence\": \"Cryo-EM of CUL1-RBX1-SKP1-FBXO3 at 3.70 Å; TBK1-dependent phospho-FBXO3 promoting TMEM192 ubiquitination and TAX1BP1-mediated lysophagy\",\n      \"pmids\": [\"39921442\", \"40083080\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structure lacks mutagenesis/activity validation and neddylation-state confirmation\", \"How TBK1 phosphorylation alters FBXO3 substrate selectivity not defined\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Established a selective oncogenic dependency by showing FBXO3 degrades DUSP9 to sustain MAPK signaling and CML leukemia stem cell maintenance with minimal effect on normal HSCs.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, in vitro/in vivo CML LSC knockdown with DUSP9 rescue epistasis and pharmacological inhibition\",\n      \"pmids\": [\"41850237\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"DUSP9-binding determinants on FBXO3 unmapped\", \"Basis for selectivity toward LSCs over normal HSCs unexplained\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How FBXO3 achieves its strikingly broad and partly non-catalytic substrate repertoire, and how upstream phosphorylation and m6A inputs are integrated to select among them, remains unresolved.\",\n      \"evidence\": \"No single study reconciles substrate breadth with the ApaG recognition mechanism or defines a unifying recruitment logic\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No common substrate-recognition motif identified across the diverse targets\", \"Linkage specificity and neddylation dependence not systematically compared across substrates\", \"Catalytic versus scaffolding functions not delineated genome-wide\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 4, 8, 9, 15]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 13]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 11]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 4, 8, 13]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 1, 7]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 6, 15]}\n    ],\n    \"complexes\": [\"SCF(SKP1-CUL1-RBX1-FBXO3) E3 ubiquitin ligase\"],\n    \"partners\": [\"FBXL2\", \"SKP1\", \"CUL1\", \"AIRE\", \"C21ORF2\", \"USP4\", \"DUSP9\", \"TMEM192\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}