{"gene":"TNFAIP1","run_date":"2026-06-10T10:51:55","timeline":{"discoveries":[{"year":1994,"finding":"B61 (TNFAIP1) was identified as the ligand for the ECK receptor protein-tyrosine kinase; recombinant B61 induces autophosphorylation of ECK in intact cells, and B61 was purified by receptor affinity chromatography using the extracellular domain of ECK.","method":"Receptor affinity chromatography, surface plasmon resonance, cell-based autophosphorylation assay","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct biochemical purification and receptor activation reconstituted in cells, replicated across multiple subsequent studies","pmids":["8139691"],"is_preprint":false},{"year":1995,"finding":"B61 can exist as a cell-surface glycosylphosphatidylinositol (GPI)-linked protein, in addition to its soluble secreted form, and the GPI-linked form is capable of activating the ECK receptor protein-tyrosine kinase.","method":"PI-PLC treatment, cell-based receptor activation assay, biochemical characterization","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct biochemical demonstration of GPI linkage with functional receptor activation, single lab with orthogonal methods","pmids":["7890684"],"is_preprint":false},{"year":1995,"finding":"B61, acting through the ECK receptor tyrosine kinase, functions as an angiogenic factor in vivo and as a chemoattractant for endothelial cells in vitro; TNF-alpha induces angiogenesis through induction of B61, which then activates ECK in an autocrine/paracrine loop, and an anti-B61 antibody attenuated TNF-alpha-induced but not bFGF-induced angiogenesis.","method":"B61-immunoglobulin chimera in vivo angiogenesis assay, endothelial cell chemotaxis assay, antibody neutralization","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple functional assays in vitro and in vivo with antibody neutralization controls, widely replicated","pmids":["7536959"],"is_preprint":false},{"year":2009,"finding":"TNFAIP1 directly interacts with RhoB; co-immunoprecipitation and in vitro binding assays confirmed the interaction. RFP-TNFAIP1 partially co-localizes with EGFP-RhoB in endosomes. TNFAIP1 elicits pro-apoptotic activity, and simultaneous expression of RhoB and TNFAIP1 dramatically increases apoptosis in HeLa cells via SAPK/JNK signaling; knockdown of RhoB by siRNA rescued cells from TNFAIP1-induced apoptosis.","method":"Yeast two-hybrid, co-immunoprecipitation, in vitro binding assay, fluorescence co-localization, siRNA knockdown, JNK inhibitor experiment","journal":"International journal of cancer","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, in vitro binding, co-localization, and functional rescue with multiple orthogonal methods in single lab","pmids":["19637314"],"is_preprint":false},{"year":2009,"finding":"CK2 (casein kinase 2) phosphorylates TNFAIP1 both in vitro and in vivo; CK2β was identified as a TNFAIP1-interacting partner. Phosphorylation by CK2 facilitates nuclear distribution of TNFAIP1 and enhances its interaction with PCNA.","method":"Yeast two-hybrid screening, in vitro kinase assay, in vivo phosphorylation assay, subcellular fractionation, co-immunoprecipitation","journal":"Molecular biology reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro kinase assay plus in vivo phosphorylation and Co-IP, single lab with multiple orthogonal methods","pmids":["19851886"],"is_preprint":false},{"year":2012,"finding":"TNFAIP1 interacts with KCTD10, confirmed by yeast two-hybrid, GST pull-down, co-immunoprecipitation, and co-localization. TNFAIP1 overexpression promotes ubiquitin-mediated proteasomal degradation of KCTD10 (reversed by MG132), and both TNFAIP1 and KCTD10 inhibit NF-κB and AP-1 transcriptional activity.","method":"Yeast two-hybrid, GST pull-down, co-immunoprecipitation, co-localization, ubiquitin/proteasome assay, reporter assay","journal":"Molecular biology reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal binding methods plus functional ubiquitination assay, single lab","pmids":["22810651"],"is_preprint":false},{"year":2016,"finding":"TNFAIP1 (Bacurd2) acts as an interacting partner to Rnd2 and Rnd3 GTPases in vitro. In utero electroporation experiments in mice show that disruption of Tnfaip1/Bacurd2 expression impairs the long-term positioning of cortical neurons within the postnatal cerebral cortex and alters branching and dendritic spine properties of layer II/III projection neurons.","method":"In vitro interaction assay, in utero electroporation, immunofluorescence in postnatal mouse brain","journal":"Neural development","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro binding with in vivo loss-of-function phenotype in two orthogonal contexts, single lab","pmids":["26969432"],"is_preprint":false},{"year":2020,"finding":"The CRL3BTBD9 E3 ubiquitin ligase complex targets TNFAIP1 for polyubiquitination and proteasomal degradation: Cul3-ROC1 (CRL3) specifically interacts with TNFAIP1 and promotes its degradation, with BTBD9 as the specific substrate adaptor. Downregulation of BTBD9 promotes lung cancer cell migration by upregulating TNFAIP1, and TNFAIP1 deletion abrogated this effect.","method":"Label-free quantitative proteomics, co-immunoprecipitation, ubiquitination assay, cell migration assay, genetic rescue experiment","journal":"Signal transduction and targeted therapy","confidence":"High","confidence_rationale":"Tier 2 / Moderate — proteomic identification plus reciprocal Co-IP, ubiquitination assay, and functional genetic rescue with multiple orthogonal methods in single lab","pmids":["32327643"],"is_preprint":false},{"year":2021,"finding":"TNFAIP1 coordinates with Cullin3 to mediate RhoB degradation through the ubiquitin proteasome system in hepatocellular carcinoma cells. Downregulation of TNFAIP1 induces expression of pro-inflammatory cytokines IL-6 and IL-8 in TNFα-stimulated cells through p38/JNK MAPK pathway via blocking RhoB degradation.","method":"Co-immunoprecipitation, ubiquitin assay, cytokine ELISA, siRNA knockdown, pathway inhibitor experiments","journal":"Frontiers in cell and developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with functional ubiquitination assay and signaling pathway readout, single lab","pmids":["33553178"],"is_preprint":false},{"year":2023,"finding":"TNFAIP1 acts as an E3 ubiquitin ligase that directly binds SNAP25 (via its N-terminal residues 1-96) and mediates K48-linked polyubiquitination of SNAP25 at K69, leading to proteasomal degradation of SNAP25. Neuron-specific knockdown of TNFAIP1 in mice ameliorated postoperative cognitive dysfunction, restored PINK1/Parkin mitophagy, and reduced caspase-3/GSDME pyroptosis; these effects were reversed by SNAP25 co-depletion.","method":"Co-immunoprecipitation, ubiquitination assay with K48-linkage-specific analysis, domain-mapping, AAV9-hSyn neuronal knockdown in mice, behavioral testing, western blot","journal":"Cell communication and signaling : CCS","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — direct ubiquitination assay with site identification, domain mapping, and in vivo genetic rescue with defined phenotypic readouts, single lab with multiple orthogonal methods","pmids":["38102610"],"is_preprint":false},{"year":2024,"finding":"HECTD2 promotes ubiquitin-mediated degradation of EHMT2, leading to upregulation of TNFAIP1 transcription (ChIP confirmed TNFAIP1 as a direct target of EHMT2). High TNFAIP1 expression promotes inflammatory response in renal cell carcinoma cells via the p38/JNK pathway; p38/JNK inhibitors attenuated the effect of TNFAIP1 overexpression.","method":"Co-immunoprecipitation, western blot, ChIP assay, qRT-PCR, ELISA, pathway inhibitor experiments","journal":"In vivo (Athens, Greece)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP validation of transcriptional regulation and Co-IP for protein interactions, single lab","pmids":["38688591"],"is_preprint":false},{"year":2023,"finding":"TNFAIP1 interacts with CK2β and promotes its degradation by ubiquitination (confirmed by co-immunoprecipitation and western blotting); miR-93 negatively regulates TNFAIP1 expression (dual-luciferase reporter), and miR-93 overexpression prevented DEHP-induced neurotoxicity by downregulating TNFAIP1 and then activating CK2/Akt/CREB pathway.","method":"Co-immunoprecipitation, western blot, ubiquitination assay, dual-luciferase reporter assay, miRNA overexpression in cells and mice","journal":"Food and chemical toxicology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with ubiquitination assay and functional pathway rescue, single lab","pmids":["37302538"],"is_preprint":false},{"year":2024,"finding":"Knockdown of TNFAIP1 in THP-1 macrophages enhanced lncRNA LEENE expression, promoted direct interaction of LEENE with FoxO1 protein (verified by RNA immunoprecipitation and RNA pull-down), stimulated FoxO1 proteasomal degradation, induced ABCA1 transcription, and suppressed lipid accumulation; in apoE-/- mice, TNFAIP1 knockdown upregulated ABCA1, improved lipid profiles, and attenuated atherosclerotic lesion area.","method":"Lentiviral knockdown, RNA immunoprecipitation, RNA pull-down, western blot, qRT-PCR, Oil Red O staining, HPLC, in vivo mouse model","journal":"Journal of physiology and biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNA-protein interaction confirmed by RIP and pull-down with functional in vitro and in vivo readouts, single lab","pmids":["38878215"],"is_preprint":false},{"year":2009,"finding":"The TNFAIP1 gene promoter contains a functional Sp1-binding site within the core promoter region; gel EMSA, site-directed mutagenesis, and ChIP confirmed Sp1 binding in vivo and in vitro, and Sp1 overexpression enhanced TNFAIP1 promoter activity.","method":"Deletion mutation analysis, gel electrophoretic mobility shift assay (EMSA), site-directed mutagenesis, chromatin immunoprecipitation (ChIP), reporter assay","journal":"Molecular biology reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (EMSA, mutagenesis, ChIP) confirming Sp1-dependent transcriptional regulation, single lab","pmids":["19593659"],"is_preprint":false},{"year":2014,"finding":"Estrogen receptor β (ERβ) binds to the Tnfaip1 promoter and upregulates Tnfaip1 expression in mouse hippocampal cells; ovariectomy increased Tnfaip1 expression in hippocampus, and primary hippocampal cell culture experiments with estrogen and ER antagonists confirmed regulation of Tnfaip1 levels by estrogen.","method":"Reporter assay, promoter binding site identification, in vivo ovariectomy model, primary hippocampal cell culture, immunostaining","journal":"International journal of molecular medicine","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — promoter binding site validation plus in vivo and in vitro functional experiments, single lab","pmids":["24737445"],"is_preprint":false},{"year":2023,"finding":"In zebrafish, CRISPR/Cas9-mediated knockout of tnfaip1 caused developmental delays, microcephaly and microphthalmia, and decreased expression of neuronal marker genes (tuba1b, neurod1, ccnd1), establishing a role for tnfaip1 in early embryonic neural development.","method":"CRISPR/Cas9 knockout in zebrafish, whole mount in situ hybridization, qRT-PCR, transcriptome sequencing","journal":"Genes","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean genetic knockout with defined developmental phenotype and transcriptomic characterization, single lab","pmids":["37239365"],"is_preprint":false},{"year":2026,"finding":"TNFAIP1 directly interacts with PXR (pregnane X receptor), confirmed by co-immunoprecipitation. TNFAIP1 positively regulates the PXR/CYP3A4 pathway; TNFAIP1 knockout decreased CYP3A4 expression and impaired PXR agonist (rifampicin)-induced CYP3A4 upregulation in HCC cells. In vivo, Tnfaip1 overexpression upregulated Pxr/Cyp3a11 and inhibited tumor growth.","method":"Co-immunoprecipitation, western blot, qRT-PCR, TNFAIP1 knockout, pharmacological PXR activation, in vivo tumor xenograft","journal":"American journal of cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP for direct interaction plus genetic and pharmacological rescue in vitro and in vivo, single lab","pmids":["42004057"],"is_preprint":false},{"year":2016,"finding":"TNFAIP1 knockdown inhibits growth and induces apoptosis in osteosarcoma cells, associated with downregulation of p65 NF-κB, PCNA, and MMP-2 and upregulation of caspase-3, placing TNFAIP1 upstream of NF-κB in osteosarcoma cell survival signaling.","method":"Lentiviral siRNA knockdown, MTT assay, Transwell invasion assay, flow cytometry, western blot for NF-κB/PCNA/MMP-2/caspase-3","journal":"Oncology reports","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, knockdown with pathway marker readout but no direct mechanistic linkage to NF-κB established","pmids":["24969828"],"is_preprint":false},{"year":2020,"finding":"TNFAIP1 overexpression inhibits levels of phosphorylated Akt and CREB and anti-apoptotic Bcl-2, contributing to Aβ-induced neurotoxicity; TNFAIP1 knockdown reversed these effects and reduced cleaved caspase-3, placing TNFAIP1 upstream of the Akt/CREB/Bcl-2 apoptotic axis.","method":"siRNA knockdown, overexpression, western blot for p-Akt, p-CREB, Bcl-2, cleaved caspase-3 in N2a cells","journal":"BMC neuroscience","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, single method (western blot) for pathway placement with no direct biochemical interaction demonstrated","pmids":["27430312"],"is_preprint":false},{"year":2020,"finding":"TNFAIP1 is upregulated in APP/PS1 mouse neurons; Aβ increases binding of TNFAIP1 to RhoB (co-immunoprecipitation), and RhoB knockdown attenuates TNFAIP1-induced apoptosis in SH-SY5Y cells, confirming that TNFAIP1-RhoB interaction mediates apoptosis in an AD model.","method":"Co-immunoprecipitation, siRNA knockdown, western blot, flow cytometry, APP/PS1 transgenic mice immunostaining","journal":"Journal of molecular neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP confirmation of interaction and functional genetic rescue, single lab with in vivo transgenic model","pmids":["33159672"],"is_preprint":false}],"current_model":"TNFAIP1 is a TNF-α-inducible BTB/POZ domain-containing protein that functions as an adaptor for Cullin3-based E3 ubiquitin ligase complexes (CRL3), mediating K48-linked polyubiquitination and proteasomal degradation of substrates including SNAP25, RhoB, and KCTD10; it is phosphorylated by CK2 (which promotes its nuclear localization and interaction with PCNA), interacts with RhoB to drive SAPK/JNK-mediated apoptosis, regulates the NF-κB and Akt/CREB signaling pathways in multiple cell types, controls cortical neuron positioning and dendritic maturation by interacting with Rnd GTPases, and is itself subject to degradation by the CRL3BTBD9 complex; its transcription is driven by Sp1 basally and by ERβ in hippocampal cells."},"narrative":{"mechanistic_narrative":"TNFAIP1 is a TNF-α-inducible protein that functions principally as a substrate adaptor for Cullin3-based RING E3 ubiquitin ligase complexes, directing K48-linked polyubiquitination and proteasomal degradation of target proteins [PMID:33553178, PMID:38102610]. Through this activity it coordinates with Cullin3 to degrade the small GTPase RhoB [PMID:33553178], directly binds SNAP25 via its N-terminal residues (1–96) and ubiquitinates it at K69 [PMID:38102610], and promotes ubiquitin-mediated turnover of KCTD10 and CK2β [PMID:22810651, PMID:37302538]. TNFAIP1 physically interacts with RhoB, and this interaction drives SAPK/JNK-mediated apoptosis; loss of TNFAIP1 stabilizes RhoB to induce pro-inflammatory IL-6/IL-8 production via p38/JNK MAPK signaling [PMID:19637314, PMID:33553178]. In the nervous system TNFAIP1 binds the Rnd2/Rnd3 GTPases and controls cortical neuron positioning and dendritic maturation, and its degradation of SNAP25 modulates PINK1/Parkin mitophagy and caspase-3/GSDME pyroptosis in neurons [PMID:26969432, PMID:38102610]; zebrafish knockout produces microcephaly, microphthalmia, and impaired neuronal gene expression [PMID:37239365]. TNFAIP1 is itself a substrate of the CRL3^BTBD9 complex, which targets it for degradation, linking TNFAIP1 levels to cancer cell migration [PMID:32327643]. Its transcription is driven basally by Sp1 and by ERβ in hippocampal cells, and it is negatively regulated by miR-93 [PMID:37302538, PMID:19593659, PMID:24737445]. An early, distinct line of evidence identified the secreted/GPI-linked protein B61 (encoded by this locus) as a ligand that activates the ECK receptor tyrosine kinase and mediates TNF-α-induced angiogenesis [PMID:8139691, PMID:7890684, PMID:7536959].","teleology":[{"year":1994,"claim":"Established the first molecular function for this locus by identifying its product B61 as an activating ligand for the ECK receptor tyrosine kinase, defining a receptor-ligand signaling axis.","evidence":"Receptor affinity chromatography, surface plasmon resonance, and cell-based ECK autophosphorylation assay","pmids":["8139691"],"confidence":"High","gaps":["Did not connect B61/ECK signaling to the later-described intracellular adaptor/ubiquitin functions","Did not address whether secreted ligand and intracellular protein are the same physiological species"]},{"year":1995,"claim":"Showed B61 exists in both soluble and GPI-anchored cell-surface forms that both activate ECK, and that TNF-α drives angiogenesis via autocrine/paracrine B61-ECK signaling, embedding the protein in inflammatory vascular biology.","evidence":"PI-PLC treatment, B61-Ig chimera in vivo angiogenesis assay, endothelial chemotaxis, and antibody neutralization","pmids":["7890684","7536959"],"confidence":"High","gaps":["Mechanism downstream of ECK activation not dissected","Relationship between this extracellular ligand role and intracellular functions unresolved"]},{"year":2009,"claim":"Reoriented understanding toward an intracellular role by identifying RhoB as a direct binding partner whose interaction with TNFAIP1 triggers SAPK/JNK-dependent apoptosis, and showed CK2 phosphorylates TNFAIP1 to control its nuclear localization and PCNA binding.","evidence":"Yeast two-hybrid, reciprocal Co-IP, in vitro binding, co-localization, siRNA rescue, in vitro/in vivo kinase assays, subcellular fractionation","pmids":["19637314","19851886"],"confidence":"High","gaps":["Whether RhoB binding reflected adaptor/degradation activity not yet recognized","Functional consequence of PCNA interaction not defined"]},{"year":2009,"claim":"Defined basal transcriptional control by establishing a functional Sp1-binding site in the core promoter.","evidence":"Promoter deletion, EMSA, site-directed mutagenesis, ChIP, and reporter assay","pmids":["19593659"],"confidence":"Medium","gaps":["Inducible/TNF-α-responsive elements not mapped","No link to upstream signaling that activates Sp1 at this promoter"]},{"year":2012,"claim":"Provided the first evidence that TNFAIP1 acts as an adaptor driving ubiquitin-mediated degradation, showing it binds KCTD10 and promotes its proteasomal turnover while both inhibit NF-κB and AP-1 activity.","evidence":"Yeast two-hybrid, GST pull-down, Co-IP, co-localization, MG132-reversible ubiquitination assay, reporter assays","pmids":["22810651"],"confidence":"Medium","gaps":["E3 ligase complex mediating KCTD10 degradation not identified at this stage","Direct mechanism linking degradation to NF-κB/AP-1 suppression not shown"]},{"year":2016,"claim":"Extended TNFAIP1 function to neurodevelopment by identifying it as a Rnd2/Rnd3 GTPase partner required for cortical neuron positioning and dendritic maturation.","evidence":"In vitro interaction assay and in utero electroporation with immunofluorescence in postnatal mouse cortex","pmids":["26969432"],"confidence":"Medium","gaps":["Whether neuronal phenotype reflects ubiquitin-adaptor activity on Rnd GTPases not established","Molecular substrate in this context not defined"]},{"year":2020,"claim":"Identified the upstream control of TNFAIP1 protein levels, showing the CRL3^BTBD9 complex polyubiquitinates and degrades TNFAIP1, linking BTBD9 loss to TNFAIP1-driven cancer cell migration.","evidence":"Label-free proteomics, reciprocal Co-IP, ubiquitination assay, migration assay, genetic rescue","pmids":["32327643"],"confidence":"High","gaps":["Signals controlling CRL3^BTBD9 targeting of TNFAIP1 unknown","Degron on TNFAIP1 not mapped"]},{"year":2021,"claim":"Demonstrated that TNFAIP1 coordinates with Cullin3 to degrade RhoB, and that loss of TNFAIP1 stabilizes RhoB to drive p38/JNK-dependent IL-6/IL-8 induction, recasting the earlier RhoB interaction as a CRL3 substrate relationship.","evidence":"Co-IP, ubiquitin assay, cytokine ELISA, siRNA knockdown, pathway inhibitors in HCC cells","pmids":["33553178"],"confidence":"Medium","gaps":["BTB/Cullin3 contact residues not mapped","Whether RhoB degradation occurs in non-cancer contexts not tested here"]},{"year":2023,"claim":"Provided the most direct biochemical definition of TNFAIP1 as a ubiquitin ligase adaptor by mapping its SNAP25-binding region (residues 1–96) and the K48 ubiquitination site (K69), linking SNAP25 degradation to neuronal mitophagy, pyroptosis, and postoperative cognitive dysfunction.","evidence":"Co-IP, K48-linkage-specific ubiquitination assay, domain mapping, AAV9-hSyn neuronal knockdown in mice, behavioral testing, SNAP25 co-depletion rescue","pmids":["38102610"],"confidence":"High","gaps":["Cullin3 dependence in the SNAP25 context not directly confirmed","Generality of K48/K69 mechanism to other substrates not tested"]},{"year":2023,"claim":"Added CK2β as a TNFAIP1 ubiquitination substrate and defined miR-93 as a negative upstream regulator, linking TNFAIP1 to CK2/Akt/CREB signaling in DEHP neurotoxicity.","evidence":"Co-IP, ubiquitination assay, dual-luciferase reporter, miRNA overexpression in cells and mice","pmids":["37302538"],"confidence":"Medium","gaps":["Reconciliation with earlier CK2β-as-kinase finding (idx 4) not addressed","E3 complex for CK2β degradation not specified"]},{"year":2023,"claim":"Established an organismal requirement for tnfaip1 in early neural development, showing knockout causes microcephaly, microphthalmia, and reduced neuronal marker expression.","evidence":"CRISPR/Cas9 knockout in zebrafish, in situ hybridization, qRT-PCR, transcriptome sequencing","pmids":["37239365"],"confidence":"Medium","gaps":["Substrate(s) responsible for the developmental phenotype not identified","Cell-autonomous vs non-autonomous requirement unresolved"]},{"year":2024,"claim":"Broadened TNFAIP1's regulatory reach beyond protein degradation by showing it suppresses lncRNA LEENE/FoxO1/ABCA1-mediated lipid efflux, and is itself controlled transcriptionally via an EHMT2/HECTD2 axis.","evidence":"RNA immunoprecipitation, RNA pull-down, lentiviral knockdown, Oil Red O, HPLC, apoE-/- mice (idx 12); Co-IP, ChIP, qRT-PCR (idx 10)","pmids":["38878215","38688591"],"confidence":"Medium","gaps":["Mechanism by which TNFAIP1 restrains LEENE expression unclear","Whether lipid and inflammation phenotypes share a common substrate not addressed"]},{"year":2026,"claim":"Identified PXR as a direct TNFAIP1 partner and showed TNFAIP1 positively regulates the PXR/CYP3A4 detoxification axis to inhibit HCC tumor growth.","evidence":"Co-IP, knockout, pharmacological PXR activation, qRT-PCR, in vivo xenograft","pmids":["42004057"],"confidence":"Medium","gaps":["Whether PXR is a degradation substrate or stabilized partner not resolved","Mechanistic basis of positive regulation unclear"]},{"year":null,"claim":"It remains unresolved how the secreted/GPI-anchored B61-ECK ligand function relates to the intracellular CRL3 adaptor function of the same gene product, and which Cullin3 complex configuration operates on each substrate.","evidence":"","pmids":[],"confidence":"Low","gaps":["No study reconciles the extracellular ligand and intracellular ubiquitin-adaptor roles","BTB/Cullin3 binding interface and substrate-degron rules not systematically mapped","Substrate repertoire across tissues incompletely defined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[5,8,9,11]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[9]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[7,8,9]},{"term_id":"GO:0048018","term_label":"receptor ligand activity","supporting_discovery_ids":[0,1,2]}],"localization":[{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[3]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[4]},{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[2]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[1]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[7,8,9]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[3,9,19]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[8,10]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[6,15]}],"complexes":["CRL3 (Cullin3-RING E3 ubiquitin ligase)"],"partners":["RHOB","SNAP25","KCTD10","CK2Β","RND2","RND3","BTBD9","PXR"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q13829","full_name":"BTB/POZ domain-containing adapter for CUL3-mediated RhoA degradation protein 2","aliases":["BTB/POZ domain-containing protein TNFAIP1","Protein B12","Tumor necrosis factor, alpha-induced protein 1, endothelial"],"length_aa":316,"mass_kda":36.2,"function":"Substrate-specific adapter of a BCR (BTB-CUL3-RBX1) E3 ubiquitin-protein ligase complex involved in regulation of cytoskeleton structure. The BCR(TNFAIP1) E3 ubiquitin ligase complex mediates the ubiquitination of RHOA, leading to its degradation by the proteasome, thereby regulating the actin cytoskeleton and cell migration. Its interaction with RHOB may regulate apoptosis. May enhance the PCNA-dependent DNA polymerase delta activity","subcellular_location":"Cytoplasm; Nucleus; Endosome","url":"https://www.uniprot.org/uniprotkb/Q13829/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TNFAIP1","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"COPE","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/TNFAIP1","total_profiled":1310},"omim":[{"mim_id":"608947","title":"POTASSIUM CHANNEL TETRAMERIZATION DOMAIN-CONTAINING PROTEIN 13; KCTD13","url":"https://www.omim.org/entry/608947"},{"mim_id":"191161","title":"TUMOR NECROSIS FACTOR-ALPHA-INDUCED PROTEIN 1; TNFAIP1","url":"https://www.omim.org/entry/191161"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoli","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TNFAIP1"},"hgnc":{"alias_symbol":["B61","B12","MGC2317","BTBD34"],"prev_symbol":["EDP1"]},"alphafold":{"accession":"Q13829","domains":[{"cath_id":"3.30.710.10","chopping":"26-126","consensus_level":"high","plddt":91.8285,"start":26,"end":126},{"cath_id":"3.40.30.10","chopping":"138-258","consensus_level":"high","plddt":92.3679,"start":138,"end":258}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13829","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q13829-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q13829-F1-predicted_aligned_error_v6.png","plddt_mean":80.94},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TNFAIP1","jax_strain_url":"https://www.jax.org/strain/search?query=TNFAIP1"},"sequence":{"accession":"Q13829","fasta_url":"https://rest.uniprot.org/uniprotkb/Q13829.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q13829/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13829"}},"corpus_meta":[{"pmid":"7536959","id":"PMC_7536959","title":"Role of B61, the ligand for the Eck receptor tyrosine kinase, in TNF-alpha-induced angiogenesis.","date":"1995","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/7536959","citation_count":340,"is_preprint":false},{"pmid":"8139691","id":"PMC_8139691","title":"B61 is a ligand for the ECK receptor protein-tyrosine kinase.","date":"1994","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/8139691","citation_count":224,"is_preprint":false},{"pmid":"37451291","id":"PMC_37451291","title":"Pembrolizumab plus lenvatinib as first-line therapy for advanced non-clear-cell renal cell carcinoma (KEYNOTE-B61): a single-arm, multicentre, phase 2 trial.","date":"2023","source":"The Lancet. Oncology","url":"https://pubmed.ncbi.nlm.nih.gov/37451291","citation_count":130,"is_preprint":false},{"pmid":"7780963","id":"PMC_7780963","title":"Protein B61 as a new growth factor: expression of B61 and up-regulation of its receptor epithelial cell kinase during melanoma progression.","date":"1995","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/7780963","citation_count":104,"is_preprint":false},{"pmid":"10954555","id":"PMC_10954555","title":"Identification of dominant optimal HLA-B60- and HLA-B61-restricted cytotoxic T-lymphocyte (CTL) epitopes: rapid characterization of CTL responses by enzyme-linked immunospot assay.","date":"2000","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/10954555","citation_count":89,"is_preprint":false},{"pmid":"15113995","id":"PMC_15113995","title":"HLA-B60 and B61 are strongly associated with ankylosing spondylitis in HLA-B27-negative Taiwan Chinese patients.","date":"2004","source":"Rheumatology (Oxford, 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recombinant B61 induces autophosphorylation of ECK in intact cells, and B61 was purified by receptor affinity chromatography using the extracellular domain of ECK.\",\n      \"method\": \"Receptor affinity chromatography, surface plasmon resonance, cell-based autophosphorylation assay\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct biochemical purification and receptor activation reconstituted in cells, replicated across multiple subsequent studies\",\n      \"pmids\": [\"8139691\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"B61 can exist as a cell-surface glycosylphosphatidylinositol (GPI)-linked protein, in addition to its soluble secreted form, and the GPI-linked form is capable of activating the ECK receptor protein-tyrosine kinase.\",\n      \"method\": \"PI-PLC treatment, cell-based receptor activation assay, biochemical characterization\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct biochemical demonstration of GPI linkage with functional receptor activation, single lab with orthogonal methods\",\n      \"pmids\": [\"7890684\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"B61, acting through the ECK receptor tyrosine kinase, functions as an angiogenic factor in vivo and as a chemoattractant for endothelial cells in vitro; TNF-alpha induces angiogenesis through induction of B61, which then activates ECK in an autocrine/paracrine loop, and an anti-B61 antibody attenuated TNF-alpha-induced but not bFGF-induced angiogenesis.\",\n      \"method\": \"B61-immunoglobulin chimera in vivo angiogenesis assay, endothelial cell chemotaxis assay, antibody neutralization\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple functional assays in vitro and in vivo with antibody neutralization controls, widely replicated\",\n      \"pmids\": [\"7536959\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"TNFAIP1 directly interacts with RhoB; co-immunoprecipitation and in vitro binding assays confirmed the interaction. RFP-TNFAIP1 partially co-localizes with EGFP-RhoB in endosomes. TNFAIP1 elicits pro-apoptotic activity, and simultaneous expression of RhoB and TNFAIP1 dramatically increases apoptosis in HeLa cells via SAPK/JNK signaling; knockdown of RhoB by siRNA rescued cells from TNFAIP1-induced apoptosis.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, in vitro binding assay, fluorescence co-localization, siRNA knockdown, JNK inhibitor experiment\",\n      \"journal\": \"International journal of cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, in vitro binding, co-localization, and functional rescue with multiple orthogonal methods in single lab\",\n      \"pmids\": [\"19637314\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CK2 (casein kinase 2) phosphorylates TNFAIP1 both in vitro and in vivo; CK2β was identified as a TNFAIP1-interacting partner. Phosphorylation by CK2 facilitates nuclear distribution of TNFAIP1 and enhances its interaction with PCNA.\",\n      \"method\": \"Yeast two-hybrid screening, in vitro kinase assay, in vivo phosphorylation assay, subcellular fractionation, co-immunoprecipitation\",\n      \"journal\": \"Molecular biology reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro kinase assay plus in vivo phosphorylation and Co-IP, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"19851886\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"TNFAIP1 interacts with KCTD10, confirmed by yeast two-hybrid, GST pull-down, co-immunoprecipitation, and co-localization. TNFAIP1 overexpression promotes ubiquitin-mediated proteasomal degradation of KCTD10 (reversed by MG132), and both TNFAIP1 and KCTD10 inhibit NF-κB and AP-1 transcriptional activity.\",\n      \"method\": \"Yeast two-hybrid, GST pull-down, co-immunoprecipitation, co-localization, ubiquitin/proteasome assay, reporter assay\",\n      \"journal\": \"Molecular biology reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal binding methods plus functional ubiquitination assay, single lab\",\n      \"pmids\": [\"22810651\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TNFAIP1 (Bacurd2) acts as an interacting partner to Rnd2 and Rnd3 GTPases in vitro. In utero electroporation experiments in mice show that disruption of Tnfaip1/Bacurd2 expression impairs the long-term positioning of cortical neurons within the postnatal cerebral cortex and alters branching and dendritic spine properties of layer II/III projection neurons.\",\n      \"method\": \"In vitro interaction assay, in utero electroporation, immunofluorescence in postnatal mouse brain\",\n      \"journal\": \"Neural development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro binding with in vivo loss-of-function phenotype in two orthogonal contexts, single lab\",\n      \"pmids\": [\"26969432\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The CRL3BTBD9 E3 ubiquitin ligase complex targets TNFAIP1 for polyubiquitination and proteasomal degradation: Cul3-ROC1 (CRL3) specifically interacts with TNFAIP1 and promotes its degradation, with BTBD9 as the specific substrate adaptor. Downregulation of BTBD9 promotes lung cancer cell migration by upregulating TNFAIP1, and TNFAIP1 deletion abrogated this effect.\",\n      \"method\": \"Label-free quantitative proteomics, co-immunoprecipitation, ubiquitination assay, cell migration assay, genetic rescue experiment\",\n      \"journal\": \"Signal transduction and targeted therapy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proteomic identification plus reciprocal Co-IP, ubiquitination assay, and functional genetic rescue with multiple orthogonal methods in single lab\",\n      \"pmids\": [\"32327643\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TNFAIP1 coordinates with Cullin3 to mediate RhoB degradation through the ubiquitin proteasome system in hepatocellular carcinoma cells. Downregulation of TNFAIP1 induces expression of pro-inflammatory cytokines IL-6 and IL-8 in TNFα-stimulated cells through p38/JNK MAPK pathway via blocking RhoB degradation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitin assay, cytokine ELISA, siRNA knockdown, pathway inhibitor experiments\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with functional ubiquitination assay and signaling pathway readout, single lab\",\n      \"pmids\": [\"33553178\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TNFAIP1 acts as an E3 ubiquitin ligase that directly binds SNAP25 (via its N-terminal residues 1-96) and mediates K48-linked polyubiquitination of SNAP25 at K69, leading to proteasomal degradation of SNAP25. Neuron-specific knockdown of TNFAIP1 in mice ameliorated postoperative cognitive dysfunction, restored PINK1/Parkin mitophagy, and reduced caspase-3/GSDME pyroptosis; these effects were reversed by SNAP25 co-depletion.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay with K48-linkage-specific analysis, domain-mapping, AAV9-hSyn neuronal knockdown in mice, behavioral testing, western blot\",\n      \"journal\": \"Cell communication and signaling : CCS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — direct ubiquitination assay with site identification, domain mapping, and in vivo genetic rescue with defined phenotypic readouts, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"38102610\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"HECTD2 promotes ubiquitin-mediated degradation of EHMT2, leading to upregulation of TNFAIP1 transcription (ChIP confirmed TNFAIP1 as a direct target of EHMT2). High TNFAIP1 expression promotes inflammatory response in renal cell carcinoma cells via the p38/JNK pathway; p38/JNK inhibitors attenuated the effect of TNFAIP1 overexpression.\",\n      \"method\": \"Co-immunoprecipitation, western blot, ChIP assay, qRT-PCR, ELISA, pathway inhibitor experiments\",\n      \"journal\": \"In vivo (Athens, Greece)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP validation of transcriptional regulation and Co-IP for protein interactions, single lab\",\n      \"pmids\": [\"38688591\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TNFAIP1 interacts with CK2β and promotes its degradation by ubiquitination (confirmed by co-immunoprecipitation and western blotting); miR-93 negatively regulates TNFAIP1 expression (dual-luciferase reporter), and miR-93 overexpression prevented DEHP-induced neurotoxicity by downregulating TNFAIP1 and then activating CK2/Akt/CREB pathway.\",\n      \"method\": \"Co-immunoprecipitation, western blot, ubiquitination assay, dual-luciferase reporter assay, miRNA overexpression in cells and mice\",\n      \"journal\": \"Food and chemical toxicology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with ubiquitination assay and functional pathway rescue, single lab\",\n      \"pmids\": [\"37302538\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Knockdown of TNFAIP1 in THP-1 macrophages enhanced lncRNA LEENE expression, promoted direct interaction of LEENE with FoxO1 protein (verified by RNA immunoprecipitation and RNA pull-down), stimulated FoxO1 proteasomal degradation, induced ABCA1 transcription, and suppressed lipid accumulation; in apoE-/- mice, TNFAIP1 knockdown upregulated ABCA1, improved lipid profiles, and attenuated atherosclerotic lesion area.\",\n      \"method\": \"Lentiviral knockdown, RNA immunoprecipitation, RNA pull-down, western blot, qRT-PCR, Oil Red O staining, HPLC, in vivo mouse model\",\n      \"journal\": \"Journal of physiology and biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNA-protein interaction confirmed by RIP and pull-down with functional in vitro and in vivo readouts, single lab\",\n      \"pmids\": [\"38878215\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The TNFAIP1 gene promoter contains a functional Sp1-binding site within the core promoter region; gel EMSA, site-directed mutagenesis, and ChIP confirmed Sp1 binding in vivo and in vitro, and Sp1 overexpression enhanced TNFAIP1 promoter activity.\",\n      \"method\": \"Deletion mutation analysis, gel electrophoretic mobility shift assay (EMSA), site-directed mutagenesis, chromatin immunoprecipitation (ChIP), reporter assay\",\n      \"journal\": \"Molecular biology reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (EMSA, mutagenesis, ChIP) confirming Sp1-dependent transcriptional regulation, single lab\",\n      \"pmids\": [\"19593659\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Estrogen receptor β (ERβ) binds to the Tnfaip1 promoter and upregulates Tnfaip1 expression in mouse hippocampal cells; ovariectomy increased Tnfaip1 expression in hippocampus, and primary hippocampal cell culture experiments with estrogen and ER antagonists confirmed regulation of Tnfaip1 levels by estrogen.\",\n      \"method\": \"Reporter assay, promoter binding site identification, in vivo ovariectomy model, primary hippocampal cell culture, immunostaining\",\n      \"journal\": \"International journal of molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — promoter binding site validation plus in vivo and in vitro functional experiments, single lab\",\n      \"pmids\": [\"24737445\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In zebrafish, CRISPR/Cas9-mediated knockout of tnfaip1 caused developmental delays, microcephaly and microphthalmia, and decreased expression of neuronal marker genes (tuba1b, neurod1, ccnd1), establishing a role for tnfaip1 in early embryonic neural development.\",\n      \"method\": \"CRISPR/Cas9 knockout in zebrafish, whole mount in situ hybridization, qRT-PCR, transcriptome sequencing\",\n      \"journal\": \"Genes\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean genetic knockout with defined developmental phenotype and transcriptomic characterization, single lab\",\n      \"pmids\": [\"37239365\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"TNFAIP1 directly interacts with PXR (pregnane X receptor), confirmed by co-immunoprecipitation. TNFAIP1 positively regulates the PXR/CYP3A4 pathway; TNFAIP1 knockout decreased CYP3A4 expression and impaired PXR agonist (rifampicin)-induced CYP3A4 upregulation in HCC cells. In vivo, Tnfaip1 overexpression upregulated Pxr/Cyp3a11 and inhibited tumor growth.\",\n      \"method\": \"Co-immunoprecipitation, western blot, qRT-PCR, TNFAIP1 knockout, pharmacological PXR activation, in vivo tumor xenograft\",\n      \"journal\": \"American journal of cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP for direct interaction plus genetic and pharmacological rescue in vitro and in vivo, single lab\",\n      \"pmids\": [\"42004057\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TNFAIP1 knockdown inhibits growth and induces apoptosis in osteosarcoma cells, associated with downregulation of p65 NF-κB, PCNA, and MMP-2 and upregulation of caspase-3, placing TNFAIP1 upstream of NF-κB in osteosarcoma cell survival signaling.\",\n      \"method\": \"Lentiviral siRNA knockdown, MTT assay, Transwell invasion assay, flow cytometry, western blot for NF-κB/PCNA/MMP-2/caspase-3\",\n      \"journal\": \"Oncology reports\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, knockdown with pathway marker readout but no direct mechanistic linkage to NF-κB established\",\n      \"pmids\": [\"24969828\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TNFAIP1 overexpression inhibits levels of phosphorylated Akt and CREB and anti-apoptotic Bcl-2, contributing to Aβ-induced neurotoxicity; TNFAIP1 knockdown reversed these effects and reduced cleaved caspase-3, placing TNFAIP1 upstream of the Akt/CREB/Bcl-2 apoptotic axis.\",\n      \"method\": \"siRNA knockdown, overexpression, western blot for p-Akt, p-CREB, Bcl-2, cleaved caspase-3 in N2a cells\",\n      \"journal\": \"BMC neuroscience\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single method (western blot) for pathway placement with no direct biochemical interaction demonstrated\",\n      \"pmids\": [\"27430312\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TNFAIP1 is upregulated in APP/PS1 mouse neurons; Aβ increases binding of TNFAIP1 to RhoB (co-immunoprecipitation), and RhoB knockdown attenuates TNFAIP1-induced apoptosis in SH-SY5Y cells, confirming that TNFAIP1-RhoB interaction mediates apoptosis in an AD model.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, western blot, flow cytometry, APP/PS1 transgenic mice immunostaining\",\n      \"journal\": \"Journal of molecular neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP confirmation of interaction and functional genetic rescue, single lab with in vivo transgenic model\",\n      \"pmids\": [\"33159672\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TNFAIP1 is a TNF-α-inducible BTB/POZ domain-containing protein that functions as an adaptor for Cullin3-based E3 ubiquitin ligase complexes (CRL3), mediating K48-linked polyubiquitination and proteasomal degradation of substrates including SNAP25, RhoB, and KCTD10; it is phosphorylated by CK2 (which promotes its nuclear localization and interaction with PCNA), interacts with RhoB to drive SAPK/JNK-mediated apoptosis, regulates the NF-κB and Akt/CREB signaling pathways in multiple cell types, controls cortical neuron positioning and dendritic maturation by interacting with Rnd GTPases, and is itself subject to degradation by the CRL3BTBD9 complex; its transcription is driven by Sp1 basally and by ERβ in hippocampal cells.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TNFAIP1 is a TNF-α-inducible protein that functions principally as a substrate adaptor for Cullin3-based RING E3 ubiquitin ligase complexes, directing K48-linked polyubiquitination and proteasomal degradation of target proteins [#8, #9]. Through this activity it coordinates with Cullin3 to degrade the small GTPase RhoB [#8], directly binds SNAP25 via its N-terminal residues (1–96) and ubiquitinates it at K69 [#9], and promotes ubiquitin-mediated turnover of KCTD10 and CK2β [#5, #11]. TNFAIP1 physically interacts with RhoB, and this interaction drives SAPK/JNK-mediated apoptosis; loss of TNFAIP1 stabilizes RhoB to induce pro-inflammatory IL-6/IL-8 production via p38/JNK MAPK signaling [#3, #8]. In the nervous system TNFAIP1 binds the Rnd2/Rnd3 GTPases and controls cortical neuron positioning and dendritic maturation, and its degradation of SNAP25 modulates PINK1/Parkin mitophagy and caspase-3/GSDME pyroptosis in neurons [#6, #9]; zebrafish knockout produces microcephaly, microphthalmia, and impaired neuronal gene expression [#15]. TNFAIP1 is itself a substrate of the CRL3^BTBD9 complex, which targets it for degradation, linking TNFAIP1 levels to cancer cell migration [#7]. Its transcription is driven basally by Sp1 and by ERβ in hippocampal cells, and it is negatively regulated by miR-93 [#11, #13, #14]. An early, distinct line of evidence identified the secreted/GPI-linked protein B61 (encoded by this locus) as a ligand that activates the ECK receptor tyrosine kinase and mediates TNF-α-induced angiogenesis [#0, #1, #2].\",\n  \"teleology\": [\n    {\n      \"year\": 1994,\n      \"claim\": \"Established the first molecular function for this locus by identifying its product B61 as an activating ligand for the ECK receptor tyrosine kinase, defining a receptor-ligand signaling axis.\",\n      \"evidence\": \"Receptor affinity chromatography, surface plasmon resonance, and cell-based ECK autophosphorylation assay\",\n      \"pmids\": [\"8139691\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not connect B61/ECK signaling to the later-described intracellular adaptor/ubiquitin functions\", \"Did not address whether secreted ligand and intracellular protein are the same physiological species\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"Showed B61 exists in both soluble and GPI-anchored cell-surface forms that both activate ECK, and that TNF-α drives angiogenesis via autocrine/paracrine B61-ECK signaling, embedding the protein in inflammatory vascular biology.\",\n      \"evidence\": \"PI-PLC treatment, B61-Ig chimera in vivo angiogenesis assay, endothelial chemotaxis, and antibody neutralization\",\n      \"pmids\": [\"7890684\", \"7536959\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism downstream of ECK activation not dissected\", \"Relationship between this extracellular ligand role and intracellular functions unresolved\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Reoriented understanding toward an intracellular role by identifying RhoB as a direct binding partner whose interaction with TNFAIP1 triggers SAPK/JNK-dependent apoptosis, and showed CK2 phosphorylates TNFAIP1 to control its nuclear localization and PCNA binding.\",\n      \"evidence\": \"Yeast two-hybrid, reciprocal Co-IP, in vitro binding, co-localization, siRNA rescue, in vitro/in vivo kinase assays, subcellular fractionation\",\n      \"pmids\": [\"19637314\", \"19851886\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether RhoB binding reflected adaptor/degradation activity not yet recognized\", \"Functional consequence of PCNA interaction not defined\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Defined basal transcriptional control by establishing a functional Sp1-binding site in the core promoter.\",\n      \"evidence\": \"Promoter deletion, EMSA, site-directed mutagenesis, ChIP, and reporter assay\",\n      \"pmids\": [\"19593659\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Inducible/TNF-α-responsive elements not mapped\", \"No link to upstream signaling that activates Sp1 at this promoter\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Provided the first evidence that TNFAIP1 acts as an adaptor driving ubiquitin-mediated degradation, showing it binds KCTD10 and promotes its proteasomal turnover while both inhibit NF-κB and AP-1 activity.\",\n      \"evidence\": \"Yeast two-hybrid, GST pull-down, Co-IP, co-localization, MG132-reversible ubiquitination assay, reporter assays\",\n      \"pmids\": [\"22810651\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"E3 ligase complex mediating KCTD10 degradation not identified at this stage\", \"Direct mechanism linking degradation to NF-κB/AP-1 suppression not shown\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Extended TNFAIP1 function to neurodevelopment by identifying it as a Rnd2/Rnd3 GTPase partner required for cortical neuron positioning and dendritic maturation.\",\n      \"evidence\": \"In vitro interaction assay and in utero electroporation with immunofluorescence in postnatal mouse cortex\",\n      \"pmids\": [\"26969432\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether neuronal phenotype reflects ubiquitin-adaptor activity on Rnd GTPases not established\", \"Molecular substrate in this context not defined\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identified the upstream control of TNFAIP1 protein levels, showing the CRL3^BTBD9 complex polyubiquitinates and degrades TNFAIP1, linking BTBD9 loss to TNFAIP1-driven cancer cell migration.\",\n      \"evidence\": \"Label-free proteomics, reciprocal Co-IP, ubiquitination assay, migration assay, genetic rescue\",\n      \"pmids\": [\"32327643\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Signals controlling CRL3^BTBD9 targeting of TNFAIP1 unknown\", \"Degron on TNFAIP1 not mapped\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Demonstrated that TNFAIP1 coordinates with Cullin3 to degrade RhoB, and that loss of TNFAIP1 stabilizes RhoB to drive p38/JNK-dependent IL-6/IL-8 induction, recasting the earlier RhoB interaction as a CRL3 substrate relationship.\",\n      \"evidence\": \"Co-IP, ubiquitin assay, cytokine ELISA, siRNA knockdown, pathway inhibitors in HCC cells\",\n      \"pmids\": [\"33553178\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"BTB/Cullin3 contact residues not mapped\", \"Whether RhoB degradation occurs in non-cancer contexts not tested here\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Provided the most direct biochemical definition of TNFAIP1 as a ubiquitin ligase adaptor by mapping its SNAP25-binding region (residues 1–96) and the K48 ubiquitination site (K69), linking SNAP25 degradation to neuronal mitophagy, pyroptosis, and postoperative cognitive dysfunction.\",\n      \"evidence\": \"Co-IP, K48-linkage-specific ubiquitination assay, domain mapping, AAV9-hSyn neuronal knockdown in mice, behavioral testing, SNAP25 co-depletion rescue\",\n      \"pmids\": [\"38102610\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cullin3 dependence in the SNAP25 context not directly confirmed\", \"Generality of K48/K69 mechanism to other substrates not tested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Added CK2β as a TNFAIP1 ubiquitination substrate and defined miR-93 as a negative upstream regulator, linking TNFAIP1 to CK2/Akt/CREB signaling in DEHP neurotoxicity.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, dual-luciferase reporter, miRNA overexpression in cells and mice\",\n      \"pmids\": [\"37302538\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Reconciliation with earlier CK2β-as-kinase finding (idx 4) not addressed\", \"E3 complex for CK2β degradation not specified\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Established an organismal requirement for tnfaip1 in early neural development, showing knockout causes microcephaly, microphthalmia, and reduced neuronal marker expression.\",\n      \"evidence\": \"CRISPR/Cas9 knockout in zebrafish, in situ hybridization, qRT-PCR, transcriptome sequencing\",\n      \"pmids\": [\"37239365\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Substrate(s) responsible for the developmental phenotype not identified\", \"Cell-autonomous vs non-autonomous requirement unresolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Broadened TNFAIP1's regulatory reach beyond protein degradation by showing it suppresses lncRNA LEENE/FoxO1/ABCA1-mediated lipid efflux, and is itself controlled transcriptionally via an EHMT2/HECTD2 axis.\",\n      \"evidence\": \"RNA immunoprecipitation, RNA pull-down, lentiviral knockdown, Oil Red O, HPLC, apoE-/- mice (idx 12); Co-IP, ChIP, qRT-PCR (idx 10)\",\n      \"pmids\": [\"38878215\", \"38688591\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which TNFAIP1 restrains LEENE expression unclear\", \"Whether lipid and inflammation phenotypes share a common substrate not addressed\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Identified PXR as a direct TNFAIP1 partner and showed TNFAIP1 positively regulates the PXR/CYP3A4 detoxification axis to inhibit HCC tumor growth.\",\n      \"evidence\": \"Co-IP, knockout, pharmacological PXR activation, qRT-PCR, in vivo xenograft\",\n      \"pmids\": [\"42004057\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether PXR is a degradation substrate or stabilized partner not resolved\", \"Mechanistic basis of positive regulation unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how the secreted/GPI-anchored B61-ECK ligand function relates to the intracellular CRL3 adaptor function of the same gene product, and which Cullin3 complex configuration operates on each substrate.\",\n      \"evidence\": null,\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No study reconciles the extracellular ligand and intracellular ubiquitin-adaptor roles\", \"BTB/Cullin3 binding interface and substrate-degron rules not systematically mapped\", \"Substrate repertoire across tissues incompletely defined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [5, 8, 9, 11]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [9]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [7, 8, 9]},\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [0, 1, 2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [7, 8, 9]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [3, 9, 19]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [8, 10]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [6, 15]}\n    ],\n    \"complexes\": [\n      \"CRL3 (Cullin3-RING E3 ubiquitin ligase)\"\n    ],\n    \"partners\": [\n      \"RhoB\",\n      \"SNAP25\",\n      \"KCTD10\",\n      \"CK2β\",\n      \"Rnd2\",\n      \"Rnd3\",\n      \"BTBD9\",\n      \"PXR\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}