{"gene":"INAVA","run_date":"2026-06-10T01:55:23","timeline":{"discoveries":[{"year":2018,"finding":"C1orf106/INAVA regulates adherens junction stability by controlling the degradation of cytohesin-1, a guanine nucleotide exchange factor. By limiting cytohesin-1-dependent ARF6 activation, C1orf106 stabilizes adherens junctions. The IBD risk variant increases C1orf106 ubiquitination and turnover, leading to impaired barrier function. C1orf106-/- mice exhibit defects in intestinal epithelial cell barrier.","method":"Knockout mouse model, degradation assays for cytohesin-1, ARF6 activation assays, ubiquitination assays","journal":"Science (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal mechanistic validation with KO mice, ARF6 activation assays, and ubiquitination assays; replicated across multiple methods in one rigorous study","pmids":["29420262"],"is_preprint":false},{"year":2017,"finding":"INAVA is required for optimal MAPK and NF-κB activation, cytokine secretion, and intracellular bacterial clearance upon PRR stimulation in primary human myeloid cells. INAVA recruits 14-3-3τ to form a signaling complex that amplifies downstream signals. INAVA also enhances bacterial clearance by regulating reactive oxygen species, reactive nitrogen species, and autophagy pathways.","method":"siRNA knockdown in primary human macrophages, Co-immunoprecipitation for 14-3-3τ recruitment, cytokine ELISA, MAPK/NF-κB pathway activation assays, bacterial clearance assays","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP for 14-3-3τ, loss-of-function in primary human cells, multiple orthogonal downstream readouts in single lab","pmids":["28436939"],"is_preprint":false},{"year":2018,"finding":"INAVA's DUF3338 domain (renamed CUPID) stably binds the cytohesin ARF-GEF ARNO to effect lateral membrane F-actin assembly underlying cell-cell junctions and barrier function. When bound to CUPID, ARNO affects F-actin dynamics independently of its canonical GEF activity. Upon IL-1β exposure, INAVA relocates to cytosolic puncta where CUPID amplifies TRAF6-dependent polyubiquitination and inflammatory signaling. ARNO binding to CUPID negatively regulates polyubiquitination and the inflammatory response.","method":"Domain mapping, Co-IP/pulldown of CUPID-ARNO interaction, F-actin assembly assays, GEF activity assays, polyubiquitination assays, live-cell imaging of INAVA relocalization, macrophage primary cell experiments","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (binding assays, GEF activity, ubiquitination, localization imaging) in one rigorous study; validated in both epithelial and primary macrophage contexts","pmids":["30355448"],"is_preprint":false},{"year":2018,"finding":"C1ORF106 directly interacts with cytohesins to maintain functional epithelial cell junctions. C1orf106-deficient mice are hypersensitive to TNF-α-induced increase in epithelial permeability.","method":"Direct interaction assay (C1ORF106 with cytohesins), C1orf106 KO mouse model, TNF-α-induced permeability assay","journal":"ImmunoHorizons","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse phenotype with direct interaction assay; single lab but two orthogonal methods","pmids":["31022698"],"is_preprint":false},{"year":2021,"finding":"IL-1β-induced INAVA cytosolic puncta are biomolecular condensates that rapidly assemble and physiologically resolve. The condensates contain ubiquitin and the E3 ligase βTrCP2, and their formation correlates with amplified ubiquitination, suggesting a role in cellular proteostasis regulation. Inhibitors of p38α and mTOR pathways enhanced resolution of condensates, while inhibitors of the Rho-ROCK pathway recruited INAVA to newly assembled intercellular junctions.","method":"Live-cell imaging of condensate dynamics, small-molecule screen, co-localization of βTrCP2 and ubiquitin with INAVA puncta, pharmacological pathway inhibition","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — live imaging with multiple small-molecule perturbations; condensate composition defined by co-localization; single lab","pmids":["34251416"],"is_preprint":false},{"year":2025,"finding":"C1ORF106 knockdown leads to impaired cortical actin belt dynamics, stress fiber dysregulation, increased cell constriction, impaired barrier permeability, cell polarity defects, and impaired cell migration. ROCK inhibition rescues the actin belt and cell polarity phenotypes in C1ORF106 KD cells, demonstrating that C1ORF106 regulates these phenotypes through a ROCK-dependent mechanism. Altered non-muscle myosin II phosphorylation (nmMYO2-P) localization was observed in C1ORF106 KD cells, associated with Vacuolar Apical Compartment (VAC) formation. The IBD-associated 333F variant in hiPSC-derived intestinal spheroids showed similar cell polarity impairments.","method":"siRNA knockdown in human colonic epithelial cells, hiPSC-derived intestinal spheroid cultures, immunofluorescence, western blots, permeability assays, ROCK inhibitor rescue experiments","journal":"bioRxiv : the preprint server for biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KD with pharmacological rescue (ROCK inhibitor), multiple orthogonal readouts, validated in patient-derived hiPSC model; single lab, preprint","pmids":["40161582"],"is_preprint":true},{"year":2018,"finding":"INAVA upregulation in papillary thyroid cancer cells promotes invasion, migration, and metastasis through elevated FGF1 expression, which in turn increases MMP9 expression.","method":"Gain- and loss-of-function assays, transcriptome resequencing, western blotting, in vitro and in vivo metastasis assays","journal":"Cell & bioscience","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, correlative pathway placement with gain/loss-of-function; mechanism is indirect (INAVA→FGF1→MMP9) without direct binding evidence","pmids":["29632659"],"is_preprint":false},{"year":2025,"finding":"INAVA protein (translated from EV-delivered mRNA) competitively binds HMGA2 and inhibits its interaction with vaccinia-related kinase 1 (VRK1), leading to reduced HMGA2 phosphorylation at Ser105. This reduced phosphorylation stabilizes HMGA2 by blocking TRIM21-mediated K48-linked ubiquitylation, ultimately enhancing STAT3 transcription to activate normal ovarian fibroblasts. A cell-permeable peptide disrupting the INAVA-HMGA2 interaction attenuated fibroblast activation.","method":"Co-immunoprecipitation (INAVA-HMGA2 and HMGA2-VRK1), phosphorylation site mapping, ubiquitination assays (K48-linked), STAT3 transcription assays, peptide competition assay, orthotopic xenograft mouse model","journal":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, defined phosphorylation site, ubiquitination assays, and peptide rescue; single lab but multiple orthogonal methods","pmids":["40265981"],"is_preprint":false}],"current_model":"INAVA (C1orf106) is a multifunctional scaffold protein that, at the lateral membrane, uses its CUPID (DUF3338) domain to bind the cytohesin ARF-GEF ARNO/cytohesin-1, suppressing ARF6 activation and stabilizing adherens junctions through ROCK-dependent cortical actin dynamics; upon inflammatory stimulation (IL-1β, PRR ligands), INAVA relocates to cytosolic biomolecular condensates containing βTrCP2 and ubiquitin where it amplifies TRAF6-dependent polyubiquitination and NF-κB/MAPK signaling by recruiting 14-3-3τ, while ARNO binding to CUPID negatively regulates this inflammatory output, and an IBD risk variant increases INAVA ubiquitination and turnover, impairing both barrier and immune functions."},"narrative":{"mechanistic_narrative":"INAVA (C1orf106) is a multifunctional scaffold protein that couples epithelial barrier integrity to innate inflammatory signaling, both functions implicated in inflammatory bowel disease risk [PMID:29420262, PMID:28436939]. At the lateral membrane it uses its CUPID (DUF3338) domain to stably bind the cytohesin ARF-GEF ARNO/cytohesin-1, limiting cytohesin-dependent ARF6 activation and driving lateral F-actin assembly that stabilizes adherens junctions and barrier function [PMID:29420262, PMID:30355448, PMID:31022698]. INAVA controls cortical actin belt dynamics through a ROCK-dependent mechanism, with its loss producing stress fiber dysregulation, polarity defects, and impaired migration and permeability [PMID:40161582]. Upon inflammatory stimulation (PRR ligands, IL-1β), INAVA relocates from junctions to cytosolic biomolecular condensates containing ubiquitin and the E3 ligase βTrCP2, where CUPID amplifies TRAF6-dependent polyubiquitination and downstream MAPK/NF-κB signaling, in part by recruiting 14-3-3τ, thereby enhancing cytokine secretion and bacterial clearance; ARNO binding to CUPID negatively regulates this inflammatory output [PMID:28436939, PMID:30355448, PMID:34251416]. An IBD risk variant increases INAVA ubiquitination and turnover, impairing both barrier and immune functions [PMID:29420262]. Beyond these intestinal/epithelial roles, INAVA has been linked in single-lab studies to thyroid cancer metastasis via FGF1/MMP9 [PMID:29632659] and to ovarian fibroblast activation via competitive binding of HMGA2 [PMID:40265981].","teleology":[{"year":2017,"claim":"Established that INAVA functions in innate immunity, defining its requirement for PRR-induced inflammatory signaling and its mechanism of signal amplification via a partner adaptor.","evidence":"siRNA knockdown in primary human macrophages with Co-IP, cytokine ELISA, and MAPK/NF-κB and bacterial clearance assays","pmids":["28436939"],"confidence":"High","gaps":["Did not define the structural basis of 14-3-3τ recruitment","Did not connect signaling role to epithelial barrier function"]},{"year":2018,"claim":"Identified the molecular basis of INAVA's barrier function, showing it stabilizes adherens junctions by limiting cytohesin-1/ARF6 activity, and linked the IBD risk variant to increased turnover.","evidence":"Knockout mouse model with cytohesin-1 degradation, ARF6 activation, and ubiquitination assays","pmids":["29420262"],"confidence":"High","gaps":["Did not resolve which domain mediates cytohesin binding","E3 ligase driving variant-enhanced ubiquitination not identified here"]},{"year":2018,"claim":"Mapped the CUPID (DUF3338) domain as the interaction module unifying INAVA's two roles, showing ARNO binding drives GEF-independent F-actin assembly at junctions while the same domain amplifies inflammatory polyubiquitination upon IL-1β.","evidence":"Domain mapping, CUPID-ARNO Co-IP/pulldown, GEF and F-actin assays, polyubiquitination assays, and live-cell relocalization imaging in epithelial and macrophage contexts","pmids":["30355448"],"confidence":"High","gaps":["Mechanism by which CUPID amplifies TRAF6-dependent ubiquitination not defined at atomic level","How IL-1β triggers junction-to-cytosol relocalization unresolved"]},{"year":2019,"claim":"Confirmed direct cytohesin binding and barrier relevance in vivo, showing C1orf106-deficient mice are hypersensitive to TNF-α-induced epithelial permeability.","evidence":"Direct interaction assay and TNF-α-induced permeability assay in C1orf106 KO mice","pmids":["31022698"],"confidence":"Medium","gaps":["Single lab with two orthogonal methods","Did not address inflammatory signaling arm"]},{"year":2021,"claim":"Characterized the IL-1β-induced cytosolic puncta as dynamic biomolecular condensates with defined composition, linking INAVA to regulated proteostasis and identifying pathways controlling condensate resolution and junctional recruitment.","evidence":"Live-cell imaging of condensate dynamics, small-molecule screen, and co-localization of βTrCP2 and ubiquitin with INAVA puncta","pmids":["34251416"],"confidence":"Medium","gaps":["Condensate composition defined by co-localization rather than reconstitution","Functional consequence of βTrCP2 within condensates not established"]},{"year":2025,"claim":"Defined the cytoskeletal effector pathway, demonstrating that INAVA regulates cortical actin belt dynamics, polarity, and barrier permeability through a ROCK-dependent mechanism, with the IBD-associated 333F variant recapitulating polarity defects in patient-derived spheroids.","evidence":"siRNA knockdown in colonic epithelial cells and hiPSC-derived intestinal spheroids with ROCK inhibitor rescue and immunofluorescence","pmids":["40161582"],"confidence":"Medium","gaps":["Preprint, single lab","How INAVA mechanistically engages the ROCK pathway not resolved"]},{"year":2018,"claim":"Proposed an oncogenic role outside the intestine, correlating INAVA upregulation with thyroid cancer invasion via an FGF1/MMP9 axis.","evidence":"Gain/loss-of-function assays, transcriptome resequencing, and in vitro/in vivo metastasis assays","pmids":["29632659"],"confidence":"Low","gaps":["Indirect mechanism without direct binding evidence","Not independently confirmed","Relationship to barrier/inflammatory functions unknown"]},{"year":2025,"claim":"Reported a distinct scaffold function in the tumor microenvironment, with INAVA competitively binding HMGA2 to block VRK1-mediated phosphorylation and stabilize HMGA2, activating fibroblasts via STAT3.","evidence":"Reciprocal Co-IP, phosphorylation site mapping, K48 ubiquitination assays, peptide competition, and orthotopic xenograft model","pmids":["40265981"],"confidence":"Medium","gaps":["Single lab","Relationship to canonical CUPID/cytohesin functions not addressed"]},{"year":null,"claim":"How INAVA's two opposing activities — junction stabilization versus inflammatory amplification — are switched and coordinated at the molecular level, and how the disparate cancer-associated functions relate to the core epithelial/immune mechanism, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of CUPID-ARNO or CUPID-TRAF6 engagement","Mechanism triggering junction-to-condensate relocalization undefined","Unified model spanning epithelial, immune, and cancer contexts lacking"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,2]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,2]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[2,5]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,2,3]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[2,4]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[1,2]},{"term_id":"R-HSA-1500931","term_label":"Cell-Cell communication","supporting_discovery_ids":[0,3]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,2]}],"complexes":[],"partners":["CYTH1","CYTH2","YWHAQ","TRAF6","BTRC","HMGA2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q3KP66","full_name":"Innate immunity activator protein","aliases":[],"length_aa":663,"mass_kda":72.9,"function":"Expressed in peripheral macrophages and intestinal myeloid-derived cells, is required for optimal PRR (pattern recognition receptor)-induced signaling, cytokine secretion, and bacterial clearance. Upon stimulation of a broad range of PRRs (pattern recognition receptor) such as NOD2 or TLR2, TLR3, TLR4, TLR5, TLR7 and TLR9, associates with YWHAQ/14-3-3T, which in turn leads to the recruitment and activation of MAP kinases and NF-kappa-B signaling complexes that amplifies PRR-induced downstream signals and cytokine secretion (PubMed:28436939). In the intestine, regulates adherens junction stability by regulating the degradation of CYTH1 and CYTH2, probably acting as substrate cofactor for SCF E3 ubiquitin-protein ligase complexes. Stabilizes adherens junctions by limiting CYTH1-dependent ARF6 activation (PubMed:29420262)","subcellular_location":"Nucleus; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q3KP66/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/INAVA","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/INAVA","total_profiled":1310},"omim":[{"mim_id":"618077","title":"INFLAMMATORY BOWEL DISEASE 29; IBD29","url":"https://www.omim.org/entry/618077"},{"mim_id":"618051","title":"INNATE IMMUNITY ACTIVATOR; INAVA","url":"https://www.omim.org/entry/618051"},{"mim_id":"266600","title":"INFLAMMATORY BOWEL DISEASE (CROHN DISEASE) 1; IBD1","url":"https://www.omim.org/entry/266600"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Nuclear bodies","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"esophagus","ntpm":41.7},{"tissue":"intestine","ntpm":52.2},{"tissue":"skin 1","ntpm":44.1}],"url":"https://www.proteinatlas.org/search/INAVA"},"hgnc":{"alias_symbol":["FLJ10901"],"prev_symbol":["C1orf106"]},"alphafold":{"accession":"Q3KP66","domains":[{"cath_id":"-","chopping":"112-181","consensus_level":"high","plddt":85.8934,"start":112,"end":181},{"cath_id":"1.10.287","chopping":"185-247","consensus_level":"high","plddt":93.6565,"start":185,"end":247}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q3KP66","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q3KP66-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q3KP66-F1-predicted_aligned_error_v6.png","plddt_mean":56.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=INAVA","jax_strain_url":"https://www.jax.org/strain/search?query=INAVA"},"sequence":{"accession":"Q3KP66","fasta_url":"https://rest.uniprot.org/uniprotkb/Q3KP66.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q3KP66/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q3KP66"}},"corpus_meta":[{"pmid":"29420262","id":"PMC_29420262","title":"C1orf106 is a colitis risk gene that regulates stability of epithelial adherens junctions.","date":"2018","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/29420262","citation_count":102,"is_preprint":false},{"pmid":"28436939","id":"PMC_28436939","title":"An inflammatory bowel disease-risk variant in INAVA decreases pattern recognition receptor-induced outcomes.","date":"2017","source":"The Journal of clinical investigation","url":"https://pubmed.ncbi.nlm.nih.gov/28436939","citation_count":32,"is_preprint":false},{"pmid":"30355448","id":"PMC_30355448","title":"INAVA-ARNO complexes bridge mucosal barrier function with inflammatory signaling.","date":"2018","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/30355448","citation_count":23,"is_preprint":false},{"pmid":"29632659","id":"PMC_29632659","title":"INAVA promotes aggressiveness of papillary thyroid cancer by upregulating MMP9 expression.","date":"2018","source":"Cell & bioscience","url":"https://pubmed.ncbi.nlm.nih.gov/29632659","citation_count":18,"is_preprint":false},{"pmid":"31376015","id":"PMC_31376015","title":"C1orf106, an innate immunity activator, is amplified in breast cancer and is required for basal-like/luminal progenitor fate decision.","date":"2019","source":"Science China. Life sciences","url":"https://pubmed.ncbi.nlm.nih.gov/31376015","citation_count":14,"is_preprint":false},{"pmid":"31022698","id":"PMC_31022698","title":"Inflammatory Bowel Disease Susceptibility Gene C1ORF106 Regulates Intestinal Epithelial Permeability.","date":"2018","source":"ImmunoHorizons","url":"https://pubmed.ncbi.nlm.nih.gov/31022698","citation_count":13,"is_preprint":false},{"pmid":"34251416","id":"PMC_34251416","title":"Small-molecule modulators of INAVA cytosolic condensate and cell-cell junction assemblies.","date":"2021","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/34251416","citation_count":10,"is_preprint":false},{"pmid":"33163192","id":"PMC_33163192","title":"Association of polymorphisms in C1orf106, IL1RN, and IL10 with post-induction infliximab trough level in Crohn's disease patients.","date":"2019","source":"Gastroenterology report","url":"https://pubmed.ncbi.nlm.nih.gov/33163192","citation_count":9,"is_preprint":false},{"pmid":"40265981","id":"PMC_40265981","title":"The INAVA mRNA in Extracellular Vesicles Activates Normal Ovarian Fibroblasts by Phosphorylation-Ubiquitylation Crosstalk of HMGA2.","date":"2025","source":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/40265981","citation_count":3,"is_preprint":false},{"pmid":"41126651","id":"PMC_41126651","title":"The Expression and Clinical Significance of C1orf106 in Low-Grade Serous Ovarian Cancer.","date":"2025","source":"The journal of obstetrics and gynaecology research","url":"https://pubmed.ncbi.nlm.nih.gov/41126651","citation_count":1,"is_preprint":false},{"pmid":"40161582","id":"PMC_40161582","title":"Inflammatory bowel disease risk gene C1ORF106 regulates actin dynamics in intestinal epithelial cells.","date":"2025","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/40161582","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7472,"output_tokens":2490,"usd":0.029883,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9726,"output_tokens":3185,"usd":0.064128,"stage2_stop_reason":"end_turn"},"total_usd":0.094011,"stage1_batch_id":"msgbatch_01DUPACZi7AAWwuZiUjseQF4","stage2_batch_id":"msgbatch_018yt7chE3f48PEFtDwtj6AE","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2018,\n      \"finding\": \"C1orf106/INAVA regulates adherens junction stability by controlling the degradation of cytohesin-1, a guanine nucleotide exchange factor. By limiting cytohesin-1-dependent ARF6 activation, C1orf106 stabilizes adherens junctions. The IBD risk variant increases C1orf106 ubiquitination and turnover, leading to impaired barrier function. C1orf106-/- mice exhibit defects in intestinal epithelial cell barrier.\",\n      \"method\": \"Knockout mouse model, degradation assays for cytohesin-1, ARF6 activation assays, ubiquitination assays\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal mechanistic validation with KO mice, ARF6 activation assays, and ubiquitination assays; replicated across multiple methods in one rigorous study\",\n      \"pmids\": [\"29420262\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"INAVA is required for optimal MAPK and NF-κB activation, cytokine secretion, and intracellular bacterial clearance upon PRR stimulation in primary human myeloid cells. INAVA recruits 14-3-3τ to form a signaling complex that amplifies downstream signals. INAVA also enhances bacterial clearance by regulating reactive oxygen species, reactive nitrogen species, and autophagy pathways.\",\n      \"method\": \"siRNA knockdown in primary human macrophages, Co-immunoprecipitation for 14-3-3τ recruitment, cytokine ELISA, MAPK/NF-κB pathway activation assays, bacterial clearance assays\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP for 14-3-3τ, loss-of-function in primary human cells, multiple orthogonal downstream readouts in single lab\",\n      \"pmids\": [\"28436939\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"INAVA's DUF3338 domain (renamed CUPID) stably binds the cytohesin ARF-GEF ARNO to effect lateral membrane F-actin assembly underlying cell-cell junctions and barrier function. When bound to CUPID, ARNO affects F-actin dynamics independently of its canonical GEF activity. Upon IL-1β exposure, INAVA relocates to cytosolic puncta where CUPID amplifies TRAF6-dependent polyubiquitination and inflammatory signaling. ARNO binding to CUPID negatively regulates polyubiquitination and the inflammatory response.\",\n      \"method\": \"Domain mapping, Co-IP/pulldown of CUPID-ARNO interaction, F-actin assembly assays, GEF activity assays, polyubiquitination assays, live-cell imaging of INAVA relocalization, macrophage primary cell experiments\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (binding assays, GEF activity, ubiquitination, localization imaging) in one rigorous study; validated in both epithelial and primary macrophage contexts\",\n      \"pmids\": [\"30355448\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"C1ORF106 directly interacts with cytohesins to maintain functional epithelial cell junctions. C1orf106-deficient mice are hypersensitive to TNF-α-induced increase in epithelial permeability.\",\n      \"method\": \"Direct interaction assay (C1ORF106 with cytohesins), C1orf106 KO mouse model, TNF-α-induced permeability assay\",\n      \"journal\": \"ImmunoHorizons\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse phenotype with direct interaction assay; single lab but two orthogonal methods\",\n      \"pmids\": [\"31022698\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"IL-1β-induced INAVA cytosolic puncta are biomolecular condensates that rapidly assemble and physiologically resolve. The condensates contain ubiquitin and the E3 ligase βTrCP2, and their formation correlates with amplified ubiquitination, suggesting a role in cellular proteostasis regulation. Inhibitors of p38α and mTOR pathways enhanced resolution of condensates, while inhibitors of the Rho-ROCK pathway recruited INAVA to newly assembled intercellular junctions.\",\n      \"method\": \"Live-cell imaging of condensate dynamics, small-molecule screen, co-localization of βTrCP2 and ubiquitin with INAVA puncta, pharmacological pathway inhibition\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live imaging with multiple small-molecule perturbations; condensate composition defined by co-localization; single lab\",\n      \"pmids\": [\"34251416\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"C1ORF106 knockdown leads to impaired cortical actin belt dynamics, stress fiber dysregulation, increased cell constriction, impaired barrier permeability, cell polarity defects, and impaired cell migration. ROCK inhibition rescues the actin belt and cell polarity phenotypes in C1ORF106 KD cells, demonstrating that C1ORF106 regulates these phenotypes through a ROCK-dependent mechanism. Altered non-muscle myosin II phosphorylation (nmMYO2-P) localization was observed in C1ORF106 KD cells, associated with Vacuolar Apical Compartment (VAC) formation. The IBD-associated 333F variant in hiPSC-derived intestinal spheroids showed similar cell polarity impairments.\",\n      \"method\": \"siRNA knockdown in human colonic epithelial cells, hiPSC-derived intestinal spheroid cultures, immunofluorescence, western blots, permeability assays, ROCK inhibitor rescue experiments\",\n      \"journal\": \"bioRxiv : the preprint server for biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KD with pharmacological rescue (ROCK inhibitor), multiple orthogonal readouts, validated in patient-derived hiPSC model; single lab, preprint\",\n      \"pmids\": [\"40161582\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"INAVA upregulation in papillary thyroid cancer cells promotes invasion, migration, and metastasis through elevated FGF1 expression, which in turn increases MMP9 expression.\",\n      \"method\": \"Gain- and loss-of-function assays, transcriptome resequencing, western blotting, in vitro and in vivo metastasis assays\",\n      \"journal\": \"Cell & bioscience\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, correlative pathway placement with gain/loss-of-function; mechanism is indirect (INAVA→FGF1→MMP9) without direct binding evidence\",\n      \"pmids\": [\"29632659\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"INAVA protein (translated from EV-delivered mRNA) competitively binds HMGA2 and inhibits its interaction with vaccinia-related kinase 1 (VRK1), leading to reduced HMGA2 phosphorylation at Ser105. This reduced phosphorylation stabilizes HMGA2 by blocking TRIM21-mediated K48-linked ubiquitylation, ultimately enhancing STAT3 transcription to activate normal ovarian fibroblasts. A cell-permeable peptide disrupting the INAVA-HMGA2 interaction attenuated fibroblast activation.\",\n      \"method\": \"Co-immunoprecipitation (INAVA-HMGA2 and HMGA2-VRK1), phosphorylation site mapping, ubiquitination assays (K48-linked), STAT3 transcription assays, peptide competition assay, orthotopic xenograft mouse model\",\n      \"journal\": \"Advanced science (Weinheim, Baden-Wurttemberg, Germany)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, defined phosphorylation site, ubiquitination assays, and peptide rescue; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"40265981\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"INAVA (C1orf106) is a multifunctional scaffold protein that, at the lateral membrane, uses its CUPID (DUF3338) domain to bind the cytohesin ARF-GEF ARNO/cytohesin-1, suppressing ARF6 activation and stabilizing adherens junctions through ROCK-dependent cortical actin dynamics; upon inflammatory stimulation (IL-1β, PRR ligands), INAVA relocates to cytosolic biomolecular condensates containing βTrCP2 and ubiquitin where it amplifies TRAF6-dependent polyubiquitination and NF-κB/MAPK signaling by recruiting 14-3-3τ, while ARNO binding to CUPID negatively regulates this inflammatory output, and an IBD risk variant increases INAVA ubiquitination and turnover, impairing both barrier and immune functions.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"INAVA (C1orf106) is a multifunctional scaffold protein that couples epithelial barrier integrity to innate inflammatory signaling, both functions implicated in inflammatory bowel disease risk [#0, #1]. At the lateral membrane it uses its CUPID (DUF3338) domain to stably bind the cytohesin ARF-GEF ARNO/cytohesin-1, limiting cytohesin-dependent ARF6 activation and driving lateral F-actin assembly that stabilizes adherens junctions and barrier function [#0, #2, #3]. INAVA controls cortical actin belt dynamics through a ROCK-dependent mechanism, with its loss producing stress fiber dysregulation, polarity defects, and impaired migration and permeability [#5]. Upon inflammatory stimulation (PRR ligands, IL-1\\u03b2), INAVA relocates from junctions to cytosolic biomolecular condensates containing ubiquitin and the E3 ligase \\u03b2TrCP2, where CUPID amplifies TRAF6-dependent polyubiquitination and downstream MAPK/NF-\\u03baB signaling, in part by recruiting 14-3-3\\u03c4, thereby enhancing cytokine secretion and bacterial clearance; ARNO binding to CUPID negatively regulates this inflammatory output [#1, #2, #4]. An IBD risk variant increases INAVA ubiquitination and turnover, impairing both barrier and immune functions [#0]. Beyond these intestinal/epithelial roles, INAVA has been linked in single-lab studies to thyroid cancer metastasis via FGF1/MMP9 [#6] and to ovarian fibroblast activation via competitive binding of HMGA2 [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 2017,\n      \"claim\": \"Established that INAVA functions in innate immunity, defining its requirement for PRR-induced inflammatory signaling and its mechanism of signal amplification via a partner adaptor.\",\n      \"evidence\": \"siRNA knockdown in primary human macrophages with Co-IP, cytokine ELISA, and MAPK/NF-\\u03baB and bacterial clearance assays\",\n      \"pmids\": [\"28436939\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the structural basis of 14-3-3\\u03c4 recruitment\", \"Did not connect signaling role to epithelial barrier function\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identified the molecular basis of INAVA's barrier function, showing it stabilizes adherens junctions by limiting cytohesin-1/ARF6 activity, and linked the IBD risk variant to increased turnover.\",\n      \"evidence\": \"Knockout mouse model with cytohesin-1 degradation, ARF6 activation, and ubiquitination assays\",\n      \"pmids\": [\"29420262\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve which domain mediates cytohesin binding\", \"E3 ligase driving variant-enhanced ubiquitination not identified here\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Mapped the CUPID (DUF3338) domain as the interaction module unifying INAVA's two roles, showing ARNO binding drives GEF-independent F-actin assembly at junctions while the same domain amplifies inflammatory polyubiquitination upon IL-1\\u03b2.\",\n      \"evidence\": \"Domain mapping, CUPID-ARNO Co-IP/pulldown, GEF and F-actin assays, polyubiquitination assays, and live-cell relocalization imaging in epithelial and macrophage contexts\",\n      \"pmids\": [\"30355448\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which CUPID amplifies TRAF6-dependent ubiquitination not defined at atomic level\", \"How IL-1\\u03b2 triggers junction-to-cytosol relocalization unresolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Confirmed direct cytohesin binding and barrier relevance in vivo, showing C1orf106-deficient mice are hypersensitive to TNF-\\u03b1-induced epithelial permeability.\",\n      \"evidence\": \"Direct interaction assay and TNF-\\u03b1-induced permeability assay in C1orf106 KO mice\",\n      \"pmids\": [\"31022698\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab with two orthogonal methods\", \"Did not address inflammatory signaling arm\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Characterized the IL-1\\u03b2-induced cytosolic puncta as dynamic biomolecular condensates with defined composition, linking INAVA to regulated proteostasis and identifying pathways controlling condensate resolution and junctional recruitment.\",\n      \"evidence\": \"Live-cell imaging of condensate dynamics, small-molecule screen, and co-localization of \\u03b2TrCP2 and ubiquitin with INAVA puncta\",\n      \"pmids\": [\"34251416\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Condensate composition defined by co-localization rather than reconstitution\", \"Functional consequence of \\u03b2TrCP2 within condensates not established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined the cytoskeletal effector pathway, demonstrating that INAVA regulates cortical actin belt dynamics, polarity, and barrier permeability through a ROCK-dependent mechanism, with the IBD-associated 333F variant recapitulating polarity defects in patient-derived spheroids.\",\n      \"evidence\": \"siRNA knockdown in colonic epithelial cells and hiPSC-derived intestinal spheroids with ROCK inhibitor rescue and immunofluorescence\",\n      \"pmids\": [\"40161582\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, single lab\", \"How INAVA mechanistically engages the ROCK pathway not resolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Proposed an oncogenic role outside the intestine, correlating INAVA upregulation with thyroid cancer invasion via an FGF1/MMP9 axis.\",\n      \"evidence\": \"Gain/loss-of-function assays, transcriptome resequencing, and in vitro/in vivo metastasis assays\",\n      \"pmids\": [\"29632659\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Indirect mechanism without direct binding evidence\", \"Not independently confirmed\", \"Relationship to barrier/inflammatory functions unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Reported a distinct scaffold function in the tumor microenvironment, with INAVA competitively binding HMGA2 to block VRK1-mediated phosphorylation and stabilize HMGA2, activating fibroblasts via STAT3.\",\n      \"evidence\": \"Reciprocal Co-IP, phosphorylation site mapping, K48 ubiquitination assays, peptide competition, and orthotopic xenograft model\",\n      \"pmids\": [\"40265981\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Relationship to canonical CUPID/cytohesin functions not addressed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How INAVA's two opposing activities — junction stabilization versus inflammatory amplification — are switched and coordinated at the molecular level, and how the disparate cancer-associated functions relate to the core epithelial/immune mechanism, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of CUPID-ARNO or CUPID-TRAF6 engagement\", \"Mechanism triggering junction-to-condensate relocalization undefined\", \"Unified model spanning epithelial, immune, and cancer contexts lacking\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 2]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [2, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 2, 3]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [2, 4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"R-HSA-1500931\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 2]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"CYTH1\", \"CYTH2\", \"YWHAQ\", \"TRAF6\", \"BTRC\", \"HMGA2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":6,"faith_pct":83.33333333333333}}