{"gene":"SNX16","run_date":"2026-06-10T07:46:37","timeline":{"discoveries":[{"year":2003,"finding":"SNX16 associates with membranes via its PX domain, which binds phosphatidylinositol 3-phosphate (PI3P). The coiled-coil domain is required for localization to late endosomal structures (mutants lacking it are restricted to early endosomes) and for homo-oligomerization. Overexpression of the coiled-coil deletion mutant delays trafficking of internalized EGF from early endosomes to later compartments.","method":"Biochemical fractionation, cellular imaging, phospholipid-binding assay, dominant-negative mutant analysis, EGF trafficking assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (lipid-binding assay, domain deletion mutants, trafficking assay, homo-oligomerization assay) in a single focused study","pmids":["12813048"],"is_preprint":false},{"year":2011,"finding":"SNX16 localizes selectively to tubulo-cisternal elements of late endosomes (not to LBPA-positive vacuolar/multivesicular regions), and this localization depends on intact microtubules. SNX16 is involved in tubule formation, cholesterol transport, and trafficking of the tetraspanin CD81 at late endosomes.","method":"Fluorescence microscopy, microtubule disruption experiments, CD81 trafficking assay, cholesterol transport assay","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization experiments with functional readouts (cholesterol transport, CD81 trafficking), single lab","pmids":["21754999"],"is_preprint":false},{"year":2017,"finding":"SNX16 regulates recycling of E-cadherin back to the cell surface. Crystal structure of the PX-CC unit reveals a unique shear-shaped homodimer with a novel PI3P-binding pocket formed by both PX and CC domains. The PPII/α2 loop, normally implicated in membrane insertion in PX proteins, was found to mediate direct binding to E-cadherin cargo.","method":"Crystal structure determination, PI3P binding assay, E-cadherin recycling trafficking assay, mutagenesis","journal":"Structure (London, England : 1993)","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with functional validation (cargo binding, PI3P binding) and mutagenesis, single lab but multiple orthogonal methods","pmids":["28712807"],"is_preprint":false},{"year":2013,"finding":"SNX16 localizes to Rab5-positive endosomes at cell cortex adjacent to focal adhesions. This cortical distribution requires SNX23, intact microtubules, and PI3-kinase activity. Ectopic overexpression of SNX16 reduces migration and tumor formation of MCF-7 cells.","method":"Fluorescence microscopy, pharmacological inhibition (PI3-kinase inhibitor, microtubule drugs), SNX23 knockdown, cell migration assay, soft agar/tumor formation assay","journal":"Cell regeneration (London, England)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization experiments combined with functional knockdown/overexpression assays, multiple pathway perturbations, single lab","pmids":["25408875"],"is_preprint":false},{"year":2020,"finding":"SNX16 interacts with eEF1A2 and inhibits its ubiquitin-mediated proteasomal degradation, thereby stabilizing eEF1A2 and activating downstream c-Myc signaling in colorectal cancer cells.","method":"Co-immunoprecipitation, ubiquitination assay, proteasome inhibitor experiments, SNX16 knockdown/overexpression with c-Myc pathway readouts","journal":"Molecular oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ubiquitination assay and downstream signaling readouts, single lab","pmids":["31876369"],"is_preprint":false},{"year":2020,"finding":"miR-196a-5p binds the 3'-UTR of SNX16 mRNA to suppress its expression. SNX16 knockdown restores trophoblast cell viability, migration, invasion, and MMP-2/MMP-9 expression under hypoxia, placing SNX16 downstream of BHLHE40/miR-196a-5p in the inhibition of trophoblast migration.","method":"Luciferase reporter assay (miR-196a-5p binding to SNX16 3'-UTR), ChIP assay, SNX16 knockdown in hypoxic trophoblasts, cell migration/invasion assays","journal":"Molecular human reproduction","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — luciferase reporter for direct miRNA-mRNA interaction, knockdown with multiple functional readouts, single lab","pmids":["32579212"],"is_preprint":false},{"year":2022,"finding":"SNX16 amino acid residue R144 is responsible for its two-band expression phenotype and affects its cellular distribution. The R144A mutation alters subcellular localization in A549 cells and partially reduces the inhibitory effect of SNX16 on influenza A virus (IAV) replication, with SNX16 acting at an early stage of the IAV replication cycle.","method":"Site-directed mutagenesis (R144A), Western blot, fluorescence microscopy, viral replication assay","journal":"Viruses","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — mutagenesis combined with localization and functional (antiviral) assay, single lab, single study","pmids":["35458555"],"is_preprint":false},{"year":2024,"finding":"SNX16 knockdown reduces phospho-EGFR levels and dampens AKT signaling in hepatocellular carcinoma cells. EGFR suppression counters the proliferation, motility, and invasiveness induced by SNX16 overexpression, placing SNX16 upstream of EGFR-AKT signaling.","method":"SNX16 knockdown/overexpression, phospho-EGFR and AKT immunoblotting, EGFR inhibitor epistasis experiment","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — epistasis experiment with EGFR inhibitor plus knockdown/overexpression and signaling readouts, single lab","pmids":["38849490"],"is_preprint":false},{"year":2025,"finding":"SNX16 promotes EGFR transactivation in cardiomyocytes and is required for AngII- or EGF-induced EGFR recycling through endosomal trafficking. Cardiac-specific SNX16 deletion inhibits AngII-induced cardiac hypertrophy and cardiomyocyte enlargement in mice; effects of SNX16 overexpression are abolished by the EGFR inhibitor AZD9291.","method":"Cardiac-specific knockout mouse, cardiomyocyte overexpression/knockdown, EGFR recycling assay in endosomal fractions, EGFR pathway inhibitor (AZD9291) epistasis, phospho-EGFR/Src immunoblotting","journal":"Communications biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — in vivo cardiac-specific KO combined with EGFR recycling assay, pharmacological epistasis, and multiple orthogonal readouts in a single study","pmids":["41318855"],"is_preprint":false}],"current_model":"SNX16 is a PI3P-binding sorting nexin that homodimerizes via its coiled-coil domain and localizes to early and late endosomal tubulo-cisternal membranes, where it regulates cargo trafficking (including EGF receptor recycling, E-cadherin recycling, CD81 trafficking, and cholesterol transport); it stabilizes eEF1A2 against ubiquitin-mediated degradation to activate c-Myc signaling, promotes EGFR recycling and transactivation downstream of AngII to drive cardiac hypertrophy, and negatively modulates cell migration and influenza A virus replication in a manner dependent on residue R144."},"narrative":{"mechanistic_narrative":"SNX16 is a phosphatidylinositol 3-phosphate (PI3P)-binding sorting nexin that controls cargo trafficking through the endosomal system [PMID:12813048, PMID:28712807]. Its PX domain binds PI3P to associate with membranes, while its coiled-coil domain mediates homo-oligomerization and is required for localization to late endosomal structures; loss of the coiled-coil restricts SNX16 to early endosomes and delays trafficking of internalized EGF [PMID:12813048]. SNX16 occupies tubulo-cisternal elements of late endosomes in a microtubule-dependent manner and acts there in tubule formation, cholesterol transport, and trafficking of the tetraspanin CD81 [PMID:21754999]. A crystal structure of the PX-CC unit defines a shear-shaped homodimer with a PI3P-binding pocket formed jointly by the PX and CC domains, and reveals that the PPII/α2 loop directly engages E-cadherin cargo to drive its recycling to the cell surface [PMID:28712807]. Through this trafficking activity SNX16 governs EGFR signaling: it is required for AngII- and EGF-induced EGFR recycling and transactivation, and cardiac-specific deletion blocks AngII-induced cardiac hypertrophy [PMID:41318855], while in hepatocellular carcinoma cells it sustains phospho-EGFR and downstream AKT signaling [PMID:38849490]. Beyond receptor trafficking, SNX16 stabilizes eEF1A2 against ubiquitin-mediated proteasomal degradation to activate c-Myc signaling [PMID:31876369], and modulates cell migration and influenza A virus replication, the latter dependent on residue R144 [PMID:25408875, PMID:35458555].","teleology":[{"year":2003,"claim":"Established the molecular basis of SNX16 membrane targeting and identified it as an endosomal trafficking regulator, answering how a sorting nexin engages endosomal membranes and influences cargo movement.","evidence":"Phospholipid-binding assays, domain-deletion/dominant-negative mutants, oligomerization and EGF trafficking assays","pmids":["12813048"],"confidence":"High","gaps":["Did not identify specific cargo bound directly by SNX16","Did not resolve the structural basis of PI3P recognition or dimerization"]},{"year":2011,"claim":"Refined SNX16 localization to tubulo-cisternal late-endosomal subdomains and linked it to specific functional outputs, addressing where within the late endosome SNX16 acts and what it traffics.","evidence":"Fluorescence microscopy with microtubule disruption, CD81 trafficking and cholesterol transport assays","pmids":["21754999"],"confidence":"Medium","gaps":["Mechanism coupling SNX16 to microtubules not defined","Direct interaction with CD81 or cholesterol machinery not demonstrated"]},{"year":2013,"claim":"Connected SNX16 endosomal positioning to cell behavior, showing cortical Rab5-endosome localization near focal adhesions and a suppressive effect on migration and tumor formation.","evidence":"Microscopy, PI3-kinase/microtubule pharmacology, SNX23 knockdown, migration and soft-agar assays in MCF-7 cells","pmids":["25408875"],"confidence":"Medium","gaps":["Cargo mediating the anti-migratory effect not identified","Reconciliation with pro-tumorigenic roles in other cell types unresolved"]},{"year":2017,"claim":"Provided the structural mechanism of SNX16, revealing a shear-shaped homodimer with a composite PX-CC PI3P pocket and direct E-cadherin cargo binding via the PPII/α2 loop, answering how SNX16 simultaneously reads lipid and selects cargo.","evidence":"Crystal structure of PX-CC unit, PI3P binding assays, mutagenesis, E-cadherin recycling assay","pmids":["28712807"],"confidence":"High","gaps":["Structure of full-length protein on membranes not determined","Whether the same loop binds other cargoes unknown"]},{"year":2020,"claim":"Extended SNX16 function beyond membrane trafficking to protein stability control, showing it shields eEF1A2 from ubiquitin-mediated degradation to activate c-Myc signaling.","evidence":"Co-IP, ubiquitination and proteasome-inhibitor assays, knockdown/overexpression with c-Myc readouts in colorectal cancer cells","pmids":["31876369"],"confidence":"Medium","gaps":["Direct vs indirect protection of eEF1A2 not distinguished","Reciprocal validation and link to SNX16's endosomal role absent"]},{"year":2020,"claim":"Placed SNX16 within a regulatory circuit in trophoblasts, identifying it as a miR-196a-5p target whose suppression restores migration and invasion under hypoxia.","evidence":"Luciferase reporter for miR-196a-5p binding, ChIP, SNX16 knockdown with migration/invasion and MMP readouts","pmids":["32579212"],"confidence":"Medium","gaps":["Molecular mechanism by which SNX16 inhibits trophoblast migration not defined","Cell-type-specific direction of effect unexplained"]},{"year":2022,"claim":"Mapped a single residue (R144) controlling SNX16 expression isoforms and localization, and tied SNX16 to early-stage restriction of influenza A virus.","evidence":"Site-directed R144A mutagenesis, Western blot, microscopy, viral replication assay in A549 cells","pmids":["35458555"],"confidence":"Medium","gaps":["Step in the IAV replication cycle targeted not pinpointed","Mechanistic basis of the two-band phenotype unresolved"]},{"year":2024,"claim":"Positioned SNX16 upstream of EGFR-AKT signaling in cancer, showing knockdown reduces phospho-EGFR and EGFR inhibition reverses SNX16-driven proliferation and invasion.","evidence":"Knockdown/overexpression, phospho-EGFR/AKT immunoblotting, EGFR-inhibitor epistasis in hepatocellular carcinoma cells","pmids":["38849490"],"confidence":"Medium","gaps":["Whether the effect operates through SNX16-dependent EGFR recycling not directly tested here","Direct EGFR-SNX16 interaction not shown"]},{"year":2025,"claim":"Demonstrated in vivo that SNX16 drives EGFR recycling and transactivation to promote pathological cardiac hypertrophy, integrating its trafficking activity with a physiological disease phenotype.","evidence":"Cardiac-specific knockout mice, endosomal EGFR recycling assay, AZD9291 epistasis, phospho-EGFR/Src immunoblotting","pmids":["41318855"],"confidence":"High","gaps":["Direct molecular contact between SNX16 and the EGFR/recycling machinery not defined","Cargo selectivity determining which receptors SNX16 recycles unresolved"]},{"year":null,"claim":"How SNX16's single PI3P-binding endosomal trafficking activity is mechanistically reconciled with its context-dependent roles (anti- vs pro-migratory, eEF1A2 stabilization, antiviral restriction) remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying model linking endosomal cargo sorting to non-trafficking outputs","Direct partners for most functional roles not biochemically defined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[0,2]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[2]}],"localization":[{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[0,1,3]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[0,1,2,8]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[7,8]}],"complexes":[],"partners":["EEF1A2","CDH1","CD81","SNX23","EGFR"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P57768","full_name":"Sorting nexin-16","aliases":[],"length_aa":344,"mass_kda":39.2,"function":"May be involved in several stages of intracellular trafficking. Plays a role in protein transport from early to late endosomes. Plays a role in protein transport to the lysosome. Promotes degradation of EGFR after EGF signaling. Plays a role in intracellular transport of vesicular stomatitis virus nucleocapsids from the endosome to the cytoplasm","subcellular_location":"Early endosome membrane; Late endosome membrane; Cytoplasm; Lysosome","url":"https://www.uniprot.org/uniprotkb/P57768/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SNX16","classification":"Not Classified","n_dependent_lines":5,"n_total_lines":1208,"dependency_fraction":0.0041390728476821195},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/SNX16","total_profiled":1310},"omim":[{"mim_id":"614903","title":"SORTING NEXIN 16; SNX16","url":"https://www.omim.org/entry/614903"},{"mim_id":"607553","title":"EPIPLAKIN 1; EPPK1","url":"https://www.omim.org/entry/607553"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Vesicles","reliability":"Approved"},{"location":"Plasma membrane","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/SNX16"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"P57768","domains":[{"cath_id":"3.30.1520.10","chopping":"112-219","consensus_level":"high","plddt":91.4228,"start":112,"end":219}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P57768","model_url":"https://alphafold.ebi.ac.uk/files/AF-P57768-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P57768-F1-predicted_aligned_error_v6.png","plddt_mean":70.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SNX16","jax_strain_url":"https://www.jax.org/strain/search?query=SNX16"},"sequence":{"accession":"P57768","fasta_url":"https://rest.uniprot.org/uniprotkb/P57768.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P57768/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P57768"}},"corpus_meta":[{"pmid":"12813048","id":"PMC_12813048","title":"Evidence for a role of SNX16 in regulating traffic between the early and later endosomal compartments.","date":"2003","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12813048","citation_count":45,"is_preprint":false},{"pmid":"31876369","id":"PMC_31876369","title":"SNX16 activates c-Myc signaling by inhibiting ubiquitin-mediated proteasomal degradation of eEF1A2 in colorectal cancer development.","date":"2020","source":"Molecular oncology","url":"https://pubmed.ncbi.nlm.nih.gov/31876369","citation_count":31,"is_preprint":false},{"pmid":"21754999","id":"PMC_21754999","title":"Role of SNX16 in the dynamics of tubulo-cisternal membrane domains of late endosomes.","date":"2011","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/21754999","citation_count":26,"is_preprint":false},{"pmid":"28712807","id":"PMC_28712807","title":"SNX16 Regulates the Recycling of E-Cadherin through a Unique Mechanism of Coordinated Membrane and Cargo Binding.","date":"2017","source":"Structure (London, England : 1993)","url":"https://pubmed.ncbi.nlm.nih.gov/28712807","citation_count":23,"is_preprint":false},{"pmid":"35482720","id":"PMC_35482720","title":"Comprehensive analysis of GSEC/miR-101-3p/SNX16/PAPOLG axis in hepatocellular carcinoma.","date":"2022","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/35482720","citation_count":16,"is_preprint":false},{"pmid":"32579212","id":"PMC_32579212","title":"BHLHE40 plays a pathological role in pre-eclampsia through upregulating SNX16 by transcriptional inhibition of miR-196a-5p.","date":"2020","source":"Molecular human reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/32579212","citation_count":15,"is_preprint":false},{"pmid":"25408875","id":"PMC_25408875","title":"SNX16 negatively regulates the migration and tumorigenesis of MCF-7 cells.","date":"2013","source":"Cell regeneration (London, England)","url":"https://pubmed.ncbi.nlm.nih.gov/25408875","citation_count":12,"is_preprint":false},{"pmid":"38849490","id":"PMC_38849490","title":"SNX16 is required for hepatocellular carcinoma survival via modulating the EGFR-AKT signaling pathway.","date":"2024","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/38849490","citation_count":1,"is_preprint":false},{"pmid":"35458555","id":"PMC_35458555","title":"A Single Amino Acid Residue R144 of SNX16 Affects Its Ability to Inhibit the Replication of Influenza A Virus.","date":"2022","source":"Viruses","url":"https://pubmed.ncbi.nlm.nih.gov/35458555","citation_count":1,"is_preprint":false},{"pmid":"41318855","id":"PMC_41318855","title":"SNX16 aggravates AngII-induced cardiac hypertrophy in mice via EGFR transactivation.","date":"2025","source":"Communications biology","url":"https://pubmed.ncbi.nlm.nih.gov/41318855","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":6408,"output_tokens":2335,"usd":0.027124,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9405,"output_tokens":3179,"usd":0.06325,"stage2_stop_reason":"end_turn"},"total_usd":0.090374,"stage1_batch_id":"msgbatch_013LWZZT6MWaqB9qyTSeQBmU","stage2_batch_id":"msgbatch_0194ZzQaTGNpruoNJBbEMbt3","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2003,\n      \"finding\": \"SNX16 associates with membranes via its PX domain, which binds phosphatidylinositol 3-phosphate (PI3P). The coiled-coil domain is required for localization to late endosomal structures (mutants lacking it are restricted to early endosomes) and for homo-oligomerization. Overexpression of the coiled-coil deletion mutant delays trafficking of internalized EGF from early endosomes to later compartments.\",\n      \"method\": \"Biochemical fractionation, cellular imaging, phospholipid-binding assay, dominant-negative mutant analysis, EGF trafficking assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (lipid-binding assay, domain deletion mutants, trafficking assay, homo-oligomerization assay) in a single focused study\",\n      \"pmids\": [\"12813048\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"SNX16 localizes selectively to tubulo-cisternal elements of late endosomes (not to LBPA-positive vacuolar/multivesicular regions), and this localization depends on intact microtubules. SNX16 is involved in tubule formation, cholesterol transport, and trafficking of the tetraspanin CD81 at late endosomes.\",\n      \"method\": \"Fluorescence microscopy, microtubule disruption experiments, CD81 trafficking assay, cholesterol transport assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization experiments with functional readouts (cholesterol transport, CD81 trafficking), single lab\",\n      \"pmids\": [\"21754999\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"SNX16 regulates recycling of E-cadherin back to the cell surface. Crystal structure of the PX-CC unit reveals a unique shear-shaped homodimer with a novel PI3P-binding pocket formed by both PX and CC domains. The PPII/α2 loop, normally implicated in membrane insertion in PX proteins, was found to mediate direct binding to E-cadherin cargo.\",\n      \"method\": \"Crystal structure determination, PI3P binding assay, E-cadherin recycling trafficking assay, mutagenesis\",\n      \"journal\": \"Structure (London, England : 1993)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with functional validation (cargo binding, PI3P binding) and mutagenesis, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"28712807\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"SNX16 localizes to Rab5-positive endosomes at cell cortex adjacent to focal adhesions. This cortical distribution requires SNX23, intact microtubules, and PI3-kinase activity. Ectopic overexpression of SNX16 reduces migration and tumor formation of MCF-7 cells.\",\n      \"method\": \"Fluorescence microscopy, pharmacological inhibition (PI3-kinase inhibitor, microtubule drugs), SNX23 knockdown, cell migration assay, soft agar/tumor formation assay\",\n      \"journal\": \"Cell regeneration (London, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization experiments combined with functional knockdown/overexpression assays, multiple pathway perturbations, single lab\",\n      \"pmids\": [\"25408875\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SNX16 interacts with eEF1A2 and inhibits its ubiquitin-mediated proteasomal degradation, thereby stabilizing eEF1A2 and activating downstream c-Myc signaling in colorectal cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, proteasome inhibitor experiments, SNX16 knockdown/overexpression with c-Myc pathway readouts\",\n      \"journal\": \"Molecular oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ubiquitination assay and downstream signaling readouts, single lab\",\n      \"pmids\": [\"31876369\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"miR-196a-5p binds the 3'-UTR of SNX16 mRNA to suppress its expression. SNX16 knockdown restores trophoblast cell viability, migration, invasion, and MMP-2/MMP-9 expression under hypoxia, placing SNX16 downstream of BHLHE40/miR-196a-5p in the inhibition of trophoblast migration.\",\n      \"method\": \"Luciferase reporter assay (miR-196a-5p binding to SNX16 3'-UTR), ChIP assay, SNX16 knockdown in hypoxic trophoblasts, cell migration/invasion assays\",\n      \"journal\": \"Molecular human reproduction\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — luciferase reporter for direct miRNA-mRNA interaction, knockdown with multiple functional readouts, single lab\",\n      \"pmids\": [\"32579212\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SNX16 amino acid residue R144 is responsible for its two-band expression phenotype and affects its cellular distribution. The R144A mutation alters subcellular localization in A549 cells and partially reduces the inhibitory effect of SNX16 on influenza A virus (IAV) replication, with SNX16 acting at an early stage of the IAV replication cycle.\",\n      \"method\": \"Site-directed mutagenesis (R144A), Western blot, fluorescence microscopy, viral replication assay\",\n      \"journal\": \"Viruses\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — mutagenesis combined with localization and functional (antiviral) assay, single lab, single study\",\n      \"pmids\": [\"35458555\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"SNX16 knockdown reduces phospho-EGFR levels and dampens AKT signaling in hepatocellular carcinoma cells. EGFR suppression counters the proliferation, motility, and invasiveness induced by SNX16 overexpression, placing SNX16 upstream of EGFR-AKT signaling.\",\n      \"method\": \"SNX16 knockdown/overexpression, phospho-EGFR and AKT immunoblotting, EGFR inhibitor epistasis experiment\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — epistasis experiment with EGFR inhibitor plus knockdown/overexpression and signaling readouts, single lab\",\n      \"pmids\": [\"38849490\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SNX16 promotes EGFR transactivation in cardiomyocytes and is required for AngII- or EGF-induced EGFR recycling through endosomal trafficking. Cardiac-specific SNX16 deletion inhibits AngII-induced cardiac hypertrophy and cardiomyocyte enlargement in mice; effects of SNX16 overexpression are abolished by the EGFR inhibitor AZD9291.\",\n      \"method\": \"Cardiac-specific knockout mouse, cardiomyocyte overexpression/knockdown, EGFR recycling assay in endosomal fractions, EGFR pathway inhibitor (AZD9291) epistasis, phospho-EGFR/Src immunoblotting\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo cardiac-specific KO combined with EGFR recycling assay, pharmacological epistasis, and multiple orthogonal readouts in a single study\",\n      \"pmids\": [\"41318855\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SNX16 is a PI3P-binding sorting nexin that homodimerizes via its coiled-coil domain and localizes to early and late endosomal tubulo-cisternal membranes, where it regulates cargo trafficking (including EGF receptor recycling, E-cadherin recycling, CD81 trafficking, and cholesterol transport); it stabilizes eEF1A2 against ubiquitin-mediated degradation to activate c-Myc signaling, promotes EGFR recycling and transactivation downstream of AngII to drive cardiac hypertrophy, and negatively modulates cell migration and influenza A virus replication in a manner dependent on residue R144.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SNX16 is a phosphatidylinositol 3-phosphate (PI3P)-binding sorting nexin that controls cargo trafficking through the endosomal system [#0, #2]. Its PX domain binds PI3P to associate with membranes, while its coiled-coil domain mediates homo-oligomerization and is required for localization to late endosomal structures; loss of the coiled-coil restricts SNX16 to early endosomes and delays trafficking of internalized EGF [#0]. SNX16 occupies tubulo-cisternal elements of late endosomes in a microtubule-dependent manner and acts there in tubule formation, cholesterol transport, and trafficking of the tetraspanin CD81 [#1]. A crystal structure of the PX-CC unit defines a shear-shaped homodimer with a PI3P-binding pocket formed jointly by the PX and CC domains, and reveals that the PPII/\\u03b12 loop directly engages E-cadherin cargo to drive its recycling to the cell surface [#2]. Through this trafficking activity SNX16 governs EGFR signaling: it is required for AngII- and EGF-induced EGFR recycling and transactivation, and cardiac-specific deletion blocks AngII-induced cardiac hypertrophy [#8], while in hepatocellular carcinoma cells it sustains phospho-EGFR and downstream AKT signaling [#7]. Beyond receptor trafficking, SNX16 stabilizes eEF1A2 against ubiquitin-mediated proteasomal degradation to activate c-Myc signaling [#4], and modulates cell migration and influenza A virus replication, the latter dependent on residue R144 [#3, #6].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Established the molecular basis of SNX16 membrane targeting and identified it as an endosomal trafficking regulator, answering how a sorting nexin engages endosomal membranes and influences cargo movement.\",\n      \"evidence\": \"Phospholipid-binding assays, domain-deletion/dominant-negative mutants, oligomerization and EGF trafficking assays\",\n      \"pmids\": [\"12813048\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify specific cargo bound directly by SNX16\", \"Did not resolve the structural basis of PI3P recognition or dimerization\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Refined SNX16 localization to tubulo-cisternal late-endosomal subdomains and linked it to specific functional outputs, addressing where within the late endosome SNX16 acts and what it traffics.\",\n      \"evidence\": \"Fluorescence microscopy with microtubule disruption, CD81 trafficking and cholesterol transport assays\",\n      \"pmids\": [\"21754999\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism coupling SNX16 to microtubules not defined\", \"Direct interaction with CD81 or cholesterol machinery not demonstrated\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Connected SNX16 endosomal positioning to cell behavior, showing cortical Rab5-endosome localization near focal adhesions and a suppressive effect on migration and tumor formation.\",\n      \"evidence\": \"Microscopy, PI3-kinase/microtubule pharmacology, SNX23 knockdown, migration and soft-agar assays in MCF-7 cells\",\n      \"pmids\": [\"25408875\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cargo mediating the anti-migratory effect not identified\", \"Reconciliation with pro-tumorigenic roles in other cell types unresolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Provided the structural mechanism of SNX16, revealing a shear-shaped homodimer with a composite PX-CC PI3P pocket and direct E-cadherin cargo binding via the PPII/\\u03b12 loop, answering how SNX16 simultaneously reads lipid and selects cargo.\",\n      \"evidence\": \"Crystal structure of PX-CC unit, PI3P binding assays, mutagenesis, E-cadherin recycling assay\",\n      \"pmids\": [\"28712807\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of full-length protein on membranes not determined\", \"Whether the same loop binds other cargoes unknown\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Extended SNX16 function beyond membrane trafficking to protein stability control, showing it shields eEF1A2 from ubiquitin-mediated degradation to activate c-Myc signaling.\",\n      \"evidence\": \"Co-IP, ubiquitination and proteasome-inhibitor assays, knockdown/overexpression with c-Myc readouts in colorectal cancer cells\",\n      \"pmids\": [\"31876369\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs indirect protection of eEF1A2 not distinguished\", \"Reciprocal validation and link to SNX16's endosomal role absent\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Placed SNX16 within a regulatory circuit in trophoblasts, identifying it as a miR-196a-5p target whose suppression restores migration and invasion under hypoxia.\",\n      \"evidence\": \"Luciferase reporter for miR-196a-5p binding, ChIP, SNX16 knockdown with migration/invasion and MMP readouts\",\n      \"pmids\": [\"32579212\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular mechanism by which SNX16 inhibits trophoblast migration not defined\", \"Cell-type-specific direction of effect unexplained\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Mapped a single residue (R144) controlling SNX16 expression isoforms and localization, and tied SNX16 to early-stage restriction of influenza A virus.\",\n      \"evidence\": \"Site-directed R144A mutagenesis, Western blot, microscopy, viral replication assay in A549 cells\",\n      \"pmids\": [\"35458555\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Step in the IAV replication cycle targeted not pinpointed\", \"Mechanistic basis of the two-band phenotype unresolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Positioned SNX16 upstream of EGFR-AKT signaling in cancer, showing knockdown reduces phospho-EGFR and EGFR inhibition reverses SNX16-driven proliferation and invasion.\",\n      \"evidence\": \"Knockdown/overexpression, phospho-EGFR/AKT immunoblotting, EGFR-inhibitor epistasis in hepatocellular carcinoma cells\",\n      \"pmids\": [\"38849490\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether the effect operates through SNX16-dependent EGFR recycling not directly tested here\", \"Direct EGFR-SNX16 interaction not shown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrated in vivo that SNX16 drives EGFR recycling and transactivation to promote pathological cardiac hypertrophy, integrating its trafficking activity with a physiological disease phenotype.\",\n      \"evidence\": \"Cardiac-specific knockout mice, endosomal EGFR recycling assay, AZD9291 epistasis, phospho-EGFR/Src immunoblotting\",\n      \"pmids\": [\"41318855\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct molecular contact between SNX16 and the EGFR/recycling machinery not defined\", \"Cargo selectivity determining which receptors SNX16 recycles unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How SNX16's single PI3P-binding endosomal trafficking activity is mechanistically reconciled with its context-dependent roles (anti- vs pro-migratory, eEF1A2 stabilization, antiviral restriction) remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying model linking endosomal cargo sorting to non-trafficking outputs\", \"Direct partners for most functional roles not biochemically defined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [0, 2]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [0, 1, 3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [0, 1, 2, 8]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [7, 8]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"eEF1A2\", \"CDH1\", \"CD81\", \"SNX23\", \"EGFR\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}