{"gene":"CNPY4","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":2005,"finding":"PRAT4B (CNPY4) associates with the hypoglycosylated, immature form of TLR4 but not with MD-2 or TLR2, and siRNA-mediated knockdown of PRAT4B decreases cell surface TLR4 expression on HEK293 cells, establishing PRAT4B as a regulator of TLR4 subcellular trafficking.","method":"Co-immunoprecipitation, siRNA knockdown, flow cytometry","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal association shown by co-IP with immature TLR4, siRNA knockdown with defined surface trafficking phenotype, single lab but two orthogonal methods","pmids":["16338228"],"is_preprint":false},{"year":2012,"finding":"PRAT4B (CNPY4) acts as a negative regulator of TLR1 cell surface trafficking, in contrast to PRAT4A which is a positive regulator; knockdown of PRAT4B rescues cell surface expression of the TLR1 I602S trafficking-deficient variant.","method":"siRNA knockdown, over-expression, flow cytometry, TLR1 deletion/point mutant analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic manipulation (KD and OE) with defined cell surface trafficking phenotype, single lab with multiple mutant constructs","pmids":["22447933"],"is_preprint":false},{"year":2022,"finding":"CNPY4, a Saposin-like protein, negatively regulates the Hedgehog (HH) signaling pathway by modulating levels of accessible membrane sterol lipids including cholesterol; Cnpy4 knockout embryos display digit number defects consistent with HH perturbation, and Cnpy4 knockdown hyperactivates HH signaling in vitro while elevating plasma membrane cholesterol levels.","method":"Cnpy4 knockout mouse model, siRNA knockdown, filipin staining/membrane cholesterol measurement, in vitro HH pathway reporter assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo knockout with defined developmental phenotype plus in vitro knockdown with pathway activation and membrane lipid measurement, multiple orthogonal methods in one study","pmids":["35504891"],"is_preprint":false},{"year":2018,"finding":"CNPY4 was identified as a novel interacting protein of LRIG1 in a yeast two-hybrid screen and confirmed in a BioPlex protein interaction dataset, placing CNPY4 in an LRIG1-centered protein interaction network that regulates growth factor receptors.","method":"Yeast two-hybrid screen, BioPlex protein interaction database mining","journal":"The Journal of biological chemistry","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single yeast two-hybrid identification with database corroboration but no biochemical validation of the specific CNPY4-LRIG1 interaction","pmids":["29317492"],"is_preprint":false},{"year":2025,"finding":"CNPY4 is a sphingolipid-binding protein (Saposin-like) that interacts with ceramide and sphingomyelin in vitro and with ceramide in cells; CNPY4 knockdown elevates sphingomyelin levels at the plasma membrane, disrupts ceramide localization and abundance, modulates neutral sphingomyelinase activity, and impacts over 150 cellular lipids, establishing CNPY4 as a sphingolipid chaperone that regulates sphingolipid homeostasis and, consequently, cholesterol distribution and cellular signaling.","method":"Lipid-binding assays in vitro, siRNA knockdown, lipidomics, filipin staining, sphingomyelinase activity assay, cell-based ceramide localization","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (in vitro lipid binding, cell-based knockdown, lipidomics, enzyme activity assay) in a single lab preprint study","pmids":["41279395"],"is_preprint":true},{"year":2025,"finding":"CNPY4 is a substrate of the secretory kinase FAM20C; FAM20C-dependent phosphorylation of CNPY4 contributes to proinflammatory adipocyte signaling in obesity.","method":"Phosphoproteomic analysis of FAM20C gain- and loss-of-function adipocytes","journal":"The Journal of clinical investigation","confidence":"Low","confidence_rationale":"Tier 3 / Weak — phosphoproteomic identification of CNPY4 as FAM20C substrate with functional attribution, single lab, no direct mutagenesis or reconstitution of specific phosphorylation site","pmids":["41148235"],"is_preprint":false}],"current_model":"CNPY4 (PRAT4B) is a Saposin-like ER-resident chaperone that regulates subcellular trafficking of TLR4 and TLR1 (acting as a negative regulator of TLR1 surface expression), negatively modulates Hedgehog signaling by controlling accessible membrane sterol lipid levels, and functions as a sphingolipid chaperone that directly binds ceramide and sphingomyelin to regulate sphingolipid homeostasis and plasma membrane composition; additionally, CNPY4 is phosphorylated by the secretory kinase FAM20C, linking it to proinflammatory adipocyte signaling."},"narrative":{"mechanistic_narrative":"CNPY4 (PRAT4B) is a Saposin-like protein that regulates membrane composition and the trafficking of signaling receptors [PMID:35504891]. As an ER-associated chaperone it controls the subcellular trafficking of Toll-like receptors: it associates selectively with the immature, hypoglycosylated form of TLR4 and is required for normal cell-surface TLR4 expression [PMID:16338228], while acting as a negative regulator of TLR1 surface trafficking, since its knockdown rescues surface expression of a trafficking-deficient TLR1 variant [PMID:22447933]. CNPY4 also acts as a negative regulator of Hedgehog signaling by limiting levels of accessible membrane sterol lipids including cholesterol; its loss hyperactivates Hedgehog signaling and elevates plasma membrane cholesterol, and Cnpy4 knockout embryos show digit-number defects consistent with Hedgehog perturbation [PMID:35504891]. Mechanistically, this lipid control reflects a direct sphingolipid-chaperone activity: CNPY4 binds ceramide and sphingomyelin, and its depletion raises plasma membrane sphingomyelin, disrupts ceramide localization, alters neutral sphingomyelinase activity, and broadly remodels the cellular lipidome, thereby coupling sphingolipid homeostasis to cholesterol distribution and downstream signaling [PMID:41279395].","teleology":[{"year":2005,"claim":"Established that CNPY4/PRAT4B participates in receptor trafficking by linking it physically to an immature receptor pool and to surface receptor levels.","evidence":"Co-immunoprecipitation with immature TLR4 plus siRNA knockdown and flow cytometry in HEK293 cells","pmids":["16338228"],"confidence":"Medium","gaps":["Did not define the molecular basis of CNPY4 binding to immature TLR4","Whether CNPY4 acts directly in the ER or at another trafficking step was not resolved","No structural or domain-level characterization of the interaction"]},{"year":2012,"claim":"Showed that CNPY4 can act as a negative trafficking regulator, contrasting with the positive regulator PRAT4A and revealing receptor-specific, directionally opposite roles.","evidence":"siRNA knockdown and overexpression with TLR1 deletion/point mutant analysis and flow cytometry","pmids":["22447933"],"confidence":"Medium","gaps":["Mechanism by which CNPY4 restrains TLR1 surface delivery not defined","Whether the same activity governs other TLRs untested","No reconciliation of positive (TLR4) vs negative (TLR1) trafficking roles"]},{"year":2022,"claim":"Connected CNPY4 to a developmental signaling pathway and to membrane lipid control, identifying it as a negative Hedgehog regulator acting through accessible sterol levels.","evidence":"Cnpy4 knockout mouse with digit phenotype, siRNA knockdown, HH reporter assays, and filipin/membrane cholesterol measurement","pmids":["35504891"],"confidence":"High","gaps":["Direct molecular mechanism linking CNPY4 to accessible cholesterol not fully resolved","Whether the Hedgehog effect is mediated by direct lipid binding was unaddressed here","Relationship between TLR trafficking role and sterol regulation unclear"]},{"year":2025,"claim":"Provided a unifying biochemical mechanism: CNPY4 is a direct sphingolipid chaperone whose lipid-handling activity remodels membrane sphingolipid and sterol distribution.","evidence":"In vitro lipid-binding assays, siRNA knockdown, lipidomics, filipin staining, and sphingomyelinase activity assays (preprint)","pmids":["41279395"],"confidence":"Medium","gaps":["Preprint not yet peer-reviewed","Structural basis of ceramide/sphingomyelin binding not determined","Causal chain from sphingolipid binding to cholesterol distribution and signaling not fully dissected"]},{"year":2025,"claim":"Placed CNPY4 downstream of a secretory kinase, identifying it as a FAM20C phosphorylation substrate with a possible inflammatory signaling output.","evidence":"Phosphoproteomics of FAM20C gain- and loss-of-function adipocytes","pmids":["41148235"],"confidence":"Low","gaps":["No direct mutagenesis or reconstitution of the specific phosphosite","Functional consequence of CNPY4 phosphorylation not mechanistically established","Link to its chaperone/lipid functions unknown"]},{"year":null,"claim":"How CNPY4's Saposin-like lipid-binding activity mechanistically unifies receptor trafficking, sterol/sphingolipid homeostasis, and developmental signaling remains open.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of CNPY4 bound to lipid or to a receptor client","Whether sphingolipid binding directly drives TLR trafficking and Hedgehog effects untested","Physiological regulation of CNPY4 activity (e.g. by phosphorylation) not characterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[4]},{"term_id":"GO:0044183","term_label":"protein folding chaperone","supporting_discovery_ids":[2,4]}],"localization":[{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[0]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[2,4]}],"pathway":[],"complexes":[],"partners":["TLR4","TLR1","LRIG1","FAM20C"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8N129","full_name":"Protein canopy homolog 4","aliases":[],"length_aa":248,"mass_kda":28.3,"function":"Plays a role in the regulation of the cell surface expression of TLR4","subcellular_location":"Secreted","url":"https://www.uniprot.org/uniprotkb/Q8N129/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CNPY4","classification":"Not Classified","n_dependent_lines":27,"n_total_lines":1208,"dependency_fraction":0.022350993377483443},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CNPY4","total_profiled":1310},"omim":[{"mim_id":"610047","title":"CANOPY FGF SIGNALING REGULATOR 4; CNPY4","url":"https://www.omim.org/entry/610047"},{"mim_id":"606868","title":"HOMEODOMAIN-INTERACTING PROTEIN KINASE 2; HIPK2","url":"https://www.omim.org/entry/606868"},{"mim_id":"605861","title":"CANOPY FGF SIGNALING REGULATOR 2; CNPY2","url":"https://www.omim.org/entry/605861"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Vesicles","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CNPY4"},"hgnc":{"alias_symbol":["MGC40499","PRAT4B"],"prev_symbol":[]},"alphafold":{"accession":"Q8N129","domains":[{"cath_id":"-","chopping":"35-115_146-200","consensus_level":"high","plddt":91.1557,"start":35,"end":200}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8N129","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8N129-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8N129-F1-predicted_aligned_error_v6.png","plddt_mean":79.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CNPY4","jax_strain_url":"https://www.jax.org/strain/search?query=CNPY4"},"sequence":{"accession":"Q8N129","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8N129.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8N129/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8N129"}},"corpus_meta":[{"pmid":"16338228","id":"PMC_16338228","title":"A molecule that is associated with Toll-like receptor 4 and regulates its cell surface expression.","date":"2005","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/16338228","citation_count":36,"is_preprint":false},{"pmid":"22447933","id":"PMC_22447933","title":"Cell surface trafficking of TLR1 is differentially regulated by the chaperones PRAT4A and PRAT4B.","date":"2012","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/22447933","citation_count":29,"is_preprint":false},{"pmid":"29317492","id":"PMC_29317492","title":"A protein interaction network centered on leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) regulates growth factor receptors.","date":"2018","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/29317492","citation_count":28,"is_preprint":false},{"pmid":"30612956","id":"PMC_30612956","title":"Endometrial stromal cell proteome mapping in repeated implantation failure and recurrent pregnancy loss cases and fertile women.","date":"2018","source":"Reproductive biomedicine online","url":"https://pubmed.ncbi.nlm.nih.gov/30612956","citation_count":27,"is_preprint":false},{"pmid":"35504891","id":"PMC_35504891","title":"CNPY4 inhibits the Hedgehog pathway by modulating membrane sterol lipids.","date":"2022","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/35504891","citation_count":10,"is_preprint":false},{"pmid":"18353240","id":"PMC_18353240","title":"[Changes and significance for regulatory factors for signal pathways of Toll-like receptors in immunological pathogenesis of Kawasaki disease].","date":"2008","source":"Zhonghua er ke za zhi = Chinese journal of pediatrics","url":"https://pubmed.ncbi.nlm.nih.gov/18353240","citation_count":10,"is_preprint":false},{"pmid":"37259932","id":"PMC_37259932","title":"Progress in Research on CNPY2 in Diseases.","date":"2024","source":"Mini reviews in medicinal chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/37259932","citation_count":7,"is_preprint":false},{"pmid":"37760635","id":"PMC_37760635","title":"Identification of Potentially Novel Molecular Targets of Endometrial Cancer Using a Non-Biased Proteomic Approach.","date":"2023","source":"Cancers","url":"https://pubmed.ncbi.nlm.nih.gov/37760635","citation_count":5,"is_preprint":false},{"pmid":"40951528","id":"PMC_40951528","title":"Biomarker Discovery for Metabolic Dysfunction-associated Steatotic Liver Disease Utilizing Mendelian Randomization, Machine Learning, and External Validation.","date":"2025","source":"Journal of clinical and translational hepatology","url":"https://pubmed.ncbi.nlm.nih.gov/40951528","citation_count":4,"is_preprint":false},{"pmid":"39869231","id":"PMC_39869231","title":"The role of canopy family proteins: biological mechanism and disease function.","date":"2025","source":"Molecular biology reports","url":"https://pubmed.ncbi.nlm.nih.gov/39869231","citation_count":4,"is_preprint":false},{"pmid":"41279395","id":"PMC_41279395","title":"CNPY4 is a Lipid-Binding Regulator of Sphingolipid Homeostasis.","date":"2025","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/41279395","citation_count":2,"is_preprint":false},{"pmid":"18786322","id":"PMC_18786322","title":"[Role of transduce molecules and modulatory factors of signal pathways of Toll-like receptor in inflammatory response of children with sepsis].","date":"2008","source":"Zhongguo wei zhong bing ji jiu yi xue = Chinese critical care medicine = Zhongguo weizhongbing jijiuyixue","url":"https://pubmed.ncbi.nlm.nih.gov/18786322","citation_count":2,"is_preprint":false},{"pmid":"41148235","id":"PMC_41148235","title":"Secretory kinase FAM20C triggers adipocyte dysfunction, inciting insulin resistance and inflammation in obesity.","date":"2025","source":"The Journal of clinical investigation","url":"https://pubmed.ncbi.nlm.nih.gov/41148235","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9458,"output_tokens":1571,"usd":0.025969,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":8360,"output_tokens":2298,"usd":0.049625,"stage2_stop_reason":"end_turn"},"total_usd":0.075594,"stage1_batch_id":"msgbatch_01ACAxJQabDsht39JXeWG22d","stage2_batch_id":"msgbatch_01QEU22A9U5sbPTgsMzTmvSf","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2005,\n      \"finding\": \"PRAT4B (CNPY4) associates with the hypoglycosylated, immature form of TLR4 but not with MD-2 or TLR2, and siRNA-mediated knockdown of PRAT4B decreases cell surface TLR4 expression on HEK293 cells, establishing PRAT4B as a regulator of TLR4 subcellular trafficking.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, flow cytometry\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal association shown by co-IP with immature TLR4, siRNA knockdown with defined surface trafficking phenotype, single lab but two orthogonal methods\",\n      \"pmids\": [\"16338228\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"PRAT4B (CNPY4) acts as a negative regulator of TLR1 cell surface trafficking, in contrast to PRAT4A which is a positive regulator; knockdown of PRAT4B rescues cell surface expression of the TLR1 I602S trafficking-deficient variant.\",\n      \"method\": \"siRNA knockdown, over-expression, flow cytometry, TLR1 deletion/point mutant analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic manipulation (KD and OE) with defined cell surface trafficking phenotype, single lab with multiple mutant constructs\",\n      \"pmids\": [\"22447933\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CNPY4, a Saposin-like protein, negatively regulates the Hedgehog (HH) signaling pathway by modulating levels of accessible membrane sterol lipids including cholesterol; Cnpy4 knockout embryos display digit number defects consistent with HH perturbation, and Cnpy4 knockdown hyperactivates HH signaling in vitro while elevating plasma membrane cholesterol levels.\",\n      \"method\": \"Cnpy4 knockout mouse model, siRNA knockdown, filipin staining/membrane cholesterol measurement, in vitro HH pathway reporter assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo knockout with defined developmental phenotype plus in vitro knockdown with pathway activation and membrane lipid measurement, multiple orthogonal methods in one study\",\n      \"pmids\": [\"35504891\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CNPY4 was identified as a novel interacting protein of LRIG1 in a yeast two-hybrid screen and confirmed in a BioPlex protein interaction dataset, placing CNPY4 in an LRIG1-centered protein interaction network that regulates growth factor receptors.\",\n      \"method\": \"Yeast two-hybrid screen, BioPlex protein interaction database mining\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single yeast two-hybrid identification with database corroboration but no biochemical validation of the specific CNPY4-LRIG1 interaction\",\n      \"pmids\": [\"29317492\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CNPY4 is a sphingolipid-binding protein (Saposin-like) that interacts with ceramide and sphingomyelin in vitro and with ceramide in cells; CNPY4 knockdown elevates sphingomyelin levels at the plasma membrane, disrupts ceramide localization and abundance, modulates neutral sphingomyelinase activity, and impacts over 150 cellular lipids, establishing CNPY4 as a sphingolipid chaperone that regulates sphingolipid homeostasis and, consequently, cholesterol distribution and cellular signaling.\",\n      \"method\": \"Lipid-binding assays in vitro, siRNA knockdown, lipidomics, filipin staining, sphingomyelinase activity assay, cell-based ceramide localization\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (in vitro lipid binding, cell-based knockdown, lipidomics, enzyme activity assay) in a single lab preprint study\",\n      \"pmids\": [\"41279395\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CNPY4 is a substrate of the secretory kinase FAM20C; FAM20C-dependent phosphorylation of CNPY4 contributes to proinflammatory adipocyte signaling in obesity.\",\n      \"method\": \"Phosphoproteomic analysis of FAM20C gain- and loss-of-function adipocytes\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — phosphoproteomic identification of CNPY4 as FAM20C substrate with functional attribution, single lab, no direct mutagenesis or reconstitution of specific phosphorylation site\",\n      \"pmids\": [\"41148235\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CNPY4 (PRAT4B) is a Saposin-like ER-resident chaperone that regulates subcellular trafficking of TLR4 and TLR1 (acting as a negative regulator of TLR1 surface expression), negatively modulates Hedgehog signaling by controlling accessible membrane sterol lipid levels, and functions as a sphingolipid chaperone that directly binds ceramide and sphingomyelin to regulate sphingolipid homeostasis and plasma membrane composition; additionally, CNPY4 is phosphorylated by the secretory kinase FAM20C, linking it to proinflammatory adipocyte signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CNPY4 (PRAT4B) is a Saposin-like protein that regulates membrane composition and the trafficking of signaling receptors [#2]. As an ER-associated chaperone it controls the subcellular trafficking of Toll-like receptors: it associates selectively with the immature, hypoglycosylated form of TLR4 and is required for normal cell-surface TLR4 expression [#0], while acting as a negative regulator of TLR1 surface trafficking, since its knockdown rescues surface expression of a trafficking-deficient TLR1 variant [#1]. CNPY4 also acts as a negative regulator of Hedgehog signaling by limiting levels of accessible membrane sterol lipids including cholesterol; its loss hyperactivates Hedgehog signaling and elevates plasma membrane cholesterol, and Cnpy4 knockout embryos show digit-number defects consistent with Hedgehog perturbation [#2]. Mechanistically, this lipid control reflects a direct sphingolipid-chaperone activity: CNPY4 binds ceramide and sphingomyelin, and its depletion raises plasma membrane sphingomyelin, disrupts ceramide localization, alters neutral sphingomyelinase activity, and broadly remodels the cellular lipidome, thereby coupling sphingolipid homeostasis to cholesterol distribution and downstream signaling [#4].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Established that CNPY4/PRAT4B participates in receptor trafficking by linking it physically to an immature receptor pool and to surface receptor levels.\",\n      \"evidence\": \"Co-immunoprecipitation with immature TLR4 plus siRNA knockdown and flow cytometry in HEK293 cells\",\n      \"pmids\": [\"16338228\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Did not define the molecular basis of CNPY4 binding to immature TLR4\",\n        \"Whether CNPY4 acts directly in the ER or at another trafficking step was not resolved\",\n        \"No structural or domain-level characterization of the interaction\"\n      ]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Showed that CNPY4 can act as a negative trafficking regulator, contrasting with the positive regulator PRAT4A and revealing receptor-specific, directionally opposite roles.\",\n      \"evidence\": \"siRNA knockdown and overexpression with TLR1 deletion/point mutant analysis and flow cytometry\",\n      \"pmids\": [\"22447933\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Mechanism by which CNPY4 restrains TLR1 surface delivery not defined\",\n        \"Whether the same activity governs other TLRs untested\",\n        \"No reconciliation of positive (TLR4) vs negative (TLR1) trafficking roles\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Connected CNPY4 to a developmental signaling pathway and to membrane lipid control, identifying it as a negative Hedgehog regulator acting through accessible sterol levels.\",\n      \"evidence\": \"Cnpy4 knockout mouse with digit phenotype, siRNA knockdown, HH reporter assays, and filipin/membrane cholesterol measurement\",\n      \"pmids\": [\"35504891\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Direct molecular mechanism linking CNPY4 to accessible cholesterol not fully resolved\",\n        \"Whether the Hedgehog effect is mediated by direct lipid binding was unaddressed here\",\n        \"Relationship between TLR trafficking role and sterol regulation unclear\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Provided a unifying biochemical mechanism: CNPY4 is a direct sphingolipid chaperone whose lipid-handling activity remodels membrane sphingolipid and sterol distribution.\",\n      \"evidence\": \"In vitro lipid-binding assays, siRNA knockdown, lipidomics, filipin staining, and sphingomyelinase activity assays (preprint)\",\n      \"pmids\": [\"41279395\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Preprint not yet peer-reviewed\",\n        \"Structural basis of ceramide/sphingomyelin binding not determined\",\n        \"Causal chain from sphingolipid binding to cholesterol distribution and signaling not fully dissected\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Placed CNPY4 downstream of a secretory kinase, identifying it as a FAM20C phosphorylation substrate with a possible inflammatory signaling output.\",\n      \"evidence\": \"Phosphoproteomics of FAM20C gain- and loss-of-function adipocytes\",\n      \"pmids\": [\"41148235\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No direct mutagenesis or reconstitution of the specific phosphosite\",\n        \"Functional consequence of CNPY4 phosphorylation not mechanistically established\",\n        \"Link to its chaperone/lipid functions unknown\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How CNPY4's Saposin-like lipid-binding activity mechanistically unifies receptor trafficking, sterol/sphingolipid homeostasis, and developmental signaling remains open.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No structure of CNPY4 bound to lipid or to a receptor client\",\n        \"Whether sphingolipid binding directly drives TLR trafficking and Hedgehog effects untested\",\n        \"Physiological regulation of CNPY4 activity (e.g. by phosphorylation) not characterized\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"GO:0044183\", \"supporting_discovery_ids\": [2, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [2, 4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0009536\", \"supporting_discovery_ids\": []}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"TLR4\",\n      \"TLR1\",\n      \"LRIG1\",\n      \"FAM20C\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":4,"faith_total":4,"faith_pct":100.0}}