{"gene":"TM9SF2","run_date":"2026-06-10T10:51:55","timeline":{"discoveries":[{"year":2017,"finding":"TM9SF2 is required for proper localization and stability of NDST1 (the enzyme catalyzing N-sulfation of heparan sulfate), and TM9SF2 knockout reduces N-sulfation of HS, thereby decreasing CHIKV binding and infectivity in HAP1 cells.","method":"Genome-wide haploid insertional mutagenesis screen; TM9SF2 knockout cells; CHIKV binding and infectivity assays","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genome-wide screen with KO validation and functional readout (viral infectivity and NDST1 localization/stability), single lab, two orthogonal approaches","pmids":["28404855"],"is_preprint":false},{"year":2018,"finding":"TM9SF2 knockout reduces levels of Gb3 and other glycosphingolipids, and TM9SF2 KO cells also show defective endosomal trafficking, indicating TM9SF2 is required for maintaining proper glycosylation in the Golgi and for normal endosomal trafficking.","method":"Genome-wide CRISPR-Cas9 screen; TM9SF2 KO cells; mass spectrometry analysis of glycolipids; functional trafficking assays","journal":"PLoS biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — CRISPR KO with mass spectrometry glycolipid profiling and trafficking assays, replicated across multiple independent CRISPR screens in different labs","pmids":["30481169"],"is_preprint":false},{"year":2019,"finding":"Loss of TM9SF2 does not affect Gb3 synthase enzymatic activity but disrupts the localization of Gb3 synthase, resulting in accumulation of its precursor lactosylceramide; this Gb3-regulating activity is conserved across the TM9SF family.","method":"Genome-wide CRISPR/Cas9 KO screen in HeLa cells; Gb3 synthase activity assays; localization studies; glycolipid biosynthesis analysis","journal":"iScience","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO with enzymatic activity assay plus localization studies and glycolipid profiling, two orthogonal methods distinguishing activity vs. localization effects","pmids":["30660999"],"is_preprint":false},{"year":2019,"finding":"TM9SF2 mediates heparan sulfate expression (as validated independently), explaining its contribution to vaccinia virus infection of HAP1 cells.","method":"Genome-wide haploid insertional mutagenesis screen; TM9SF2 KO validation; heparan sulfate expression assays","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO with functional validation of HS expression, single lab, corroborates prior findings from PMID:28404855","pmids":["30996093"],"is_preprint":false},{"year":2020,"finding":"TM9SF2 is required for efficient AAV2 transduction across multiple AAV serotypes (validated in U-2 OS cells and confirmed for at least 8 serotypes), suggesting a role in AAV intracellular trafficking.","method":"Genome-wide pooled CRISPR screens; flow cytometry and imaging validation; multi-serotype AAV transduction assays","journal":"Molecular therapy. Methods & clinical development","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR screen with multi-serotype validation by flow cytometry and imaging, single lab, multiple orthogonal readouts","pmids":["32280726"],"is_preprint":false},{"year":2008,"finding":"Drosophila TM9SF2 (paralogue of TM9SF4) contributes to bacterial internalisation in S2 cells with partial redundancy with TM9SF4; both proteins control the actin cytoskeleton in larval haemocytes and S2 cells.","method":"RNAi knockdown in Drosophila S2 cells; phagocytosis assays; actin cytoskeleton analysis; genetic null mutant flies","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNAi KD with phagocytosis and actin readouts in Drosophila model, replicated across cell and in vivo contexts","pmids":["18796536"],"is_preprint":false},{"year":2014,"finding":"Drosophila TM9SF2 co-immunoprecipitates with the peptidoglycan recognition receptor PGRP-LC and co-localizes with it in intracellular vesicles and at the plasma membrane; silencing TM9SF2 (unlike TM9SF4) does not prevent plasma membrane localization of PGRP-LC, suggesting TM9SF2 can suppress inappropriate signalling from the unstimulated receptor but is not required for PGRP-LC trafficking.","method":"Co-immunoprecipitation; co-localization imaging; RNAi knockdown in Drosophila S2 cells and fat body","journal":"Journal of innate immunity","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — reciprocal Co-IP plus co-localization and RNAi, Drosophila ortholog, single lab","pmids":["25139117"],"is_preprint":false},{"year":2018,"finding":"TM9SF2 mutations in intestinal epithelial cells reduce binding of Shiga toxin and prevent entry of EHEC T3SS effectors into host cells, placing TM9SF2 at early steps of both Stx and T3SS pathogenicity, likely via its role in sphingolipid/Gb3 biosynthesis.","method":"Genome-wide CRISPR/Cas9 screen; TM9SF2 KO validation; Stx binding assays; T3SS effector entry assays in intestinal epithelial cells","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR KO with multiple functional readouts (toxin binding, effector entry), single lab, two orthogonal assays","pmids":["29921669"],"is_preprint":false},{"year":2024,"finding":"Silencing TM9SF2 in THP-1-derived M2 macrophages significantly reduces phagocytosis (of IgG-coated beads and apoptotic Jurkat cells) and attenuates activation of PLC-γ1, placing TM9SF2 upstream of PLC-γ1 signalling in macrophage phagocytosis.","method":"siRNA knockdown; phagocytosis assays with IgG-coated beads and apoptotic cells; PLC-γ1 activation assay","journal":"Journal of orthopaedic surgery and research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, siRNA KD with phagocytosis and signalling readout, limited mechanistic follow-up","pmids":["38218914"],"is_preprint":false},{"year":2025,"finding":"TM9SF2 knockdown causes abnormal cholesterol accumulation in Golgi compartments and Golgi fragmentation; this disruption of Golgi integrity impedes retrograde transport of ricin, attenuating ricin-induced cytotoxicity. Pharmacological restoration of cholesterol metabolism rescues Golgi integrity and reverses the ricin-resistant phenotype.","method":"TM9SF2 KD; cholesterol trafficking assays; Golgi morphology imaging; ricin cytotoxicity assays; pharmacological manipulation (A939572, avasimibe)","journal":"Toxins","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KD combined with pharmacological rescue and multiple orthogonal readouts (cholesterol, Golgi morphology, toxin trafficking), single lab","pmids":["40423301"],"is_preprint":false},{"year":2026,"finding":"TM9SF2 recruits phosphoglycerate kinase 1 (PGK1) to promote recycling of PD-L1 to the plasma membrane while also dismantling HIP1R-mediated lysosomal degradation of PD-L1; disruption of the TM9SF2-PGK1 complex depletes surface PD-L1. The endogenous ceramide species Cer(d18:1/26:0) destabilizes the TM9SF2-PGK1 complex, triggering PD-L1 lysosomal destruction.","method":"CRISPR screen targeting glycosphingolipid metabolism; Co-IP of TM9SF2-PGK1; PD-L1 trafficking assays; lysosomal degradation assays; ceramide treatment experiments","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR screen plus Co-IP and trafficking/degradation assays with lipid perturbation, single lab, multiple orthogonal methods","pmids":["41888515"],"is_preprint":false},{"year":2025,"finding":"TM9SF2 knockdown in A549 cells inhibits VSV replication, upregulates IFN-β mRNA and IRF3 phosphorylation after poly(I:C) stimulation, indicating TM9SF2 negatively regulates the type I interferon signalling pathway.","method":"siRNA knockdown; VSV-GFP infection model; plaque assay; RT-qPCR; Western blot for IRF3 phosphorylation","journal":"Xi bao yu fen zi mian yi xue za zhi","confidence":"Low","confidence_rationale":"Tier 3 / Weak — siRNA KD, single lab, limited mechanistic depth beyond signalling readouts","pmids":["40525335"],"is_preprint":false}],"current_model":"TM9SF2 is a conserved nonaspanin (nine-transmembrane domain) Golgi-localized protein that maintains Golgi integrity and regulates glycosylation by controlling the localization (but not enzymatic activity) of glycosyltransferases such as Gb3 synthase; it is required for proper heparan sulfate N-sulfation through NDST1 stability/localization, for glycosphingolipid biosynthesis, and for normal endosomal and retrograde trafficking — functions that are exploited by diverse pathogens and toxins — and it also regulates PD-L1 trafficking by recruiting PGK1 to promote plasma membrane recycling while suppressing HIP1R-mediated lysosomal degradation, a complex disrupted by ceramide."},"narrative":{"mechanistic_narrative":"TM9SF2 is a conserved Golgi-resident nonaspanin that maintains Golgi integrity and governs glycosphingolipid and glycosaminoglycan biosynthesis by controlling the localization—rather than the catalytic activity—of relevant enzymes [PMID:30481169, PMID:30660999]. It is required for the proper localization and stability of NDST1, the enzyme that N-sulfates heparan sulfate, so its loss reduces HS N-sulfation [PMID:28404855], and it positions Gb3 synthase correctly within the Golgi; in its absence Gb3 synthase activity is preserved but mislocalized, causing accumulation of the precursor lactosylceramide and depletion of Gb3 and other glycosphingolipids [PMID:30660999]. TM9SF2 sustains Golgi architecture in part by regulating Golgi cholesterol, since its depletion drives abnormal Golgi cholesterol accumulation and Golgi fragmentation [PMID:40423301]. Through these glycosylation, lipid, and trafficking functions TM9SF2 is co-opted by diverse pathogens and toxins, supporting CHIKV and vaccinia virus infection via heparan sulfate [PMID:28404855, PMID:30996093], AAV transduction [PMID:32280726], and Shiga toxin/EHEC T3SS effector entry and ricin retrograde transport [PMID:29921669, PMID:40423301]. TM9SF2 additionally controls PD-L1 surface levels by recruiting PGK1 to promote PD-L1 plasma-membrane recycling while dismantling HIP1R-mediated lysosomal degradation, a complex destabilized by the ceramide species Cer(d18:1/26:0) [PMID:41888515]. The Drosophila ortholog contributes to bacterial phagocytosis and actin regulation and interacts with the receptor PGRP-LC to restrain unstimulated signaling [PMID:18796536, PMID:25139117].","teleology":[{"year":2008,"claim":"Established the first cellular role for TM9SF2, showing it acts in pathogen internalization and actin cytoskeleton control, partially redundant with its paralog TM9SF4.","evidence":"RNAi knockdown and null mutants in Drosophila S2 cells and haemocytes with phagocytosis and actin readouts","pmids":["18796536"],"confidence":"Medium","gaps":["Molecular mechanism linking TM9SF2 to actin remodeling not defined","Findings from Drosophila ortholog, not human protein","Redundancy with TM9SF4 not fully resolved"]},{"year":2014,"claim":"Connected TM9SF2 to immune receptor regulation by showing it physically associates with PGRP-LC and suppresses inappropriate signaling without being required for receptor trafficking, distinguishing its role from TM9SF4.","evidence":"Reciprocal Co-IP, co-localization imaging, and RNAi in Drosophila S2 cells and fat body","pmids":["25139117"],"confidence":"Medium","gaps":["Mechanism of signal suppression undefined","Drosophila ortholog only","No structural basis for the interaction"]},{"year":2017,"claim":"Identified a glycosylation-control function in human cells, showing TM9SF2 is needed for NDST1 localization/stability and thus heparan sulfate N-sulfation, which determines CHIKV binding.","evidence":"Genome-wide haploid insertional mutagenesis screen with KO validation and viral binding/infectivity assays in HAP1 cells","pmids":["28404855"],"confidence":"Medium","gaps":["Direct interaction between TM9SF2 and NDST1 not shown","How TM9SF2 stabilizes NDST1 mechanistically unknown"]},{"year":2018,"claim":"Broadened TM9SF2 to a general regulator of Golgi glycosylation and endosomal trafficking by showing its loss depletes Gb3 and other glycosphingolipids and disrupts endosomal transport.","evidence":"Genome-wide CRISPR-Cas9 screen, mass spectrometry glycolipid profiling, and trafficking assays in KO cells","pmids":["30481169"],"confidence":"High","gaps":["Direct enzyme partners not all identified","Link between Golgi and endosomal defects not mechanistically resolved"]},{"year":2018,"claim":"Extended TM9SF2's glycosphingolipid role to toxin/pathogen entry, placing it at early steps of Shiga toxin binding and EHEC T3SS effector entry via Gb3 biosynthesis.","evidence":"Genome-wide CRISPR screen with KO validation, Stx binding and T3SS effector entry assays in intestinal epithelial cells","pmids":["29921669"],"confidence":"Medium","gaps":["Whether T3SS effect is solely Gb3-dependent unresolved","No direct toxin-TM9SF2 interaction tested"]},{"year":2019,"claim":"Resolved that TM9SF2 acts by controlling enzyme localization rather than activity, demonstrating Gb3 synthase retains activity but is mislocalized with precursor accumulation, a conserved TM9SF-family function.","evidence":"Genome-wide CRISPR/Cas9 KO screen in HeLa, Gb3 synthase activity assays, localization studies, and glycolipid profiling","pmids":["30660999"],"confidence":"High","gaps":["How TM9SF2 directs glycosyltransferase localization unknown","Whether mechanism is direct binding or compartmental"]},{"year":2019,"claim":"Independently confirmed TM9SF2's role in heparan sulfate expression as the basis for its contribution to vaccinia virus infection.","evidence":"Genome-wide haploid insertional mutagenesis screen with KO validation and HS expression assays in HAP1 cells","pmids":["30996093"],"confidence":"Medium","gaps":["Mechanism overlaps with prior NDST1 finding but not further dissected"]},{"year":2020,"claim":"Implicated TM9SF2 in AAV intracellular trafficking, showing it is broadly required for efficient transduction across AAV serotypes.","evidence":"Genome-wide pooled CRISPR screens with flow cytometry and imaging validation across at least 8 AAV serotypes in U-2 OS cells","pmids":["32280726"],"confidence":"Medium","gaps":["Specific trafficking step affected not pinpointed","Whether effect is glycan- or trafficking-mediated unclear"]},{"year":2024,"claim":"Linked TM9SF2 to macrophage phagocytic signaling, placing it upstream of PLC-γ1 activation during phagocytosis.","evidence":"siRNA knockdown in THP-1-derived M2 macrophages with phagocytosis and PLC-γ1 activation assays","pmids":["38218914"],"confidence":"Low","gaps":["Single lab, siRNA only without rescue","Mechanism connecting TM9SF2 to PLC-γ1 undefined","No direct interaction shown"]},{"year":2025,"claim":"Defined a cholesterol-dependent basis for TM9SF2's Golgi maintenance, showing its loss causes Golgi cholesterol accumulation and fragmentation that impairs ricin retrograde transport, reversible by restoring cholesterol metabolism.","evidence":"TM9SF2 KD with cholesterol trafficking assays, Golgi morphology imaging, ricin cytotoxicity assays, and pharmacological rescue (A939572, avasimibe)","pmids":["40423301"],"confidence":"Medium","gaps":["How TM9SF2 regulates Golgi cholesterol mechanistically unknown","Direct cholesterol-handling activity not demonstrated"]},{"year":2025,"claim":"Identified TM9SF2 as a negative regulator of type I interferon signaling, with knockdown enhancing IFN-β induction and IRF3 phosphorylation.","evidence":"siRNA knockdown in A549 cells, VSV-GFP infection, plaque assay, RT-qPCR, and IRF3 phosphorylation Western blot","pmids":["40525335"],"confidence":"Low","gaps":["Single lab, siRNA without rescue","Mechanism of IFN pathway suppression undefined","No direct pathway-component interaction tested"]},{"year":2026,"claim":"Established a direct protein-complex function for TM9SF2 in immune checkpoint regulation, showing it recruits PGK1 to recycle PD-L1 and block HIP1R-driven lysosomal degradation, a complex lipid-sensitive to ceramide.","evidence":"CRISPR screen of glycosphingolipid metabolism, Co-IP of TM9SF2-PGK1, PD-L1 trafficking and lysosomal degradation assays, and ceramide treatment","pmids":["41888515"],"confidence":"Medium","gaps":["Structural basis of TM9SF2-PGK1 binding unknown","How ceramide destabilizes the complex mechanistically unresolved","Single lab"]},{"year":null,"claim":"The biochemical mechanism by which a multipass Golgi membrane protein directs glycosyltransferase localization, regulates Golgi cholesterol, and scaffolds cytosolic partners like PGK1 remains undefined.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure or direct biochemical activity established","Whether localization control reflects direct enzyme binding vs. compartmental effects is unresolved","Unifying molecular function across glycosylation, lipid, and trafficking roles not defined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,2,10]}],"localization":[{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[1,2,9]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[1]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[1,2]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[1,9,10]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[2,9]}],"complexes":[],"partners":["PGK1","HIP1R","NDST1","PGRP-LC"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q99805","full_name":"Transmembrane 9 superfamily member 2","aliases":["p76"],"length_aa":663,"mass_kda":75.8,"function":"In the intracellular compartments, may function as a channel or small molecule transporter","subcellular_location":"Endosome membrane; Golgi outpost; Cytoplasm, cytoskeleton, microtubule organizing center","url":"https://www.uniprot.org/uniprotkb/Q99805/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TM9SF2","classification":"Not Classified","n_dependent_lines":83,"n_total_lines":1208,"dependency_fraction":0.06870860927152318},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"SYAP1","stoichiometry":4.0},{"gene":"RAB11A","stoichiometry":0.2},{"gene":"RAB1A","stoichiometry":0.2},{"gene":"RAB2A","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/TM9SF2","total_profiled":1310},"omim":[{"mim_id":"604678","title":"TRANSMEMBRANE 9 SUPERFAMILY, MEMBER 2; TM9SF2","url":"https://www.omim.org/entry/604678"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TM9SF2"},"hgnc":{"alias_symbol":["P76","Lnc-PCIR"],"prev_symbol":[]},"alphafold":{"accession":"Q99805","domains":[{"cath_id":"-","chopping":"59-292","consensus_level":"medium","plddt":86.1206,"start":59,"end":292},{"cath_id":"-","chopping":"295-334_346-497_522-658","consensus_level":"medium","plddt":90.0157,"start":295,"end":658}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q99805","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q99805-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q99805-F1-predicted_aligned_error_v6.png","plddt_mean":84.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TM9SF2","jax_strain_url":"https://www.jax.org/strain/search?query=TM9SF2"},"sequence":{"accession":"Q99805","fasta_url":"https://rest.uniprot.org/uniprotkb/Q99805.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q99805/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q99805"}},"corpus_meta":[{"pmid":"28404855","id":"PMC_28404855","title":"Genome-Wide 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cells; CHIKV binding and infectivity assays\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genome-wide screen with KO validation and functional readout (viral infectivity and NDST1 localization/stability), single lab, two orthogonal approaches\",\n      \"pmids\": [\"28404855\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TM9SF2 knockout reduces levels of Gb3 and other glycosphingolipids, and TM9SF2 KO cells also show defective endosomal trafficking, indicating TM9SF2 is required for maintaining proper glycosylation in the Golgi and for normal endosomal trafficking.\",\n      \"method\": \"Genome-wide CRISPR-Cas9 screen; TM9SF2 KO cells; mass spectrometry analysis of glycolipids; functional trafficking assays\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — CRISPR KO with mass spectrometry glycolipid profiling and trafficking assays, replicated across multiple independent CRISPR screens in different labs\",\n      \"pmids\": [\"30481169\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Loss of TM9SF2 does not affect Gb3 synthase enzymatic activity but disrupts the localization of Gb3 synthase, resulting in accumulation of its precursor lactosylceramide; this Gb3-regulating activity is conserved across the TM9SF family.\",\n      \"method\": \"Genome-wide CRISPR/Cas9 KO screen in HeLa cells; Gb3 synthase activity assays; localization studies; glycolipid biosynthesis analysis\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO with enzymatic activity assay plus localization studies and glycolipid profiling, two orthogonal methods distinguishing activity vs. localization effects\",\n      \"pmids\": [\"30660999\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TM9SF2 mediates heparan sulfate expression (as validated independently), explaining its contribution to vaccinia virus infection of HAP1 cells.\",\n      \"method\": \"Genome-wide haploid insertional mutagenesis screen; TM9SF2 KO validation; heparan sulfate expression assays\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO with functional validation of HS expression, single lab, corroborates prior findings from PMID:28404855\",\n      \"pmids\": [\"30996093\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TM9SF2 is required for efficient AAV2 transduction across multiple AAV serotypes (validated in U-2 OS cells and confirmed for at least 8 serotypes), suggesting a role in AAV intracellular trafficking.\",\n      \"method\": \"Genome-wide pooled CRISPR screens; flow cytometry and imaging validation; multi-serotype AAV transduction assays\",\n      \"journal\": \"Molecular therapy. Methods & clinical development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR screen with multi-serotype validation by flow cytometry and imaging, single lab, multiple orthogonal readouts\",\n      \"pmids\": [\"32280726\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Drosophila TM9SF2 (paralogue of TM9SF4) contributes to bacterial internalisation in S2 cells with partial redundancy with TM9SF4; both proteins control the actin cytoskeleton in larval haemocytes and S2 cells.\",\n      \"method\": \"RNAi knockdown in Drosophila S2 cells; phagocytosis assays; actin cytoskeleton analysis; genetic null mutant flies\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNAi KD with phagocytosis and actin readouts in Drosophila model, replicated across cell and in vivo contexts\",\n      \"pmids\": [\"18796536\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Drosophila TM9SF2 co-immunoprecipitates with the peptidoglycan recognition receptor PGRP-LC and co-localizes with it in intracellular vesicles and at the plasma membrane; silencing TM9SF2 (unlike TM9SF4) does not prevent plasma membrane localization of PGRP-LC, suggesting TM9SF2 can suppress inappropriate signalling from the unstimulated receptor but is not required for PGRP-LC trafficking.\",\n      \"method\": \"Co-immunoprecipitation; co-localization imaging; RNAi knockdown in Drosophila S2 cells and fat body\",\n      \"journal\": \"Journal of innate immunity\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — reciprocal Co-IP plus co-localization and RNAi, Drosophila ortholog, single lab\",\n      \"pmids\": [\"25139117\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TM9SF2 mutations in intestinal epithelial cells reduce binding of Shiga toxin and prevent entry of EHEC T3SS effectors into host cells, placing TM9SF2 at early steps of both Stx and T3SS pathogenicity, likely via its role in sphingolipid/Gb3 biosynthesis.\",\n      \"method\": \"Genome-wide CRISPR/Cas9 screen; TM9SF2 KO validation; Stx binding assays; T3SS effector entry assays in intestinal epithelial cells\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR KO with multiple functional readouts (toxin binding, effector entry), single lab, two orthogonal assays\",\n      \"pmids\": [\"29921669\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Silencing TM9SF2 in THP-1-derived M2 macrophages significantly reduces phagocytosis (of IgG-coated beads and apoptotic Jurkat cells) and attenuates activation of PLC-γ1, placing TM9SF2 upstream of PLC-γ1 signalling in macrophage phagocytosis.\",\n      \"method\": \"siRNA knockdown; phagocytosis assays with IgG-coated beads and apoptotic cells; PLC-γ1 activation assay\",\n      \"journal\": \"Journal of orthopaedic surgery and research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, siRNA KD with phagocytosis and signalling readout, limited mechanistic follow-up\",\n      \"pmids\": [\"38218914\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TM9SF2 knockdown causes abnormal cholesterol accumulation in Golgi compartments and Golgi fragmentation; this disruption of Golgi integrity impedes retrograde transport of ricin, attenuating ricin-induced cytotoxicity. Pharmacological restoration of cholesterol metabolism rescues Golgi integrity and reverses the ricin-resistant phenotype.\",\n      \"method\": \"TM9SF2 KD; cholesterol trafficking assays; Golgi morphology imaging; ricin cytotoxicity assays; pharmacological manipulation (A939572, avasimibe)\",\n      \"journal\": \"Toxins\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KD combined with pharmacological rescue and multiple orthogonal readouts (cholesterol, Golgi morphology, toxin trafficking), single lab\",\n      \"pmids\": [\"40423301\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"TM9SF2 recruits phosphoglycerate kinase 1 (PGK1) to promote recycling of PD-L1 to the plasma membrane while also dismantling HIP1R-mediated lysosomal degradation of PD-L1; disruption of the TM9SF2-PGK1 complex depletes surface PD-L1. The endogenous ceramide species Cer(d18:1/26:0) destabilizes the TM9SF2-PGK1 complex, triggering PD-L1 lysosomal destruction.\",\n      \"method\": \"CRISPR screen targeting glycosphingolipid metabolism; Co-IP of TM9SF2-PGK1; PD-L1 trafficking assays; lysosomal degradation assays; ceramide treatment experiments\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR screen plus Co-IP and trafficking/degradation assays with lipid perturbation, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"41888515\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TM9SF2 knockdown in A549 cells inhibits VSV replication, upregulates IFN-β mRNA and IRF3 phosphorylation after poly(I:C) stimulation, indicating TM9SF2 negatively regulates the type I interferon signalling pathway.\",\n      \"method\": \"siRNA knockdown; VSV-GFP infection model; plaque assay; RT-qPCR; Western blot for IRF3 phosphorylation\",\n      \"journal\": \"Xi bao yu fen zi mian yi xue za zhi\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — siRNA KD, single lab, limited mechanistic depth beyond signalling readouts\",\n      \"pmids\": [\"40525335\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TM9SF2 is a conserved nonaspanin (nine-transmembrane domain) Golgi-localized protein that maintains Golgi integrity and regulates glycosylation by controlling the localization (but not enzymatic activity) of glycosyltransferases such as Gb3 synthase; it is required for proper heparan sulfate N-sulfation through NDST1 stability/localization, for glycosphingolipid biosynthesis, and for normal endosomal and retrograde trafficking — functions that are exploited by diverse pathogens and toxins — and it also regulates PD-L1 trafficking by recruiting PGK1 to promote plasma membrane recycling while suppressing HIP1R-mediated lysosomal degradation, a complex disrupted by ceramide.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TM9SF2 is a conserved Golgi-resident nonaspanin that maintains Golgi integrity and governs glycosphingolipid and glycosaminoglycan biosynthesis by controlling the localization—rather than the catalytic activity—of relevant enzymes [#1, #2]. It is required for the proper localization and stability of NDST1, the enzyme that N-sulfates heparan sulfate, so its loss reduces HS N-sulfation [#0], and it positions Gb3 synthase correctly within the Golgi; in its absence Gb3 synthase activity is preserved but mislocalized, causing accumulation of the precursor lactosylceramide and depletion of Gb3 and other glycosphingolipids [#2]. TM9SF2 sustains Golgi architecture in part by regulating Golgi cholesterol, since its depletion drives abnormal Golgi cholesterol accumulation and Golgi fragmentation [#9]. Through these glycosylation, lipid, and trafficking functions TM9SF2 is co-opted by diverse pathogens and toxins, supporting CHIKV and vaccinia virus infection via heparan sulfate [#0, #3], AAV transduction [#4], and Shiga toxin/EHEC T3SS effector entry and ricin retrograde transport [#7, #9]. TM9SF2 additionally controls PD-L1 surface levels by recruiting PGK1 to promote PD-L1 plasma-membrane recycling while dismantling HIP1R-mediated lysosomal degradation, a complex destabilized by the ceramide species Cer(d18:1/26:0) [#10]. The Drosophila ortholog contributes to bacterial phagocytosis and actin regulation and interacts with the receptor PGRP-LC to restrain unstimulated signaling [#5, #6].\",\n  \"teleology\": [\n    {\n      \"year\": 2008,\n      \"claim\": \"Established the first cellular role for TM9SF2, showing it acts in pathogen internalization and actin cytoskeleton control, partially redundant with its paralog TM9SF4.\",\n      \"evidence\": \"RNAi knockdown and null mutants in Drosophila S2 cells and haemocytes with phagocytosis and actin readouts\",\n      \"pmids\": [\"18796536\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular mechanism linking TM9SF2 to actin remodeling not defined\", \"Findings from Drosophila ortholog, not human protein\", \"Redundancy with TM9SF4 not fully resolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Connected TM9SF2 to immune receptor regulation by showing it physically associates with PGRP-LC and suppresses inappropriate signaling without being required for receptor trafficking, distinguishing its role from TM9SF4.\",\n      \"evidence\": \"Reciprocal Co-IP, co-localization imaging, and RNAi in Drosophila S2 cells and fat body\",\n      \"pmids\": [\"25139117\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of signal suppression undefined\", \"Drosophila ortholog only\", \"No structural basis for the interaction\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identified a glycosylation-control function in human cells, showing TM9SF2 is needed for NDST1 localization/stability and thus heparan sulfate N-sulfation, which determines CHIKV binding.\",\n      \"evidence\": \"Genome-wide haploid insertional mutagenesis screen with KO validation and viral binding/infectivity assays in HAP1 cells\",\n      \"pmids\": [\"28404855\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct interaction between TM9SF2 and NDST1 not shown\", \"How TM9SF2 stabilizes NDST1 mechanistically unknown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Broadened TM9SF2 to a general regulator of Golgi glycosylation and endosomal trafficking by showing its loss depletes Gb3 and other glycosphingolipids and disrupts endosomal transport.\",\n      \"evidence\": \"Genome-wide CRISPR-Cas9 screen, mass spectrometry glycolipid profiling, and trafficking assays in KO cells\",\n      \"pmids\": [\"30481169\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct enzyme partners not all identified\", \"Link between Golgi and endosomal defects not mechanistically resolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Extended TM9SF2's glycosphingolipid role to toxin/pathogen entry, placing it at early steps of Shiga toxin binding and EHEC T3SS effector entry via Gb3 biosynthesis.\",\n      \"evidence\": \"Genome-wide CRISPR screen with KO validation, Stx binding and T3SS effector entry assays in intestinal epithelial cells\",\n      \"pmids\": [\"29921669\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether T3SS effect is solely Gb3-dependent unresolved\", \"No direct toxin-TM9SF2 interaction tested\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Resolved that TM9SF2 acts by controlling enzyme localization rather than activity, demonstrating Gb3 synthase retains activity but is mislocalized with precursor accumulation, a conserved TM9SF-family function.\",\n      \"evidence\": \"Genome-wide CRISPR/Cas9 KO screen in HeLa, Gb3 synthase activity assays, localization studies, and glycolipid profiling\",\n      \"pmids\": [\"30660999\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How TM9SF2 directs glycosyltransferase localization unknown\", \"Whether mechanism is direct binding or compartmental\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Independently confirmed TM9SF2's role in heparan sulfate expression as the basis for its contribution to vaccinia virus infection.\",\n      \"evidence\": \"Genome-wide haploid insertional mutagenesis screen with KO validation and HS expression assays in HAP1 cells\",\n      \"pmids\": [\"30996093\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism overlaps with prior NDST1 finding but not further dissected\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Implicated TM9SF2 in AAV intracellular trafficking, showing it is broadly required for efficient transduction across AAV serotypes.\",\n      \"evidence\": \"Genome-wide pooled CRISPR screens with flow cytometry and imaging validation across at least 8 AAV serotypes in U-2 OS cells\",\n      \"pmids\": [\"32280726\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific trafficking step affected not pinpointed\", \"Whether effect is glycan- or trafficking-mediated unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Linked TM9SF2 to macrophage phagocytic signaling, placing it upstream of PLC-\\u03b31 activation during phagocytosis.\",\n      \"evidence\": \"siRNA knockdown in THP-1-derived M2 macrophages with phagocytosis and PLC-\\u03b31 activation assays\",\n      \"pmids\": [\"38218914\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single lab, siRNA only without rescue\", \"Mechanism connecting TM9SF2 to PLC-\\u03b31 undefined\", \"No direct interaction shown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined a cholesterol-dependent basis for TM9SF2's Golgi maintenance, showing its loss causes Golgi cholesterol accumulation and fragmentation that impairs ricin retrograde transport, reversible by restoring cholesterol metabolism.\",\n      \"evidence\": \"TM9SF2 KD with cholesterol trafficking assays, Golgi morphology imaging, ricin cytotoxicity assays, and pharmacological rescue (A939572, avasimibe)\",\n      \"pmids\": [\"40423301\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How TM9SF2 regulates Golgi cholesterol mechanistically unknown\", \"Direct cholesterol-handling activity not demonstrated\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified TM9SF2 as a negative regulator of type I interferon signaling, with knockdown enhancing IFN-\\u03b2 induction and IRF3 phosphorylation.\",\n      \"evidence\": \"siRNA knockdown in A549 cells, VSV-GFP infection, plaque assay, RT-qPCR, and IRF3 phosphorylation Western blot\",\n      \"pmids\": [\"40525335\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single lab, siRNA without rescue\", \"Mechanism of IFN pathway suppression undefined\", \"No direct pathway-component interaction tested\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Established a direct protein-complex function for TM9SF2 in immune checkpoint regulation, showing it recruits PGK1 to recycle PD-L1 and block HIP1R-driven lysosomal degradation, a complex lipid-sensitive to ceramide.\",\n      \"evidence\": \"CRISPR screen of glycosphingolipid metabolism, Co-IP of TM9SF2-PGK1, PD-L1 trafficking and lysosomal degradation assays, and ceramide treatment\",\n      \"pmids\": [\"41888515\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of TM9SF2-PGK1 binding unknown\", \"How ceramide destabilizes the complex mechanistically unresolved\", \"Single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The biochemical mechanism by which a multipass Golgi membrane protein directs glycosyltransferase localization, regulates Golgi cholesterol, and scaffolds cytosolic partners like PGK1 remains undefined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure or direct biochemical activity established\", \"Whether localization control reflects direct enzyme binding vs. compartmental effects is unresolved\", \"Unifying molecular function across glycosylation, lipid, and trafficking roles not defined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 2, 10]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [1, 2, 9]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [1, 9, 10]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [2, 9]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"PGK1\", \"HIP1R\", \"NDST1\", \"PGRP-LC\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}