{"gene":"TM9SF2","run_date":"2026-04-28T21:42:59","timeline":{"discoveries":[{"year":2007,"finding":"TM9SF2 (then called 'p76'/LOC196463) is a mannose-6-phosphate (Man6P)-bearing glycoprotein with six N-glycosylation sites that localizes to lysosomes, as demonstrated by Man6P-dependent interaction with mannose-6-phosphate receptors (MPR overlay), progressive PNGase F deglycosylation, immunofluorescence, and subcellular fractionation of mouse liver homogenates.","method":"MPR overlay assay, PNGase F deglycosylation, immunofluorescence microscopy, subcellular fractionation","journal":"The Biochemical Journal","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (overlay assay, biochemical fractionation, immunofluorescence) in a single study with rigorous controls","pmids":["17105447"],"is_preprint":false},{"year":2014,"finding":"Drosophila TM9SF2 (ortholog of human 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 does not prevent plasma membrane localization of PGRP-LC (unlike TM9SF4 silencing), and both TM9 proteins control actin cytoskeleton organization in haemocytes, with evidence that TM9 proteins suppress inappropriate signaling from the unstimulated PGRP-LC receptor.","method":"Co-immunoprecipitation, RNAi knockdown, immunofluorescence co-localization, actin cytoskeleton assays in Drosophila S2 cells and fat body","journal":"Journal of Innate Immunity","confidence":"Medium","confidence_rationale":"Tier 2 — reciprocal Co-IP and functional knockdown with defined phenotypic readout, but in Drosophila ortholog system","pmids":["25139117"],"is_preprint":false},{"year":2018,"finding":"CRISPR/Cas9 knockout of TM9SF2 substantially diminishes colorectal cancer (CRC) cell fitness in vitro and in vivo (anchorage-independent growth, tumor growth), and transcriptome analysis of knockout cells reveals TM9SF2 involvement in cell cycle progression, oxidative phosphorylation, and ceramide signaling.","method":"CRISPR/Cas9 knockout, RNAi silencing, anchorage-independent growth assay, in vivo tumor xenograft, transcriptome analysis","journal":"Scientific Reports","confidence":"Medium","confidence_rationale":"Tier 2 — clean KO with defined cellular and in vivo phenotype, but pathway placement is transcriptome-inferred rather than mechanistically dissected","pmids":["30333512"],"is_preprint":false},{"year":2019,"finding":"LINC01232 recruits the RNA-binding protein EIF4A3 to boost TM9SF2 mRNA stability, thereby upregulating TM9SF2 expression; additionally, the transcription factor SP1 mediates transcriptional activation of both LINC01232 and TM9SF2, placing TM9SF2 downstream of a SP1/LINC01232/EIF4A3 regulatory axis in pancreatic adenocarcinoma.","method":"RNA pulldown, RIP assay, siRNA knockdown, luciferase reporter assay, qRT-PCR, mRNA stability assay","journal":"Cell Death & Disease","confidence":"Medium","confidence_rationale":"Tier 2-3 — multiple methods identifying regulatory mechanism, but single-lab study","pmids":["31541081"],"is_preprint":false},{"year":2020,"finding":"Genome-wide CRISPR pooled screens identified TM9SF2 as a host cell factor required for AAV2 transduction across eight different AAV serotypes (including AAV1–4, AAV6–9); validation in flow cytometry and imaging studies confirmed TM9SF2's role, distinct from and complementary to GPR108 (which localizes to Golgi and shows serotype selectivity).","method":"Genome-wide CRISPR pooled screen, flow cytometry, imaging validation, multi-serotype AAV transduction assays","journal":"Molecular Therapy: Methods & Clinical Development","confidence":"High","confidence_rationale":"Tier 2 — genome-wide unbiased screen with multi-serotype validation across multiple cell types","pmids":["32280726"],"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 in the Fcγ receptor-mediated phagocytic signaling pathway.","method":"siRNA knockdown, phagocytosis assay (IgG-coated beads and apoptotic cell co-culture), Western blotting for PLC-γ1 phosphorylation","journal":"Journal of Orthopaedic Surgery and Research","confidence":"Medium","confidence_rationale":"Tier 2-3 — functional knockdown with specific signaling readout, single-lab study","pmids":["38218914"],"is_preprint":false},{"year":2025,"finding":"TM9SF2 maintains Golgi integrity by regulating intracellular cholesterol trafficking; genetic knockdown of TM9SF2 causes abnormal cholesterol accumulation in Golgi compartments, severe Golgi fragmentation, and impaired retrograde transport of ricin, thereby attenuating ricin-induced cytotoxicity. Pharmacological restoration of cholesterol homeostasis (using A939572 or avasimibe) rescues Golgi integrity and reverses the ricin-resistant phenotype of TM9SF2-knockdown cells.","method":"siRNA/genetic knockdown, cholesterol trafficking assays (filipin staining, cholesterol fractionation), Golgi morphology imaging, ricin cytotoxicity assays, pharmacological rescue with A939572/avasimibe","journal":"Toxins","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal methods with pharmacological rescue validation, single-lab study","pmids":["40423301"],"is_preprint":false},{"year":2025,"finding":"TM9SF2 knockdown in A549 cells inhibits vesicular stomatitis virus (VSV) replication and positively regulates the type I interferon signaling pathway; specifically, TM9SF2 knockdown upregulates IFN-β mRNA and increases IRF3 phosphorylation upon poly(I:C) stimulation, indicating TM9SF2 normally suppresses innate antiviral immunity.","method":"siRNA knockdown, VSV-GFP infection model, plaque assay, RT-qPCR, Western blotting for IRF3 phosphorylation, poly(I:C) stimulation","journal":"Chinese Journal of Cellular and Molecular Immunology","confidence":"Medium","confidence_rationale":"Tier 2-3 — functional knockdown with defined molecular readouts, single-lab study","pmids":["40525335"],"is_preprint":false},{"year":2026,"finding":"TM9SF2 orchestrates PD-L1 trafficking by: (1) recruiting phosphoglycerate kinase 1 (PGK1) to promote PD-L1 recycling to the plasma membrane, and (2) dismantling the HIP1R-mediated lysosomal degradation pathway for PD-L1. Genetic or pharmacological disruption of the TM9SF2-PGK1 complex depletes surface PD-L1 and enhances antitumor immunity. The endogenous ceramide species Cer(d18:1/26:0) destabilizes the TM9SF2-PGK1 complex, triggering PD-L1 lysosomal degradation; this mechanism was identified via CRISPR screen targeting glycosphingolipid metabolism.","method":"CRISPR screen (glycosphingolipid metabolism library), co-immunoprecipitation, PD-L1 trafficking assays, genetic knockdown/knockout, pharmacological disruption, ceramide treatment, antitumor immunity assays","journal":"Nature Communications","confidence":"High","confidence_rationale":"Tier 1-2 — unbiased CRISPR screen plus reciprocal Co-IP, mechanistic dissection of dual pathway with pharmacological and genetic validation","pmids":["41888515"],"is_preprint":false}],"current_model":"TM9SF2 is a lysosome-localized nonaspanin (9-transmembrane) glycoprotein that regulates intracellular vesicle trafficking and membrane receptor dynamics: it maintains Golgi integrity via cholesterol trafficking control, promotes PD-L1 recycling to the plasma membrane by recruiting PGK1 while suppressing HIP1R-mediated lysosomal degradation (a complex disrupted by ceramide Cer(d18:1/26:0)), supports phagocytosis upstream of PLC-γ1 in macrophages, acts as an essential host factor for AAV transduction across multiple serotypes, suppresses innate antiviral type I interferon signaling, and in Drosophila orthologs co-immunoprecipitates with and controls plasma membrane localization of the PGRP-LC immune receptor while regulating actin cytoskeleton organization."},"narrative":{"teleology":[{"year":2007,"claim":"Establishing the basic identity of TM9SF2 as a mannose-6-phosphate-bearing lysosomal glycoprotein with six N-glycosylation sites resolved its subcellular address and post-translational processing, framing it as a potential regulator of endolysosomal trafficking.","evidence":"MPR overlay assay, PNGase F deglycosylation, immunofluorescence, and subcellular fractionation in mouse liver","pmids":["17105447"],"confidence":"High","gaps":["No functional role or binding partners identified at this stage","Whether lysosomal localization is constitutive or regulated was not addressed","Mechanism of lysosomal targeting beyond Man6P modification unknown"]},{"year":2014,"claim":"Demonstrating that the Drosophila TM9SF2 ortholog physically associates with the innate immune receptor PGRP-LC and regulates actin organization in haemocytes established the first link between TM9SF family members and immune receptor trafficking and cytoskeletal control.","evidence":"Co-immunoprecipitation, RNAi knockdown, immunofluorescence co-localization, and actin assays in Drosophila S2 cells","pmids":["25139117"],"confidence":"Medium","gaps":["Findings are in Drosophila; conservation of PGRP-LC-like interaction in mammals unconfirmed","Molecular mechanism linking TM9SF2 to actin remodeling not identified","Whether TM9SF2 directly controls receptor surface delivery versus retention was not resolved"]},{"year":2018,"claim":"CRISPR knockout revealing that TM9SF2 loss impairs colorectal cancer cell fitness in vitro and in xenografts connected TM9SF2 to cell proliferation, while transcriptome analysis implicated it in cell cycle, oxidative phosphorylation, and ceramide signaling pathways.","evidence":"CRISPR/Cas9 knockout, anchorage-independent growth assay, xenograft tumor model, and transcriptome profiling in CRC cells","pmids":["30333512"],"confidence":"Medium","gaps":["Pathway involvement is transcriptome-inferred, not mechanistically validated","Whether growth defect is cell-autonomous or microenvironment-dependent was not dissected","Direct molecular targets mediating TM9SF2 pro-proliferative effect not identified"]},{"year":2020,"claim":"Genome-wide CRISPR screens identifying TM9SF2 as an essential host factor for AAV transduction across eight serotypes demonstrated a broad, serotype-independent role in viral entry or intracellular trafficking, distinct from the Golgi-localized factor GPR108.","evidence":"Genome-wide CRISPR pooled screen with multi-serotype AAV transduction validation by flow cytometry and imaging","pmids":["32280726"],"confidence":"High","gaps":["Precise step in the AAV entry/trafficking pathway mediated by TM9SF2 remains undefined","Whether TM9SF2 acts at the endosomal escape or uncoating stage was not resolved","Structural basis for TM9SF2 requirement across diverse AAV capsids unknown"]},{"year":2024,"claim":"Showing that TM9SF2 knockdown impairs Fcγ receptor-mediated phagocytosis and attenuates PLCγ1 activation in macrophages placed TM9SF2 upstream of a defined signaling node in innate immune effector function.","evidence":"siRNA knockdown in THP-1-derived M2 macrophages, phagocytosis assays with IgG-coated beads and apoptotic cells, Western blotting for PLCγ1 phosphorylation","pmids":["38218914"],"confidence":"Medium","gaps":["Single-lab study; independent replication in primary macrophages not reported","Whether TM9SF2 acts on Fcγ receptor surface expression, clustering, or downstream adaptor recruitment is unknown","Relationship between lysosomal localization and phagocytic function not mechanistically explained"]},{"year":2025,"claim":"Discovering that TM9SF2 maintains Golgi integrity through cholesterol trafficking control—and that pharmacological restoration of cholesterol homeostasis rescues Golgi fragmentation in TM9SF2-depleted cells—provided the first mechanistic explanation for TM9SF2's broad influence on membrane trafficking.","evidence":"siRNA knockdown, filipin staining, cholesterol fractionation, Golgi morphology imaging, ricin cytotoxicity and retrograde transport assays, pharmacological rescue with A939572/avasimibe","pmids":["40423301"],"confidence":"Medium","gaps":["Single-lab study; the cholesterol sensor or transporter that TM9SF2 directly regulates is unidentified","Whether Golgi fragmentation explains the AAV and phagocytosis phenotypes was not tested","Contribution of individual TM9SF2 transmembrane domains to cholesterol sensing unknown"]},{"year":2025,"claim":"Demonstrating that TM9SF2 knockdown enhances IRF3 phosphorylation and IFN-β induction upon poly(I:C) stimulation revealed TM9SF2 as a negative regulator of type I interferon signaling, linking its trafficking function to innate antiviral immunity.","evidence":"siRNA knockdown in A549 cells, VSV-GFP infection, plaque assay, RT-qPCR, Western blotting for phospho-IRF3","pmids":["40525335"],"confidence":"Medium","gaps":["Single-lab study; mechanism by which TM9SF2 restrains IRF3 activation (e.g. receptor turnover, adaptor sequestration) not identified","Whether this reflects altered STING or RIG-I/MAVS pathway trafficking was not examined","In vivo relevance of immune suppression not tested"]},{"year":2025,"claim":"Mechanistic dissection of TM9SF2's role in PD-L1 trafficking—recruiting PGK1 for recycling while opposing HIP1R-mediated lysosomal degradation, with ceramide Cer(d18:1/26:0) as an endogenous disruptor of the TM9SF2-PGK1 complex—provided a unified model for how TM9SF2 coordinates receptor surface expression with therapeutic implications for antitumor immunity.","evidence":"CRISPR screen targeting glycosphingolipid metabolism, co-immunoprecipitation, PD-L1 trafficking assays, genetic and pharmacological disruption, ceramide treatment, antitumor immunity assays","pmids":["41888515"],"confidence":"High","gaps":["Whether TM9SF2-PGK1 recycling applies to receptors beyond PD-L1 is untested","Structural basis of the TM9SF2-PGK1 interaction and ceramide-mediated disruption unresolved","In vivo pharmacological targeting of TM9SF2-PGK1 axis not yet evaluated in clinical models"]},{"year":null,"claim":"A unifying structural and mechanistic model explaining how TM9SF2 coordinates cholesterol trafficking, receptor recycling, Golgi maintenance, and innate immune regulation through its nine-transmembrane architecture remains to be established.","evidence":"","pmids":[],"confidence":"Low","gaps":["No crystal or cryo-EM structure of TM9SF2 exists","Whether cholesterol-binding is direct and mediates all downstream phenotypes is unknown","Relative importance of TM9SF2 in different tissue and immune cell contexts has not been systematically compared"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[8]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[7,8]}],"localization":[{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[0,8]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[6]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[1,8]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[6,8]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[5,7]}],"complexes":[],"partners":["PGK1","HIP1R","PGRP-LC","PD-L1"],"other_free_text":[]},"mechanistic_narrative":"TM9SF2 is a lysosome-localized, nine-transmembrane (nonaspanin) glycoprotein that governs intracellular vesicle trafficking, membrane receptor dynamics, and innate immune signaling. It maintains Golgi integrity by controlling intracellular cholesterol distribution, with its depletion causing cholesterol accumulation in Golgi compartments and consequent Golgi fragmentation [PMID:40423301], and it orchestrates PD-L1 surface expression by recruiting PGK1 to promote PD-L1 recycling while suppressing HIP1R-mediated lysosomal degradation—a complex destabilized by the ceramide species Cer(d18:1/26:0) [PMID:41888515]. TM9SF2 functions as a broadly required host factor for adeno-associated virus transduction across multiple serotypes [PMID:32280726], promotes Fcγ receptor-mediated phagocytosis upstream of PLCγ1 in macrophages [PMID:38218914], and suppresses type I interferon signaling by restraining IRF3 phosphorylation [PMID:40525335]. In Drosophila, the ortholog physically associates with the innate immune receptor PGRP-LC and regulates actin cytoskeleton organization in haemocytes [PMID:25139117]."},"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":"10681559","id":"PMC_10681559","title":"p76(MDM2) inhibits the ability of p90(MDM2) to destabilize p53.","date":"2000","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10681559","citation_count":64,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"31541081","id":"PMC_31541081","title":"LINC01232 exerts oncogenic activities in pancreatic adenocarcinoma via regulation of TM9SF2.","date":"2019","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/31541081","citation_count":50,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"32280726","id":"PMC_32280726","title":"Pooled Screens Identify GPR108 and TM9SF2 as Host Cell Factors Critical for AAV Transduction.","date":"2020","source":"Molecular therapy. 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Biotinylation.","date":"2020","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/32344865","citation_count":146,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"9230071","id":"PMC_9230071","title":"A novel Rab9 effector required for endosome-to-TGN transport.","date":"1997","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/9230071","citation_count":119,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"20379614","id":"PMC_20379614","title":"Personalized smoking cessation: interactions between nicotine dose, dependence and quit-success genotype score.","date":"2010","source":"Molecular medicine (Cambridge, Mass.)","url":"https://pubmed.ncbi.nlm.nih.gov/20379614","citation_count":108,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"33545068","id":"PMC_33545068","title":"Systematically defining selective autophagy receptor-specific cargo using autophagosome content profiling.","date":"2021","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/33545068","citation_count":105,"is_preprint":false,"source_track":"gene2pubmed"}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9954,"output_tokens":2419,"usd":0.033073},"stage2":{"model":"claude-opus-4-6","input_tokens":5728,"output_tokens":2672,"usd":0.14316},"total_usd":0.384006,"stage1_batch_id":"msgbatch_01RPbVc9PDH9zQmYK5Hs6srm","stage2_batch_id":"msgbatch_01JVbVLzeKN2yVrsGp3zC8aM","note":"batch pricing = 50% of standard","round2_stage1":{"model":"claude-sonnet-4-6","input_tokens":22340,"output_tokens":3220,"usd":0.05766},"round2_rules_fired":"R3","round2_stage2":{"model":"claude-opus-4-6","input_tokens":6185,"output_tokens":2766,"usd":0.150113}},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2007,\n      \"finding\": \"TM9SF2 (p76/LOC196463) is a lysosomal protein carrying mannose-6-phosphate (Man6P) tags that mediate its direct, Man6P-dependent interaction with Man6P receptors (MPRs), enabling lysosomal targeting. The protein has six N-glycosylation sites and undergoes maturation processing detectable by N- and C-terminal antibodies.\",\n      \"method\": \"MPR overlay experiment (Man6P-dependent binding), peptide-N-glycosidase F deglycosylation, immunofluorescence, subcellular fractionation of mouse liver homogenates\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods (biochemical binding assay, glycosylation mapping, immunofluorescence, fractionation) in single study\",\n      \"pmids\": [\"17105447\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Drosophila TM9SF2 co-immunoprecipitates with the peptidoglycan recognition protein PGRP-LC and co-localizes with it in intracellular vesicles and at the plasma membrane in S2 cells and fat body. TM9SF2 (together with TM9SF4) contributes to phagocytosis and controls the actin cytoskeleton in haemocytes, and both proteins can prevent inappropriate signalling from the unstimulated PGRP-LC receptor.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence co-localization, RNAi silencing with plasma membrane localization and phagocytosis readouts\",\n      \"journal\": \"Journal of innate immunity\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — reciprocal Co-IP and co-localization with functional RNAi phenotype, single lab\",\n      \"pmids\": [\"25139117\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TM9SF2 acts as a colorectal cancer oncogene whose loss via CRISPR/Cas9 knockout substantially diminishes tumor cell fitness in vitro and in vivo, and whose RNAi silencing reduces anchorage-independent growth; transcriptome analysis implicates TM9SF2 in cell cycle progression, oxidative phosphorylation, and ceramide signaling.\",\n      \"method\": \"CRISPR/Cas9 knockout, RNAi silencing, anchorage-independent growth assay, xenograft in vivo model, transcriptome (RNA-seq) analysis\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with defined cellular phenotype in vitro and in vivo, single lab\",\n      \"pmids\": [\"30333512\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TM9SF2 mRNA stability is boosted by LINC01232 through recruitment of the RNA-binding protein EIF4A3, and transcriptional activation of TM9SF2 is mediated by the transcription factor SP1.\",\n      \"method\": \"Loss-of-function RNAi assays, mRNA stability assays, RNA immunoprecipitation/co-immunoprecipitation of EIF4A3, SP1 transcription factor binding assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — mechanistic follow-up with RNA-binding protein pulldown and transcription factor assays, single lab\",\n      \"pmids\": [\"31541081\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TM9SF2 is a host cell factor required for transduction by multiple AAV serotypes (AAV1-9 tested); loss of TM9SF2 reduces AAV2 transduction efficiency, validated by genome-wide CRISPR screen and confirmed by flow cytometry and imaging in multiple cell types.\",\n      \"method\": \"Genome-wide pooled CRISPR/Cas9 screen, EGFP-AAV transduction flow cytometry, imaging validation across 8 AAV serotypes\",\n      \"journal\": \"Molecular therapy. Methods & clinical development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genome-wide screen with multi-serotype validation across cell types, single lab\",\n      \"pmids\": [\"32280726\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TM9SF2 silencing 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 in the Fcγ-mediated phagocytic signaling pathway.\",\n      \"method\": \"siRNA knockdown, phagocytosis assay with IgG-coated beads and apoptotic cells, Western blotting for PLC-γ1 phosphorylation\",\n      \"journal\": \"Journal of orthopaedic surgery and research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single lab, single knockdown approach with limited pathway placement\",\n      \"pmids\": [\"38218914\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TM9SF2 maintains Golgi integrity by regulating intracellular cholesterol trafficking; TM9SF2 knockdown causes abnormal cholesterol accumulation in Golgi compartments leading to Golgi fragmentation, which in turn impedes retrograde transport of ricin and attenuates ricin-induced cytotoxicity. Pharmacological restoration of cholesterol metabolism (via A939572 or avasimibe) rescues Golgi integrity and reverses the ricin-resistant phenotype.\",\n      \"method\": \"siRNA/genetic knockdown, cholesterol trafficking assays, Golgi morphology imaging, ricin cytotoxicity assays, pharmacological rescue with cholesterol metabolism inhibitors\",\n      \"journal\": \"Toxins\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic and pharmacological rescue with orthogonal mechanistic readouts, single lab\",\n      \"pmids\": [\"40423301\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TM9SF2 knockdown in A549 cells inhibits vesicular stomatitis virus (VSV) replication and positively regulates the type I interferon signaling pathway, evidenced by upregulated IFN-β mRNA and increased IRF3 phosphorylation upon poly(I:C) stimulation.\",\n      \"method\": \"siRNA knockdown, VSV-GFP infection model, plaque assay, RT-qPCR, Western blotting for IRF3 phosphorylation\",\n      \"journal\": \"Xi bao yu fen zi mian yi xue za zhi (Chinese journal of cellular and molecular immunology)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single lab, single knockdown approach with functional antiviral phenotype but limited pathway placement\",\n      \"pmids\": [\"40525335\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"TM9SF2 regulates PD-L1 levels by a dual mechanism: (1) recruiting phosphoglycerate kinase 1 (PGK1) to promote PD-L1 recycling to the plasma membrane via endosomal recycling, and (2) dismantling HIP1R-mediated lysosomal degradation of PD-L1. Genetic or pharmacological 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 degradation.\",\n      \"method\": \"CRISPR screen (glycosphingolipid metabolism library), Co-immunoprecipitation of TM9SF2-PGK1 complex, PD-L1 trafficking assays, genetic KO, pharmacological ceramide treatment, antitumor immunity readouts\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — CRISPR screen discovery followed by Co-IP, trafficking assays, genetic and pharmacological manipulation with multiple orthogonal readouts\",\n      \"pmids\": [\"41888515\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TM9SF2 is a lysosomal/endosomal nonaspanin protein that regulates intracellular vesicle trafficking and membrane protein sorting: it maintains Golgi integrity through cholesterol homeostasis, promotes PD-L1 plasma membrane recycling by forming a complex with PGK1 (disrupted by ceramide to redirect PD-L1 to lysosomal degradation), facilitates AAV vector intracellular trafficking, supports Fcγ-mediated phagocytosis upstream of PLC-γ1 signaling, and modulates innate antiviral immunity via the type I interferon pathway.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\nI'll classify each paper first, then extract discoveries only from KEEP papers.\n\n**Classification:**\n\n- [1] EXCLUDE — alias collision: p76(MDM2), about MDM2 gene\n- [2] KEEP — about TM9SF2 (LINC01232 regulates TM9SF2 mRNA stability via EIF4A3)\n- [3] KEEP — about TM9SF2 as host factor for AAV transduction\n- [4] KEEP — about TM9SF2 as CRC oncogene\n- [5] KEEP — about p76 = LOC196463 = TM9SF2, lysosomal localization\n- [6] KEEP — Drosophila TM9SF2/TM9SF4 ortholog study, consistent with mammalian TM9SF2\n- [7] EXCLUDE — alias collision: p76(MDM2), about MDM2 gene\n- [8] EXCLUDE — alias collision: p76(Rb), about RB1 gene\n- [9] KEEP — about TM9SF2 in macrophage phagocytosis and OA\n- [10] EXCLUDE — alias collision: p76(Rb), about RB1 gene\n- [11] EXCLUDE — about P-76 antigen (unrelated protein)\n- [12] KEEP — about TM9SF2 in Golgi integrity and ricin cytotoxicity\n- [13] KEEP — about TM9SF2-PGK1 axis and PD-L1 trafficking\n- [14] KEEP — about TM9SF2 knockdown and interferon signaling/VSV replication\n- [15] EXCLUDE — bioinformatics tool; TM9SF2 mentioned only as a marker\n- [16] EXCLUDE — about SLC35A1; TM9SF2 mentioned only incidentally\n- [17] EXCLUDE — about tau/PHF; p76 refers to a peptide, not TM9SF2\n\n**Additional gene2pubmed papers:**\n- Most are large-scale proteomics/interactome studies or unrelated. Only those directly providing mechanistic data about TM9SF2 are relevant. Papers [3,6,7,8] (BioPlex, etc.) mention TM9SF2 only as part of large datasets with no specific mechanistic data extracted — these are cataloguing studies, not mechanistic ones. I'll exclude them from Discoveries unless they provide specific TM9SF2 mechanism data.\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2007,\n      \"finding\": \"TM9SF2 (then called 'p76'/LOC196463) is a mannose-6-phosphate (Man6P)-bearing glycoprotein with six N-glycosylation sites that localizes to lysosomes, as demonstrated by Man6P-dependent interaction with mannose-6-phosphate receptors (MPR overlay), progressive PNGase F deglycosylation, immunofluorescence, and subcellular fractionation of mouse liver homogenates.\",\n      \"method\": \"MPR overlay assay, PNGase F deglycosylation, immunofluorescence microscopy, subcellular fractionation\",\n      \"journal\": \"The Biochemical Journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (overlay assay, biochemical fractionation, immunofluorescence) in a single study with rigorous controls\",\n      \"pmids\": [\"17105447\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Drosophila TM9SF2 (ortholog of human 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 does not prevent plasma membrane localization of PGRP-LC (unlike TM9SF4 silencing), and both TM9 proteins control actin cytoskeleton organization in haemocytes, with evidence that TM9 proteins suppress inappropriate signaling from the unstimulated PGRP-LC receptor.\",\n      \"method\": \"Co-immunoprecipitation, RNAi knockdown, immunofluorescence co-localization, actin cytoskeleton assays in Drosophila S2 cells and fat body\",\n      \"journal\": \"Journal of Innate Immunity\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP and functional knockdown with defined phenotypic readout, but in Drosophila ortholog system\",\n      \"pmids\": [\"25139117\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CRISPR/Cas9 knockout of TM9SF2 substantially diminishes colorectal cancer (CRC) cell fitness in vitro and in vivo (anchorage-independent growth, tumor growth), and transcriptome analysis of knockout cells reveals TM9SF2 involvement in cell cycle progression, oxidative phosphorylation, and ceramide signaling.\",\n      \"method\": \"CRISPR/Cas9 knockout, RNAi silencing, anchorage-independent growth assay, in vivo tumor xenograft, transcriptome analysis\",\n      \"journal\": \"Scientific Reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with defined cellular and in vivo phenotype, but pathway placement is transcriptome-inferred rather than mechanistically dissected\",\n      \"pmids\": [\"30333512\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"LINC01232 recruits the RNA-binding protein EIF4A3 to boost TM9SF2 mRNA stability, thereby upregulating TM9SF2 expression; additionally, the transcription factor SP1 mediates transcriptional activation of both LINC01232 and TM9SF2, placing TM9SF2 downstream of a SP1/LINC01232/EIF4A3 regulatory axis in pancreatic adenocarcinoma.\",\n      \"method\": \"RNA pulldown, RIP assay, siRNA knockdown, luciferase reporter assay, qRT-PCR, mRNA stability assay\",\n      \"journal\": \"Cell Death & Disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — multiple methods identifying regulatory mechanism, but single-lab study\",\n      \"pmids\": [\"31541081\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Genome-wide CRISPR pooled screens identified TM9SF2 as a host cell factor required for AAV2 transduction across eight different AAV serotypes (including AAV1–4, AAV6–9); validation in flow cytometry and imaging studies confirmed TM9SF2's role, distinct from and complementary to GPR108 (which localizes to Golgi and shows serotype selectivity).\",\n      \"method\": \"Genome-wide CRISPR pooled screen, flow cytometry, imaging validation, multi-serotype AAV transduction assays\",\n      \"journal\": \"Molecular Therapy: Methods & Clinical Development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genome-wide unbiased screen with multi-serotype validation across multiple cell types\",\n      \"pmids\": [\"32280726\"],\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 in the Fcγ receptor-mediated phagocytic signaling pathway.\",\n      \"method\": \"siRNA knockdown, phagocytosis assay (IgG-coated beads and apoptotic cell co-culture), Western blotting for PLC-γ1 phosphorylation\",\n      \"journal\": \"Journal of Orthopaedic Surgery and Research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — functional knockdown with specific signaling readout, single-lab study\",\n      \"pmids\": [\"38218914\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TM9SF2 maintains Golgi integrity by regulating intracellular cholesterol trafficking; genetic knockdown of TM9SF2 causes abnormal cholesterol accumulation in Golgi compartments, severe Golgi fragmentation, and impaired retrograde transport of ricin, thereby attenuating ricin-induced cytotoxicity. Pharmacological restoration of cholesterol homeostasis (using A939572 or avasimibe) rescues Golgi integrity and reverses the ricin-resistant phenotype of TM9SF2-knockdown cells.\",\n      \"method\": \"siRNA/genetic knockdown, cholesterol trafficking assays (filipin staining, cholesterol fractionation), Golgi morphology imaging, ricin cytotoxicity assays, pharmacological rescue with A939572/avasimibe\",\n      \"journal\": \"Toxins\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods with pharmacological rescue validation, single-lab study\",\n      \"pmids\": [\"40423301\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TM9SF2 knockdown in A549 cells inhibits vesicular stomatitis virus (VSV) replication and positively regulates the type I interferon signaling pathway; specifically, TM9SF2 knockdown upregulates IFN-β mRNA and increases IRF3 phosphorylation upon poly(I:C) stimulation, indicating TM9SF2 normally suppresses innate antiviral immunity.\",\n      \"method\": \"siRNA knockdown, VSV-GFP infection model, plaque assay, RT-qPCR, Western blotting for IRF3 phosphorylation, poly(I:C) stimulation\",\n      \"journal\": \"Chinese Journal of Cellular and Molecular Immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — functional knockdown with defined molecular readouts, single-lab study\",\n      \"pmids\": [\"40525335\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"TM9SF2 orchestrates PD-L1 trafficking by: (1) recruiting phosphoglycerate kinase 1 (PGK1) to promote PD-L1 recycling to the plasma membrane, and (2) dismantling the HIP1R-mediated lysosomal degradation pathway for PD-L1. Genetic or pharmacological disruption of the TM9SF2-PGK1 complex depletes surface PD-L1 and enhances antitumor immunity. The endogenous ceramide species Cer(d18:1/26:0) destabilizes the TM9SF2-PGK1 complex, triggering PD-L1 lysosomal degradation; this mechanism was identified via CRISPR screen targeting glycosphingolipid metabolism.\",\n      \"method\": \"CRISPR screen (glycosphingolipid metabolism library), co-immunoprecipitation, PD-L1 trafficking assays, genetic knockdown/knockout, pharmacological disruption, ceramide treatment, antitumor immunity assays\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — unbiased CRISPR screen plus reciprocal Co-IP, mechanistic dissection of dual pathway with pharmacological and genetic validation\",\n      \"pmids\": [\"41888515\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TM9SF2 is a lysosome-localized nonaspanin (9-transmembrane) glycoprotein that regulates intracellular vesicle trafficking and membrane receptor dynamics: it maintains Golgi integrity via cholesterol trafficking control, promotes PD-L1 recycling to the plasma membrane by recruiting PGK1 while suppressing HIP1R-mediated lysosomal degradation (a complex disrupted by ceramide Cer(d18:1/26:0)), supports phagocytosis upstream of PLC-γ1 in macrophages, acts as an essential host factor for AAV transduction across multiple serotypes, suppresses innate antiviral type I interferon signaling, and in Drosophila orthologs co-immunoprecipitates with and controls plasma membrane localization of the PGRP-LC immune receptor while regulating actin cytoskeleton organization.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"TM9SF2 is a nonaspanin transmembrane protein that functions as a regulator of intracellular vesicle trafficking, membrane protein sorting, and cholesterol homeostasis across endosomal, lysosomal, and Golgi compartments. It is a mannose-6-phosphate-tagged lysosomal glycoprotein whose targeting depends on direct interaction with mannose-6-phosphate receptors [PMID:17105447], and it maintains Golgi integrity by controlling intracellular cholesterol distribution, with its loss causing cholesterol accumulation, Golgi fragmentation, and impaired retrograde transport [PMID:40423301]. TM9SF2 promotes PD-L1 recycling to the plasma membrane by recruiting PGK1 to form a complex that opposes HIP1R-mediated lysosomal degradation; the endogenous ceramide Cer(d18:1/26:0) destabilizes this complex, redirecting PD-L1 to lysosomes and enhancing antitumor immunity [PMID:41888515]. TM9SF2 additionally supports Fcγ-mediated phagocytosis upstream of PLCγ1 signaling [PMID:38218914], facilitates intracellular trafficking of adeno-associated virus vectors across multiple serotypes [PMID:32280726], and promotes colorectal cancer cell fitness through effects on cell cycle progression and oxidative phosphorylation [PMID:30333512].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Establishing the basic identity and targeting mechanism of TM9SF2: the protein was shown to be a mannose-6-phosphate-tagged lysosomal glycoprotein that reaches lysosomes through direct, Man6P-dependent binding to mannose-6-phosphate receptors, resolving how a nonaspanin is sorted to the endo-lysosomal system.\",\n      \"evidence\": \"MPR overlay binding assay, PNGase F deglycosylation, immunofluorescence and subcellular fractionation in mouse liver\",\n      \"pmids\": [\"17105447\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Functional consequence of lysosomal localization unknown\",\n        \"Whether Man6P tagging is required for all cellular functions of TM9SF2 not tested\",\n        \"No interacting partners beyond MPRs identified\"\n      ]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Extending TM9SF2 function to innate immunity and phagocytosis: in Drosophila, TM9SF2 was found to co-immunoprecipitate with the innate immune receptor PGRP-LC and to regulate phagocytosis and the actin cytoskeleton, establishing it as a trafficking factor that controls immune receptor surface presentation.\",\n      \"evidence\": \"Co-immunoprecipitation, co-localization, and RNAi silencing with phagocytosis readouts in Drosophila S2 cells and haemocytes\",\n      \"pmids\": [\"25139117\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Mammalian conservation of PGRP-LC-like interactions not demonstrated\",\n        \"Mechanism linking TM9SF2 to actin cytoskeleton control unresolved\",\n        \"Single lab study\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Linking TM9SF2 to cancer cell fitness: CRISPR knockout and RNAi in colorectal cancer cells showed TM9SF2 is required for anchorage-independent growth and xenograft tumor formation, with transcriptomic analysis implicating cell cycle, oxidative phosphorylation, and ceramide signaling pathways.\",\n      \"evidence\": \"CRISPR/Cas9 KO and RNAi with in vitro growth and in vivo xenograft assays, RNA-seq in colorectal cancer cells\",\n      \"pmids\": [\"30333512\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct molecular targets mediating oncogenic role not identified\",\n        \"Ceramide signaling link was correlative (transcriptomic); causal mechanism not dissected at this stage\",\n        \"Not tested in non-cancer cell contexts\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Demonstrating a broad role in intracellular vesicle trafficking: a genome-wide CRISPR screen identified TM9SF2 as a host factor required for transduction by multiple AAV serotypes (AAV1–9), establishing that TM9SF2 facilitates endosomal trafficking of incoming cargo beyond its known lysosomal residence.\",\n      \"evidence\": \"Genome-wide pooled CRISPR/Cas9 screen with flow cytometry and imaging validation across 8 AAV serotypes in multiple cell types\",\n      \"pmids\": [\"32280726\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Step in AAV intracellular trafficking controlled by TM9SF2 not pinpointed (entry, endosomal escape, nuclear import)\",\n        \"No direct physical interaction between TM9SF2 and AAV particles shown\",\n        \"Whether cholesterol trafficking underlies the AAV phenotype not tested\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Revealing the mechanistic basis for Golgi integrity maintenance: TM9SF2 knockdown caused abnormal cholesterol accumulation in the Golgi leading to fragmentation and impaired retrograde transport, and pharmacological correction of cholesterol metabolism rescued Golgi morphology, establishing cholesterol trafficking as a core cellular function of TM9SF2.\",\n      \"evidence\": \"siRNA knockdown, cholesterol trafficking assays, Golgi morphology imaging, ricin cytotoxicity assays, pharmacological rescue with A939572 and avasimibe\",\n      \"pmids\": [\"40423301\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct molecular mechanism by which TM9SF2 regulates cholesterol transport not identified (transporter, sensor, or scaffolding role unclear)\",\n        \"Relationship between cholesterol trafficking function and the AAV/phagocytosis phenotypes not established\",\n        \"Single lab study\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Implicating TM9SF2 in antiviral innate immunity: TM9SF2 silencing enhanced type I interferon signaling (elevated IFN-β, increased IRF3 phosphorylation) and restricted VSV replication, suggesting TM9SF2 normally dampens innate antiviral responses.\",\n      \"evidence\": \"siRNA knockdown, VSV-GFP infection, plaque assay, RT-qPCR, Western blot for p-IRF3 in A549 cells\",\n      \"pmids\": [\"40525335\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"Single knockdown approach without genetic rescue or orthogonal loss-of-function validation\",\n        \"Mechanism connecting TM9SF2 trafficking function to IRF3 activation not dissected\",\n        \"Not tested beyond a single cell line and virus\"\n      ]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Defining a PD-L1 recycling mechanism: TM9SF2 was shown to recruit PGK1 to form a complex that promotes PD-L1 endosomal recycling to the plasma membrane while opposing HIP1R-mediated lysosomal degradation; the endogenous ceramide Cer(d18:1/26:0) destabilizes this complex, triggering PD-L1 degradation and enhancing antitumor immunity — directly connecting the earlier ceramide signaling association to a concrete trafficking mechanism.\",\n      \"evidence\": \"Glycosphingolipid-focused CRISPR screen, Co-IP of TM9SF2–PGK1, PD-L1 trafficking assays, genetic KO, pharmacological ceramide treatment with antitumor immunity readouts\",\n      \"pmids\": [\"41888515\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether TM9SF2–PGK1 complex regulates recycling of membrane proteins beyond PD-L1 is unknown\",\n        \"Structural basis of ceramide-mediated complex disruption not resolved\",\n        \"In vivo validation in immunocompetent tumor models limited\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A unifying structural and mechanistic model for how TM9SF2 coordinates cholesterol homeostasis, endosomal recycling, and phagocytosis through its nine transmembrane domains is still lacking; whether the cholesterol-trafficking and PGK1-dependent recycling functions represent a single integrated mechanism or parallel activities remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No structural information (cryo-EM, crystallography) for TM9SF2 or its complexes\",\n        \"Whether TM9SF2 cholesterol regulation and PD-L1 recycling are mechanistically coupled is untested\",\n        \"Tissue-specific and in vivo functions in normal physiology remain largely uncharacterized\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [6, 8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [1, 8]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [1, 4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [0, 4, 6, 8]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [1, 5]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"complexes\": [\n      \"TM9SF2–PGK1 complex\"\n    ],\n    \"partners\": [\n      \"PGK1\",\n      \"PGRP-LC\",\n      \"HIP1R\",\n      \"EIF4A3\",\n      \"SP1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"TM9SF2 is a lysosome-localized, nine-transmembrane (nonaspanin) glycoprotein that governs intracellular vesicle trafficking, membrane receptor dynamics, and innate immune signaling. It maintains Golgi integrity by controlling intracellular cholesterol distribution, with its depletion causing cholesterol accumulation in Golgi compartments and consequent Golgi fragmentation [PMID:40423301], and it orchestrates PD-L1 surface expression by recruiting PGK1 to promote PD-L1 recycling while suppressing HIP1R-mediated lysosomal degradation—a complex destabilized by the ceramide species Cer(d18:1/26:0) [PMID:41888515]. TM9SF2 functions as a broadly required host factor for adeno-associated virus transduction across multiple serotypes [PMID:32280726], promotes Fcγ receptor-mediated phagocytosis upstream of PLCγ1 in macrophages [PMID:38218914], and suppresses type I interferon signaling by restraining IRF3 phosphorylation [PMID:40525335]. In Drosophila, the ortholog physically associates with the innate immune receptor PGRP-LC and regulates actin cytoskeleton organization in haemocytes [PMID:25139117].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Establishing the basic identity of TM9SF2 as a mannose-6-phosphate-bearing lysosomal glycoprotein with six N-glycosylation sites resolved its subcellular address and post-translational processing, framing it as a potential regulator of endolysosomal trafficking.\",\n      \"evidence\": \"MPR overlay assay, PNGase F deglycosylation, immunofluorescence, and subcellular fractionation in mouse liver\",\n      \"pmids\": [\"17105447\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No functional role or binding partners identified at this stage\",\n        \"Whether lysosomal localization is constitutive or regulated was not addressed\",\n        \"Mechanism of lysosomal targeting beyond Man6P modification unknown\"\n      ]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Demonstrating that the Drosophila TM9SF2 ortholog physically associates with the innate immune receptor PGRP-LC and regulates actin organization in haemocytes established the first link between TM9SF family members and immune receptor trafficking and cytoskeletal control.\",\n      \"evidence\": \"Co-immunoprecipitation, RNAi knockdown, immunofluorescence co-localization, and actin assays in Drosophila S2 cells\",\n      \"pmids\": [\"25139117\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Findings are in Drosophila; conservation of PGRP-LC-like interaction in mammals unconfirmed\",\n        \"Molecular mechanism linking TM9SF2 to actin remodeling not identified\",\n        \"Whether TM9SF2 directly controls receptor surface delivery versus retention was not resolved\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"CRISPR knockout revealing that TM9SF2 loss impairs colorectal cancer cell fitness in vitro and in xenografts connected TM9SF2 to cell proliferation, while transcriptome analysis implicated it in cell cycle, oxidative phosphorylation, and ceramide signaling pathways.\",\n      \"evidence\": \"CRISPR/Cas9 knockout, anchorage-independent growth assay, xenograft tumor model, and transcriptome profiling in CRC cells\",\n      \"pmids\": [\"30333512\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Pathway involvement is transcriptome-inferred, not mechanistically validated\",\n        \"Whether growth defect is cell-autonomous or microenvironment-dependent was not dissected\",\n        \"Direct molecular targets mediating TM9SF2 pro-proliferative effect not identified\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Genome-wide CRISPR screens identifying TM9SF2 as an essential host factor for AAV transduction across eight serotypes demonstrated a broad, serotype-independent role in viral entry or intracellular trafficking, distinct from the Golgi-localized factor GPR108.\",\n      \"evidence\": \"Genome-wide CRISPR pooled screen with multi-serotype AAV transduction validation by flow cytometry and imaging\",\n      \"pmids\": [\"32280726\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Precise step in the AAV entry/trafficking pathway mediated by TM9SF2 remains undefined\",\n        \"Whether TM9SF2 acts at the endosomal escape or uncoating stage was not resolved\",\n        \"Structural basis for TM9SF2 requirement across diverse AAV capsids unknown\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Showing that TM9SF2 knockdown impairs Fcγ receptor-mediated phagocytosis and attenuates PLCγ1 activation in macrophages placed TM9SF2 upstream of a defined signaling node in innate immune effector function.\",\n      \"evidence\": \"siRNA knockdown in THP-1-derived M2 macrophages, phagocytosis assays with IgG-coated beads and apoptotic cells, Western blotting for PLCγ1 phosphorylation\",\n      \"pmids\": [\"38218914\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Single-lab study; independent replication in primary macrophages not reported\",\n        \"Whether TM9SF2 acts on Fcγ receptor surface expression, clustering, or downstream adaptor recruitment is unknown\",\n        \"Relationship between lysosomal localization and phagocytic function not mechanistically explained\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Discovering that TM9SF2 maintains Golgi integrity through cholesterol trafficking control—and that pharmacological restoration of cholesterol homeostasis rescues Golgi fragmentation in TM9SF2-depleted cells—provided the first mechanistic explanation for TM9SF2's broad influence on membrane trafficking.\",\n      \"evidence\": \"siRNA knockdown, filipin staining, cholesterol fractionation, Golgi morphology imaging, ricin cytotoxicity and retrograde transport assays, pharmacological rescue with A939572/avasimibe\",\n      \"pmids\": [\"40423301\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Single-lab study; the cholesterol sensor or transporter that TM9SF2 directly regulates is unidentified\",\n        \"Whether Golgi fragmentation explains the AAV and phagocytosis phenotypes was not tested\",\n        \"Contribution of individual TM9SF2 transmembrane domains to cholesterol sensing unknown\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrating that TM9SF2 knockdown enhances IRF3 phosphorylation and IFN-β induction upon poly(I:C) stimulation revealed TM9SF2 as a negative regulator of type I interferon signaling, linking its trafficking function to innate antiviral immunity.\",\n      \"evidence\": \"siRNA knockdown in A549 cells, VSV-GFP infection, plaque assay, RT-qPCR, Western blotting for phospho-IRF3\",\n      \"pmids\": [\"40525335\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Single-lab study; mechanism by which TM9SF2 restrains IRF3 activation (e.g. receptor turnover, adaptor sequestration) not identified\",\n        \"Whether this reflects altered STING or RIG-I/MAVS pathway trafficking was not examined\",\n        \"In vivo relevance of immune suppression not tested\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Mechanistic dissection of TM9SF2's role in PD-L1 trafficking—recruiting PGK1 for recycling while opposing HIP1R-mediated lysosomal degradation, with ceramide Cer(d18:1/26:0) as an endogenous disruptor of the TM9SF2-PGK1 complex—provided a unified model for how TM9SF2 coordinates receptor surface expression with therapeutic implications for antitumor immunity.\",\n      \"evidence\": \"CRISPR screen targeting glycosphingolipid metabolism, co-immunoprecipitation, PD-L1 trafficking assays, genetic and pharmacological disruption, ceramide treatment, antitumor immunity assays\",\n      \"pmids\": [\"41888515\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether TM9SF2-PGK1 recycling applies to receptors beyond PD-L1 is untested\",\n        \"Structural basis of the TM9SF2-PGK1 interaction and ceramide-mediated disruption unresolved\",\n        \"In vivo pharmacological targeting of TM9SF2-PGK1 axis not yet evaluated in clinical models\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A unifying structural and mechanistic model explaining how TM9SF2 coordinates cholesterol trafficking, receptor recycling, Golgi maintenance, and innate immune regulation through its nine-transmembrane architecture remains to be established.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No crystal or cryo-EM structure of TM9SF2 exists\",\n        \"Whether cholesterol-binding is direct and mediates all downstream phenotypes is unknown\",\n        \"Relative importance of TM9SF2 in different tissue and immune cell contexts has not been systematically compared\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [7, 8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [0, 8]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [1, 8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [6, 8]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [5, 7]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"PGK1\",\n      \"HIP1R\",\n      \"PGRP-LC\",\n      \"PD-L1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}