{"gene":"TMEM9","run_date":"2026-04-28T21:42:59","timeline":{"discoveries":[{"year":2002,"finding":"TMEM9 is a transmembrane protein that localizes to late endosomes and lysosomes (co-localizing with LAMP1) as well as ER, contains three N-glycosylation sites and three cysteine-rich domains, and is expressed as glycosylated forms of ~28, 31, and 33 kDa from a ~26 kDa protein backbone.","method":"Transfection of TMEM9-GFP in COS-1 cells with co-localization to LAMP1 by fluorescence microscopy; Western blot of glycosylated forms","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 — direct localization experiment with GFP fusion and LAMP1 co-localization, single lab","pmids":["12359240"],"is_preprint":false},{"year":2018,"finding":"TMEM9 binds to and facilitates assembly of vacuolar-ATPase (v-ATPase), enhancing vesicular acidification and trafficking, which leads to lysosomal degradation of APC, thereby hyperactivating Wnt/β-catenin signaling in colorectal cancer. TMEM9 is itself transcriptionally activated by β-catenin, forming a positive feedback loop.","method":"Proteomic analysis, co-immunoprecipitation (TMEM9-v-ATPase interaction), v-ATPase assembly assay, lysosomal acidification assay, APC degradation assay, genetic ablation (KO) in vitro/in vivo, v-ATPase inhibitor treatment","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal methods (proteomics, Co-IP, functional assays, KO mouse models, pharmacological inhibition), replicated across cancer contexts","pmids":["30374053"],"is_preprint":false},{"year":2018,"finding":"TMEM9 overexpression increases IL-6 and IL-1β secretion in LX-2 cells treated with TNF-α, and this effect is associated with upregulation of canonical Wnt/β-catenin signaling components (wnt2b, wnt3a, β-catenin); TMEM9 knockdown reduces these cytokines.","method":"Overexpression (pEGFP-C2-TMEM9) and siRNA knockdown in LX-2 cells; ELISA for cytokines; Western blot for Wnt pathway proteins","journal":"International immunopharmacology","confidence":"Low","confidence_rationale":"Tier 3 — single lab, gain/loss-of-function with phenotypic readout but limited mechanistic depth","pmids":["30119033"],"is_preprint":false},{"year":2020,"finding":"TMEM9 facilitates v-ATPase assembly for vesicular acidification and lysosomal protein degradation of APC in hepatocytes; Tmem9 knockout in mice impairs hepatic regeneration with aberrantly increased APC and reduced Wnt signaling; in HCC, TMEM9 maintains β-catenin hyperactivation through lysosomal APC degradation independent of β-catenin mutations.","method":"Tmem9 knockout mice (liver regeneration model), pharmacological blockade of v-ATPase/lysosomal degradation, Western blot for APC/β-catenin, HCC cell lines","journal":"Hepatology (Baltimore, Md.)","confidence":"High","confidence_rationale":"Tier 2 — KO mouse model with defined phenotype, pharmacological validation, mechanistic consistency with prior study, two independent cancer contexts","pmids":["32380568"],"is_preprint":false},{"year":2024,"finding":"TMEM9 promotes lung adenocarcinoma progression by activating the MEK/ERK/STAT3 pathway to upregulate VEGF expression; VEGF-neutralizing antibodies reversed angiogenesis and migration phenotypes caused by TMEM9 overexpression.","method":"TMEM9 knockdown and overexpression in LUAD cells; VEGF-neutralizing antibody rescue; recombinant VEGF rescue; Western blot for MEK/ERK/STAT3; in vitro/in vivo tumor models; HUVEC co-culture angiogenesis assay","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal methods (KD/OE, rescue experiments, pathway inhibition), single lab","pmids":["38664392"],"is_preprint":false},{"year":2024,"finding":"TMEM9 activates Rab9-dependent alternative autophagy through direct interaction with Beclin1 via Beclin1's Bcl-2-binding domain; this interaction dissociates Bcl-2 from Beclin1 and activates LC3-independent, Rab9-dependent autophagy. N-glycosylation of TMEM9 is required for its lysosomal localization and its interaction with Beclin1 to activate this pathway.","method":"Co-immunoprecipitation (TMEM9-Beclin1 and TMEM9-Bcl-2 interactions), domain mapping, glycosylation mutants, co-localization with Rab9/LC3, autophagy flux assays","journal":"Cellular and molecular life sciences : CMLS","confidence":"High","confidence_rationale":"Tier 1-2 — reciprocal Co-IP, domain-level mapping, glycosylation mutants, co-localization, multiple autophagy readouts, single lab with multiple orthogonal methods","pmids":["39078420"],"is_preprint":false},{"year":2025,"finding":"TMEM9 microglial protein contributes to complement activation by regulating ATP6V0D1, a V-ATPase subunit; downregulation of microglial TMEM9 restrains complement (C1q) activity and decreases microglia-mediated synaptic engulfment in an Alzheimer's disease mouse model.","method":"Tmem9 knockdown and overexpression in BV2 cells and 5xFAD mice; C1q activation assay; synaptic engulfment quantification; physical exercise intervention","journal":"Aging cell","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo KO/OE with defined complement and synaptic phenotypes, single lab","pmids":["39871402"],"is_preprint":false},{"year":2025,"finding":"TMEM9 functions as an accessory β-subunit of ClC-3, ClC-4, and ClC-5 endosomal Cl-/H+ antiporters; cryo-EM structures reveal TMEM9 inhibits ClC-3 by sealing the cytosolic entrance to the Cl- ion pathway; phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) stabilizes the TMEM9–ClC-3 interaction and is required for proper regulation of ClC-3 by TMEM9.","method":"Cryo-electron microscopy structure determination; direct interaction demonstrated; PtdIns(3,5)P2 co-factor requirement established biochemically","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 — cryo-EM structure with functional validation, lipid co-factor requirement, multiple CLC family members tested","pmids":["40670814"],"is_preprint":false},{"year":2025,"finding":"TMEM9 interacts with ClC-5 in renal proximal tubule epithelial cells; TMEM9 knockdown recapitulates Dent's Disease type 1 characteristics (defective endocytosis, epithelial dedifferentiation) but paradoxically enhances endosomal acidification; TMEM9 loss also causes enlarged endosomes and Golgi fragmentation.","method":"Interactome analysis (Co-IP), TMEM9 knockdown in renal proximal tubule cell lines, endocytosis assay, endosomal pH measurement, morphological analysis","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2-3 — interactome plus KD phenotype in relevant cell type, preprint not yet peer-reviewed","pmids":["bio_10.1101_2025.11.03.686312"],"is_preprint":true},{"year":2025,"finding":"TMEM9 and TMEM9B form structural complexes with CLCN3/4/5 chloride-proton antiporters on early endosomes, as validated by cross-linking mass spectrometry of purified human early endosomes and structural predictions.","method":"Cross-linking mass spectrometry of purified early endosomes, AlphaFold structural modeling, native gel MS","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 — cross-linking MS from native subcellular context supports the structural findings of the cryo-EM study","pmids":["bio_10.1101_2025.02.07.636106"],"is_preprint":true}],"current_model":"TMEM9 is a lysosomal/late endosomal transmembrane glycoprotein that acts as (1) an accessory β-subunit of endosomal CLC Cl-/H+ antiporters (ClC-3/4/5), inhibiting ClC-3 activity by sealing the cytosolic Cl- pathway in a PtdIns(3,5)P2-dependent manner; (2) a facilitator of v-ATPase assembly that enhances vesicular acidification and lysosomal degradation of APC, thereby hyperactivating Wnt/β-catenin signaling; and (3) an activator of Rab9-dependent alternative autophagy through its glycosylation-dependent interaction with Beclin1, which displaces Bcl-2 and induces LC3-independent autophagosome formation."},"narrative":{"teleology":[{"year":2002,"claim":"Establishing TMEM9 as a glycosylated endolysosomal transmembrane protein resolved its subcellular context and post-translational processing, setting the stage for understanding its organelle-specific functions.","evidence":"GFP-tagged TMEM9 expressed in COS-1 cells co-localized with LAMP1 by fluorescence microscopy; Western blot revealed multiple N-glycosylated forms","pmids":["12359240"],"confidence":"Medium","gaps":["No functional role assigned; localization based on overexpressed GFP fusion only","Endogenous protein localization not confirmed","Topology within endolysosomal membranes not determined"]},{"year":2018,"claim":"Discovery that TMEM9 facilitates v-ATPase assembly and promotes lysosomal APC degradation revealed TMEM9 as a critical amplifier of Wnt/β-catenin signaling, linking endosomal acidification to oncogenic pathway activation.","evidence":"Proteomics, co-IP of TMEM9–v-ATPase, v-ATPase assembly and acidification assays, APC degradation assays, TMEM9 KO in vitro/in vivo in colorectal cancer models","pmids":["30374053"],"confidence":"High","gaps":["Direct binding interface between TMEM9 and v-ATPase subunits not structurally resolved","Whether TMEM9 regulates v-ATPase independently of its CLC antiporter role is unclear","APC degradation selectivity mechanism not defined"]},{"year":2020,"claim":"Extending the v-ATPase/APC axis to liver physiology and hepatocellular carcinoma established TMEM9 as a tissue-general regulator of Wnt signaling, demonstrating its requirement for hepatic regeneration.","evidence":"Tmem9 knockout mice showed impaired liver regeneration with elevated APC and reduced Wnt signaling; v-ATPase/lysosomal inhibitor phenocopied the defect; validated in HCC cell lines","pmids":["32380568"],"confidence":"High","gaps":["Whether TMEM9's regeneration role extends to other rapidly renewing tissues is unknown","Contribution of autophagy versus lysosomal degradation to APC turnover not distinguished"]},{"year":2024,"claim":"Identification of TMEM9 as an activator of Rab9-dependent alternative autophagy via glycosylation-dependent Beclin1 binding uncovered a second major cellular function independent of classical autophagy.","evidence":"Reciprocal co-IP of TMEM9–Beclin1, domain mapping to Bcl-2-binding region, glycosylation mutants abolishing the interaction and lysosomal localization, Rab9/LC3 co-localization, autophagy flux assays","pmids":["39078420"],"confidence":"High","gaps":["Physiological stimuli triggering TMEM9-dependent alternative autophagy are not identified","Whether TMEM9's autophagy and v-ATPase functions are coordinated or mutually exclusive is unclear","In vivo validation of the alternative autophagy pathway via TMEM9 is lacking"]},{"year":2024,"claim":"Demonstration that TMEM9 activates MEK/ERK/STAT3 signaling to drive VEGF-mediated angiogenesis in lung adenocarcinoma expanded its oncogenic repertoire beyond Wnt/β-catenin.","evidence":"TMEM9 KD/OE in LUAD cells, VEGF-neutralizing antibody rescue, pathway Western blots, HUVEC co-culture angiogenesis assays, in vivo tumor models","pmids":["38664392"],"confidence":"Medium","gaps":["Whether MEK/ERK/STAT3 activation is downstream of v-ATPase assembly or a distinct mechanism is not resolved","Direct molecular link between TMEM9 and MEK/ERK pathway initiation not established"]},{"year":2025,"claim":"Cryo-EM structures of TMEM9 bound to ClC-3 established it as a bona fide accessory β-subunit of endosomal CLC antiporters, resolving its inhibitory mechanism through occlusion of the cytosolic Cl⁻ pathway and identifying PtdIns(3,5)P₂ as a stabilizing lipid cofactor.","evidence":"Cryo-EM structure determination of TMEM9–ClC-3 complex; biochemical demonstration of PtdIns(3,5)P₂ requirement; interaction validated with ClC-4 and ClC-5","pmids":["40670814"],"confidence":"High","gaps":["Whether TMEM9 regulation of CLC antiporters is dynamic in response to cellular signals is unknown","Functional consequences of TMEM9-mediated CLC inhibition for cargo sorting and endosomal maturation are not fully characterized","Structural basis for TMEM9 versus TMEM9B specificity among CLC family members not determined"]},{"year":2025,"claim":"TMEM9 regulation of complement activation via ATP6V0D1 in microglia connected its v-ATPase function to neuroinflammation and synapse elimination in Alzheimer's disease models.","evidence":"TMEM9 KD/OE in BV2 cells and 5xFAD mice; C1q activation and synaptic engulfment assays","pmids":["39871402"],"confidence":"Medium","gaps":["Whether TMEM9's microglial role involves CLC antiporter regulation in addition to v-ATPase is untested","Mechanism linking v-ATPase to C1q activation not molecularly defined","Single disease model without independent replication"]},{"year":null,"claim":"How TMEM9's dual roles as a CLC antiporter subunit and v-ATPase assembly factor are coordinated, whether these functions are mutually exclusive or operate on distinct subcellular pools, and the physiological triggers that switch between its acidification, signaling, and autophagy functions remain unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No integrated model reconciling CLC regulation and v-ATPase facilitation","Structural basis of TMEM9–v-ATPase interaction unknown","In vivo role of TMEM9-dependent alternative autophagy not tested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[1,3,7]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[7,9]}],"localization":[{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[0,1,5]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[0,7,9]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[0]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,3,4]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[5]},{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[7]}],"complexes":["ClC-3/TMEM9 complex","ClC-5/TMEM9 complex"],"partners":["CLCN3","CLCN4","CLCN5","ATP6V0D1","BECN1","BCL2"],"other_free_text":[]},"mechanistic_narrative":"TMEM9 is a glycosylated transmembrane protein of the endolysosomal system that regulates vesicular ion homeostasis, acidification, and signaling. It serves as an accessory β-subunit of endosomal ClC-3/4/5 Cl⁻/H⁺ antiporters, where cryo-EM structures show it inhibits ClC-3 by sealing the cytosolic Cl⁻ pathway in a PtdIns(3,5)P₂-dependent manner [PMID:40670814]. TMEM9 also facilitates v-ATPase assembly, promoting vesicular acidification and lysosomal degradation of APC, which hyperactivates Wnt/β-catenin signaling in colorectal cancer and hepatocellular carcinoma, with β-catenin transcriptionally activating TMEM9 to form a positive feedback loop [PMID:30374053, PMID:32380568]. Additionally, TMEM9 activates Rab9-dependent alternative autophagy by binding Beclin1 through its Bcl-2-binding domain in a glycosylation-dependent manner, displacing Bcl-2 and inducing LC3-independent autophagosome formation [PMID:39078420]."},"prefetch_data":{"uniprot":{"accession":"Q9P0T7","full_name":"Proton-transporting V-type ATPase complex assembly regulator TMEM9","aliases":["Dermal papilla-derived protein 4","Transmembrane protein 9","Protein TMEM9"],"length_aa":183,"mass_kda":20.6,"function":"Transmembrane protein that binds to and facilitates the assembly of lysosomal proton-transporting V-type ATPase (v-ATPase), resulting in enhanced lysosomal acidification and trafficking (PubMed:30374053). By bringing the v-ATPase accessory protein ATP6AP2 and the v-ATPase subunit ATP6V0D1 together, allows v-ATPase complex formation and activation (PubMed:30374053). TMEM9-controlled vesicular acidification induces hyperactivation of Wnt/beta-catenin signaling, involved in development, tissue homeostasis and tissue regeneration, through lysosomal degradation of adenomatous polyposis coli/APC (PubMed:30374053, PubMed:32380568). In the liver, involved in hepatic regeneration (PubMed:32380568)","subcellular_location":"Lysosome membrane; Late endosome membrane; Endosome, multivesicular body membrane","url":"https://www.uniprot.org/uniprotkb/Q9P0T7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TMEM9","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/TMEM9","total_profiled":1310},"omim":[{"mim_id":"620293","title":"TMEM9 DOMAIN FAMILY, MEMBER B; TMEM9B","url":"https://www.omim.org/entry/620293"},{"mim_id":"616877","title":"TRANSMEMBRANE PROTEIN 9; TMEM9","url":"https://www.omim.org/entry/616877"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Vesicles","reliability":"Approved"},{"location":"Microtubules","reliability":"Additional"},{"location":"Cytokinetic bridge","reliability":"Additional"},{"location":"Mitotic spindle","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TMEM9"},"hgnc":{"alias_symbol":["TMEM9A"],"prev_symbol":[]},"alphafold":{"accession":"Q9P0T7","domains":[{"cath_id":"3.10.20","chopping":"25-83","consensus_level":"high","plddt":87.7517,"start":25,"end":83},{"cath_id":"1.20.5","chopping":"86-117","consensus_level":"medium","plddt":82.2738,"start":86,"end":117}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9P0T7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9P0T7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9P0T7-F1-predicted_aligned_error_v6.png","plddt_mean":72.81},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TMEM9","jax_strain_url":"https://www.jax.org/strain/search?query=TMEM9"},"sequence":{"accession":"Q9P0T7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9P0T7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9P0T7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9P0T7"}},"corpus_meta":[{"pmid":"30374053","id":"PMC_30374053","title":"TMEM9 promotes intestinal tumorigenesis through vacuolar-ATPase-activated Wnt/β-catenin signalling.","date":"2018","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/30374053","citation_count":80,"is_preprint":false},{"pmid":"32380568","id":"PMC_32380568","title":"TMEM9-v-ATPase Activates Wnt/β-Catenin Signaling Via APC Lysosomal Degradation for Liver Regeneration and Tumorigenesis.","date":"2020","source":"Hepatology (Baltimore, Md.)","url":"https://pubmed.ncbi.nlm.nih.gov/32380568","citation_count":52,"is_preprint":false},{"pmid":"12359240","id":"PMC_12359240","title":"Characterization of the novel human transmembrane protein 9 (TMEM9) that localizes to lysosomes and late endosomes.","date":"2002","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/12359240","citation_count":21,"is_preprint":false},{"pmid":"30119033","id":"PMC_30119033","title":"TMEM9 mediates IL-6 and IL-1β secretion and is modulated by the Wnt pathway.","date":"2018","source":"International immunopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/30119033","citation_count":19,"is_preprint":false},{"pmid":"38664392","id":"PMC_38664392","title":"TMEM9 promotes lung adenocarcinoma progression via activating the MEK/ERK/STAT3 pathway to induce VEGF expression.","date":"2024","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/38664392","citation_count":15,"is_preprint":false},{"pmid":"27220462","id":"PMC_27220462","title":"Effects of TMEM9 gene on cell progression in hepatocellular carcinoma by RNA interference.","date":"2016","source":"Oncology reports","url":"https://pubmed.ncbi.nlm.nih.gov/27220462","citation_count":15,"is_preprint":false},{"pmid":"39078420","id":"PMC_39078420","title":"TMEM9 activates Rab9-dependent alternative autophagy through interaction with Beclin1.","date":"2024","source":"Cellular and molecular life sciences : CMLS","url":"https://pubmed.ncbi.nlm.nih.gov/39078420","citation_count":10,"is_preprint":false},{"pmid":"39871402","id":"PMC_39871402","title":"Physical Exercise Decreases Complement-Mediated Synaptic Loss and Protects Against Cognitive Impairment by Inhibiting Microglial Tmem9-ATP6V0D1 in Alzheimer's Disease.","date":"2025","source":"Aging cell","url":"https://pubmed.ncbi.nlm.nih.gov/39871402","citation_count":8,"is_preprint":false},{"pmid":"36596527","id":"PMC_36596527","title":"Tumor-Promoting Properties of TMEM9A in Breast Cancer Progression via Activating the Wnt/β-Catenin Signaling Pathway.","date":"2023","source":"Biological & pharmaceutical bulletin","url":"https://pubmed.ncbi.nlm.nih.gov/36596527","citation_count":6,"is_preprint":false},{"pmid":"40670814","id":"PMC_40670814","title":"Structural basis of ClC-3 transporter inhibition by TMEM9 and PtdIns(3,5)P2.","date":"2025","source":"Nature structural & molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/40670814","citation_count":3,"is_preprint":false},{"pmid":"40053281","id":"PMC_40053281","title":"Comprehensive analysis of TMEM9 in human tumors.","date":"2025","source":"Discover oncology","url":"https://pubmed.ncbi.nlm.nih.gov/40053281","citation_count":2,"is_preprint":false},{"pmid":"40093093","id":"PMC_40093093","title":"Structural basis of ClC-3 inhibition by TMEM9 and PI(3,5)P2.","date":"2025","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/40093093","citation_count":1,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.11.03.686312","title":"TMEM9 and SEC22B interact with ClC-5 to shape renal proximal tubule function and Dent’s Disease type I pathogenesis","date":"2025-11-04","source":"bioRxiv","url":"https://doi.org/10.1101/2025.11.03.686312","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.02.28.640562","title":"Structural basis of ClC-3 inhibition by TMEM9 and PI(3,5)P<sub>2</sub>","date":"2025-03-03","source":"bioRxiv","url":"https://doi.org/10.1101/2025.02.28.640562","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.02.07.636106","title":"EndoMAP.v1, a Structural Protein Complex Landscape of Human Endosomes","date":"2025-02-09","source":"bioRxiv","url":"https://doi.org/10.1101/2025.02.07.636106","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8529,"output_tokens":2776,"usd":0.033613},"stage2":{"model":"claude-opus-4-6","input_tokens":6135,"output_tokens":2440,"usd":0.137513},"total_usd":0.171126,"stage1_batch_id":"msgbatch_01RPbVc9PDH9zQmYK5Hs6srm","stage2_batch_id":"msgbatch_01JVbVLzeKN2yVrsGp3zC8aM","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2002,\n      \"finding\": \"TMEM9 is a transmembrane protein that localizes to late endosomes and lysosomes (co-localizing with LAMP1) as well as ER, contains three N-glycosylation sites and three cysteine-rich domains, and is expressed as glycosylated forms of ~28, 31, and 33 kDa from a ~26 kDa protein backbone.\",\n      \"method\": \"Transfection of TMEM9-GFP in COS-1 cells with co-localization to LAMP1 by fluorescence microscopy; Western blot of glycosylated forms\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct localization experiment with GFP fusion and LAMP1 co-localization, single lab\",\n      \"pmids\": [\"12359240\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TMEM9 binds to and facilitates assembly of vacuolar-ATPase (v-ATPase), enhancing vesicular acidification and trafficking, which leads to lysosomal degradation of APC, thereby hyperactivating Wnt/β-catenin signaling in colorectal cancer. TMEM9 is itself transcriptionally activated by β-catenin, forming a positive feedback loop.\",\n      \"method\": \"Proteomic analysis, co-immunoprecipitation (TMEM9-v-ATPase interaction), v-ATPase assembly assay, lysosomal acidification assay, APC degradation assay, genetic ablation (KO) in vitro/in vivo, v-ATPase inhibitor treatment\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods (proteomics, Co-IP, functional assays, KO mouse models, pharmacological inhibition), replicated across cancer contexts\",\n      \"pmids\": [\"30374053\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TMEM9 overexpression increases IL-6 and IL-1β secretion in LX-2 cells treated with TNF-α, and this effect is associated with upregulation of canonical Wnt/β-catenin signaling components (wnt2b, wnt3a, β-catenin); TMEM9 knockdown reduces these cytokines.\",\n      \"method\": \"Overexpression (pEGFP-C2-TMEM9) and siRNA knockdown in LX-2 cells; ELISA for cytokines; Western blot for Wnt pathway proteins\",\n      \"journal\": \"International immunopharmacology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single lab, gain/loss-of-function with phenotypic readout but limited mechanistic depth\",\n      \"pmids\": [\"30119033\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TMEM9 facilitates v-ATPase assembly for vesicular acidification and lysosomal protein degradation of APC in hepatocytes; Tmem9 knockout in mice impairs hepatic regeneration with aberrantly increased APC and reduced Wnt signaling; in HCC, TMEM9 maintains β-catenin hyperactivation through lysosomal APC degradation independent of β-catenin mutations.\",\n      \"method\": \"Tmem9 knockout mice (liver regeneration model), pharmacological blockade of v-ATPase/lysosomal degradation, Western blot for APC/β-catenin, HCC cell lines\",\n      \"journal\": \"Hepatology (Baltimore, Md.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO mouse model with defined phenotype, pharmacological validation, mechanistic consistency with prior study, two independent cancer contexts\",\n      \"pmids\": [\"32380568\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TMEM9 promotes lung adenocarcinoma progression by activating the MEK/ERK/STAT3 pathway to upregulate VEGF expression; VEGF-neutralizing antibodies reversed angiogenesis and migration phenotypes caused by TMEM9 overexpression.\",\n      \"method\": \"TMEM9 knockdown and overexpression in LUAD cells; VEGF-neutralizing antibody rescue; recombinant VEGF rescue; Western blot for MEK/ERK/STAT3; in vitro/in vivo tumor models; HUVEC co-culture angiogenesis assay\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (KD/OE, rescue experiments, pathway inhibition), single lab\",\n      \"pmids\": [\"38664392\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TMEM9 activates Rab9-dependent alternative autophagy through direct interaction with Beclin1 via Beclin1's Bcl-2-binding domain; this interaction dissociates Bcl-2 from Beclin1 and activates LC3-independent, Rab9-dependent autophagy. N-glycosylation of TMEM9 is required for its lysosomal localization and its interaction with Beclin1 to activate this pathway.\",\n      \"method\": \"Co-immunoprecipitation (TMEM9-Beclin1 and TMEM9-Bcl-2 interactions), domain mapping, glycosylation mutants, co-localization with Rab9/LC3, autophagy flux assays\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — reciprocal Co-IP, domain-level mapping, glycosylation mutants, co-localization, multiple autophagy readouts, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"39078420\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TMEM9 microglial protein contributes to complement activation by regulating ATP6V0D1, a V-ATPase subunit; downregulation of microglial TMEM9 restrains complement (C1q) activity and decreases microglia-mediated synaptic engulfment in an Alzheimer's disease mouse model.\",\n      \"method\": \"Tmem9 knockdown and overexpression in BV2 cells and 5xFAD mice; C1q activation assay; synaptic engulfment quantification; physical exercise intervention\",\n      \"journal\": \"Aging cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo KO/OE with defined complement and synaptic phenotypes, single lab\",\n      \"pmids\": [\"39871402\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TMEM9 functions as an accessory β-subunit of ClC-3, ClC-4, and ClC-5 endosomal Cl-/H+ antiporters; cryo-EM structures reveal TMEM9 inhibits ClC-3 by sealing the cytosolic entrance to the Cl- ion pathway; phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) stabilizes the TMEM9–ClC-3 interaction and is required for proper regulation of ClC-3 by TMEM9.\",\n      \"method\": \"Cryo-electron microscopy structure determination; direct interaction demonstrated; PtdIns(3,5)P2 co-factor requirement established biochemically\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structure with functional validation, lipid co-factor requirement, multiple CLC family members tested\",\n      \"pmids\": [\"40670814\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TMEM9 interacts with ClC-5 in renal proximal tubule epithelial cells; TMEM9 knockdown recapitulates Dent's Disease type 1 characteristics (defective endocytosis, epithelial dedifferentiation) but paradoxically enhances endosomal acidification; TMEM9 loss also causes enlarged endosomes and Golgi fragmentation.\",\n      \"method\": \"Interactome analysis (Co-IP), TMEM9 knockdown in renal proximal tubule cell lines, endocytosis assay, endosomal pH measurement, morphological analysis\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — interactome plus KD phenotype in relevant cell type, preprint not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.11.03.686312\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TMEM9 and TMEM9B form structural complexes with CLCN3/4/5 chloride-proton antiporters on early endosomes, as validated by cross-linking mass spectrometry of purified human early endosomes and structural predictions.\",\n      \"method\": \"Cross-linking mass spectrometry of purified early endosomes, AlphaFold structural modeling, native gel MS\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — cross-linking MS from native subcellular context supports the structural findings of the cryo-EM study\",\n      \"pmids\": [\"bio_10.1101_2025.02.07.636106\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"TMEM9 is a lysosomal/late endosomal transmembrane glycoprotein that acts as (1) an accessory β-subunit of endosomal CLC Cl-/H+ antiporters (ClC-3/4/5), inhibiting ClC-3 activity by sealing the cytosolic Cl- pathway in a PtdIns(3,5)P2-dependent manner; (2) a facilitator of v-ATPase assembly that enhances vesicular acidification and lysosomal degradation of APC, thereby hyperactivating Wnt/β-catenin signaling; and (3) an activator of Rab9-dependent alternative autophagy through its glycosylation-dependent interaction with Beclin1, which displaces Bcl-2 and induces LC3-independent autophagosome formation.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"TMEM9 is a glycosylated transmembrane protein of the endolysosomal system that regulates vesicular ion homeostasis, acidification, and signaling. It serves as an accessory β-subunit of endosomal ClC-3/4/5 Cl⁻/H⁺ antiporters, where cryo-EM structures show it inhibits ClC-3 by sealing the cytosolic Cl⁻ pathway in a PtdIns(3,5)P₂-dependent manner [PMID:40670814]. TMEM9 also facilitates v-ATPase assembly, promoting vesicular acidification and lysosomal degradation of APC, which hyperactivates Wnt/β-catenin signaling in colorectal cancer and hepatocellular carcinoma, with β-catenin transcriptionally activating TMEM9 to form a positive feedback loop [PMID:30374053, PMID:32380568]. Additionally, TMEM9 activates Rab9-dependent alternative autophagy by binding Beclin1 through its Bcl-2-binding domain in a glycosylation-dependent manner, displacing Bcl-2 and inducing LC3-independent autophagosome formation [PMID:39078420].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Establishing TMEM9 as a glycosylated endolysosomal transmembrane protein resolved its subcellular context and post-translational processing, setting the stage for understanding its organelle-specific functions.\",\n      \"evidence\": \"GFP-tagged TMEM9 expressed in COS-1 cells co-localized with LAMP1 by fluorescence microscopy; Western blot revealed multiple N-glycosylated forms\",\n      \"pmids\": [\"12359240\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No functional role assigned; localization based on overexpressed GFP fusion only\", \"Endogenous protein localization not confirmed\", \"Topology within endolysosomal membranes not determined\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Discovery that TMEM9 facilitates v-ATPase assembly and promotes lysosomal APC degradation revealed TMEM9 as a critical amplifier of Wnt/β-catenin signaling, linking endosomal acidification to oncogenic pathway activation.\",\n      \"evidence\": \"Proteomics, co-IP of TMEM9–v-ATPase, v-ATPase assembly and acidification assays, APC degradation assays, TMEM9 KO in vitro/in vivo in colorectal cancer models\",\n      \"pmids\": [\"30374053\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct binding interface between TMEM9 and v-ATPase subunits not structurally resolved\", \"Whether TMEM9 regulates v-ATPase independently of its CLC antiporter role is unclear\", \"APC degradation selectivity mechanism not defined\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Extending the v-ATPase/APC axis to liver physiology and hepatocellular carcinoma established TMEM9 as a tissue-general regulator of Wnt signaling, demonstrating its requirement for hepatic regeneration.\",\n      \"evidence\": \"Tmem9 knockout mice showed impaired liver regeneration with elevated APC and reduced Wnt signaling; v-ATPase/lysosomal inhibitor phenocopied the defect; validated in HCC cell lines\",\n      \"pmids\": [\"32380568\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether TMEM9's regeneration role extends to other rapidly renewing tissues is unknown\", \"Contribution of autophagy versus lysosomal degradation to APC turnover not distinguished\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identification of TMEM9 as an activator of Rab9-dependent alternative autophagy via glycosylation-dependent Beclin1 binding uncovered a second major cellular function independent of classical autophagy.\",\n      \"evidence\": \"Reciprocal co-IP of TMEM9–Beclin1, domain mapping to Bcl-2-binding region, glycosylation mutants abolishing the interaction and lysosomal localization, Rab9/LC3 co-localization, autophagy flux assays\",\n      \"pmids\": [\"39078420\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological stimuli triggering TMEM9-dependent alternative autophagy are not identified\", \"Whether TMEM9's autophagy and v-ATPase functions are coordinated or mutually exclusive is unclear\", \"In vivo validation of the alternative autophagy pathway via TMEM9 is lacking\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstration that TMEM9 activates MEK/ERK/STAT3 signaling to drive VEGF-mediated angiogenesis in lung adenocarcinoma expanded its oncogenic repertoire beyond Wnt/β-catenin.\",\n      \"evidence\": \"TMEM9 KD/OE in LUAD cells, VEGF-neutralizing antibody rescue, pathway Western blots, HUVEC co-culture angiogenesis assays, in vivo tumor models\",\n      \"pmids\": [\"38664392\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether MEK/ERK/STAT3 activation is downstream of v-ATPase assembly or a distinct mechanism is not resolved\", \"Direct molecular link between TMEM9 and MEK/ERK pathway initiation not established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Cryo-EM structures of TMEM9 bound to ClC-3 established it as a bona fide accessory β-subunit of endosomal CLC antiporters, resolving its inhibitory mechanism through occlusion of the cytosolic Cl⁻ pathway and identifying PtdIns(3,5)P₂ as a stabilizing lipid cofactor.\",\n      \"evidence\": \"Cryo-EM structure determination of TMEM9–ClC-3 complex; biochemical demonstration of PtdIns(3,5)P₂ requirement; interaction validated with ClC-4 and ClC-5\",\n      \"pmids\": [\"40670814\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether TMEM9 regulation of CLC antiporters is dynamic in response to cellular signals is unknown\", \"Functional consequences of TMEM9-mediated CLC inhibition for cargo sorting and endosomal maturation are not fully characterized\", \"Structural basis for TMEM9 versus TMEM9B specificity among CLC family members not determined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"TMEM9 regulation of complement activation via ATP6V0D1 in microglia connected its v-ATPase function to neuroinflammation and synapse elimination in Alzheimer's disease models.\",\n      \"evidence\": \"TMEM9 KD/OE in BV2 cells and 5xFAD mice; C1q activation and synaptic engulfment assays\",\n      \"pmids\": [\"39871402\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether TMEM9's microglial role involves CLC antiporter regulation in addition to v-ATPase is untested\", \"Mechanism linking v-ATPase to C1q activation not molecularly defined\", \"Single disease model without independent replication\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How TMEM9's dual roles as a CLC antiporter subunit and v-ATPase assembly factor are coordinated, whether these functions are mutually exclusive or operate on distinct subcellular pools, and the physiological triggers that switch between its acidification, signaling, and autophagy functions remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No integrated model reconciling CLC regulation and v-ATPase facilitation\", \"Structural basis of TMEM9–v-ATPase interaction unknown\", \"In vivo role of TMEM9-dependent alternative autophagy not tested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 3, 7]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [7, 9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [0, 1, 5]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [0, 7, 9]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 3, 4]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"complexes\": [\n      \"ClC-3/TMEM9 complex\",\n      \"ClC-5/TMEM9 complex\"\n    ],\n    \"partners\": [\n      \"CLCN3\",\n      \"CLCN4\",\n      \"CLCN5\",\n      \"ATP6V0D1\",\n      \"BECN1\",\n      \"BCL2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}