{"gene":"ATP6V0D1","run_date":"2026-06-09T22:02:44","timeline":{"discoveries":[{"year":2008,"finding":"RNAi knockdown of ATP6V0D1 (the human homologue of the Drosophila gene identified in a genome-wide screen) in HEK 293 cells significantly inhibited replication of H5N1 and H1N1 influenza A viruses but not vesicular stomatitis virus or vaccinia virus, establishing a specific host-factor role for ATP6V0D1 in influenza virus replication.","method":"Genome-wide RNAi screen in Drosophila followed by siRNA knockdown of human ATP6V0D1 in HEK 293 cells with influenza virus infection assay","journal":"Nature","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean knockdown with specific viral phenotype, replicated across two influenza strains, single lab","pmids":["18615018"],"is_preprint":false},{"year":2005,"finding":"ATP6V0D1 (d1 subunit) was localized by immunostaining to the apical membrane of principal cells in the rat epididymis and vas deferens in the apparent absence of other V-ATPase subunits, suggesting a physiological function distinct from its role in proton transport via the complete V-ATPase complex.","method":"Immunohistochemistry/immunofluorescence localization of V-ATPase subunit isoforms in rat epididymis and vas deferens","journal":"Biology of reproduction","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single localization study, no functional rescue or KO validation","pmids":["16192400"],"is_preprint":false},{"year":2008,"finding":"Zebrafish carrying a loss-of-function mutation in atp6v0d1 exhibited microphthalmic eyes, defective retinoblast cell cycle exit, elevated retinal apoptosis, abnormal photoreceptor outer segment morphology, RPE malformation with undigested outer segment material in vacuoles, and oculocutaneous albinism with melanosome biogenesis defects, demonstrating that ATP6V0D1 is required for v-ATPase complex function during vertebrate eye development.","method":"Genetic loss-of-function zebrafish mutant analysis with histology, BrdU incorporation, TUNEL assay, in situ hybridization, and electron microscopy","journal":"Investigative ophthalmology & visual science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal readouts in a clean genetic model, single lab","pmids":["18836173"],"is_preprint":false},{"year":2020,"finding":"siRNA knockdown of ATP6V0D1 in LLC-MK2 cells blocked the cytopathic effect of HCoV-NL63 coronavirus, and ATP6V0D1 knockdown was associated with increased lysosomal pH, implicating the subunit in lysosome-dependent coronavirus entry/replication.","method":"siRNA knockdown of ATP6V0D1 with coronavirus cytopathic effect assay and lysosomal pH measurement","journal":"ACS infectious diseases","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — clean knockdown with defined cytopathic phenotype, single lab, single method","pmids":["33346633"],"is_preprint":false},{"year":2022,"finding":"ATP6V0D1 was identified as a direct molecular target of JTC801 (the alkaliptosis-inducing drug) using mass-spectrometry-based target identification and cellular thermal shift assay. JTC801-mediated stabilization of ATP6V0D1 protein increases its interaction with STAT3, leading to increased STAT3 expression and activity that sustains lysosomal pH homeostasis. Genetic or pharmacological inhibition of STAT3 restored alkaliptosis sensitivity in ATP6V0D1-deficient pancreatic ductal adenocarcinoma cells.","method":"Mass-spectrometry-based drug target ID, cellular thermal shift assay, point mutation, co-immunoprecipitation, siRNA/gene knockout with cell death assays, mouse xenograft models","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (MS target ID, CETSA, mutagenesis, Co-IP, in vivo validation) in one study","pmids":["36640329"],"is_preprint":false},{"year":2022,"finding":"Schisandrol A (SolA) was shown to allosterically activate ATP6V0D1 by binding to cysteine 335, inducing a conformational change that activates V-ATPase-dependent lysosomal acidification. This lysosomal acidification selectively promoted mitochondrial BH3-only protein BIM degradation, preserving mitochondrial homeostasis and neuronal cell survival against AGEs-induced apoptosis.","method":"Drug target identification, cysteine-335 point mutation, lysosomal pH measurement, mitochondrial assays, cell survival assay","journal":"Acta pharmaceutica Sinica. B","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — site-directed mutagenesis identifying the binding residue, functional pH and cell survival readouts, multiple orthogonal methods in one study","pmids":["36213534"],"is_preprint":false},{"year":2022,"finding":"HPS6 (a BLOC-2 complex subunit) was shown to interact with ATP6V0D1 by co-immunoprecipitation. Knockdown of either HPS6 or ATP6V0D1 in HUVECs produced similar Weibel-Palade body defects (misshaped WPBs, decreased WPB number, impaired vWF tubulation), indicating that HPS6 transports ATP6V0D1 to the WPB limiting membrane for V-ATPase assembly and maintenance of acidic luminal pH required for WPB biogenesis.","method":"Co-immunoprecipitation, siRNA knockdown of HPS6 and ATP6V0D1, fluorescence microscopy of WPB morphology","journal":"Frontiers in cell and developmental biology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP plus parallel KD phenotypes showing convergent defects, single lab","pmids":["35252216"],"is_preprint":false},{"year":2024,"finding":"Adipose-specific deletion of Atp6v0d1 in mice caused generalized lipodystrophy, identifying ATP6V0D1 as a master regulator of adipogenesis. The resulting Atp6v0d1AKO mice developed spontaneous cardiomyopathy with cardiac insulin resistance (decreased IRS-1/2 expression), lipid accumulation, and increased FoxO1. Myocardin was downregulated in these hearts and shown by RNAi, luciferase reporter, and ChIP-qPCR to directly regulate IRS-1 transcription; restoring cardiac myocardin expression reversed metabolic gene dysregulation and improved cardiac function.","method":"Adipose-specific Atp6v0d1 knockout mice, RNA-seq, RNAi, luciferase reporter assay, ChIP-qPCR, in vivo AAV-mediated gene delivery","journal":"Theranostics","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean conditional KO with defined metabolic phenotype, multiple orthogonal mechanistic assays (RNA-seq, ChIP, reporter, in vivo rescue)","pmids":["38505620"],"is_preprint":false},{"year":2025,"finding":"Knockdown of ATP6V0D1 (but not ATP6V1H) in neuroblastoma cells enhanced sensitivity to ellipticine, suppressed proliferation and migration, decreased lysosomal drug uptake, and induced G2/M arrest. ATP6V0D1 knockdown also suppressed ellipticine-induced cytoplasmic vacuolation (ER swelling), establishing a specific role for ATP6V0D1 in lysosomal drug sequestration-mediated chemoresistance.","method":"siRNA knockdown of ATP6V0D1 and ATP6V1H, cell viability assay, migration assay, flow cytometry (cell cycle), lysosomal uptake assay, EM for vacuolation characterization","journal":"Molecular & cellular oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — parallel KD of two V-ATPase subunits as controls with multiple orthogonal phenotypic readouts, single lab","pmids":["40552114"],"is_preprint":false},{"year":2025,"finding":"Microglial Tmem9 was shown to regulate V-ATPase assembly through ATP6V0D1. In an Alzheimer's disease mouse model (5xFAD), Tmem9 downregulation (by physical exercise) inhibited C1q complement activation and reduced microglial synapse engulfment; overexpression of Tmem9 promoted complement activation. The pathway was placed as Tmem9 → ATP6V0D1 (V-ATPase subunit) → V-ATPase assembly → complement activation.","method":"In vivo exercise intervention in 5xFAD mice, Tmem9 overexpression/knockdown in BV2 cells treated with oAβ, complement activation assay, synapse engulfment assay","journal":"Aging cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo and in vitro gain/loss-of-function with defined complement and synaptic phenotypes, single lab; ATP6V0D1's precise mechanistic role is inferred from the pathway rather than directly tested","pmids":["39871402"],"is_preprint":false},{"year":2000,"finding":"The human VPATPD (ATP6V0D1) gene was characterized: it spans 19 kb, consists of 8 exons, and encodes a protein 99.5% identical to mouse subunit D at the amino acid level. The gene is located on chromosome 16q22, transcribed from the complementary strand relative to HSD11B2, with their 3' ends only 0.5 kb apart. Forskolin upregulates HSD11B2 but not VPATPD in JEG3 cells, showing distinct regulatory control despite genomic proximity.","method":"Gene cloning, sequencing, exon-intron mapping, RT-PCR, comparative sequence analysis, cell-based reporter/expression assay with forskolin","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct gene structure characterization with expression experiments; multiple methods in one study","pmids":["11118322"],"is_preprint":false},{"year":2024,"finding":"ATP6V0D1 overexpression or JTC801-mediated stabilization of ATP6V0D1 protein inhibited ABCB1 (multidrug resistance protein) upregulation in paclitaxel-resistant ovarian cancer cells, overcoming drug resistance via alkaliptosis. Increasing intracellular pH (pH 8.5 via NaOH) suppressed ABCB1 expression whereas acidification (pH 6.5 via HCl) amplified ABCB1, demonstrating that the ATP6V0D1-mediated alkaliptosis-ABCB1 axis mediates paclitaxel resistance.","method":"Gene transfection (ATP6V0D1 overexpression), JTC801 pharmacological treatment, intracellular pH manipulation, ABCB1 expression assay, cell growth inhibition assay","journal":"Molecular carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal interventions (genetic OE, pharmacological, pH manipulation) with defined resistance phenotype, single lab","pmids":["38751020"],"is_preprint":false},{"year":2009,"finding":"Control elements located within the first intron of ATP6V0D1 were shown experimentally to function as enhancers that modulate expression of the neighboring AgRP gene with spatiotemporal specificity, demonstrating that intronic sequences of ATP6V0D1 can act in trans on adjacent gene promoters.","method":"In vitro enhancer screening, transgenic mouse reporter assays with dietary/fasting challenges, comparative sequence analysis","journal":"Journal of molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct functional demonstration in transgenic mice with multiple reporter readouts; finding is about ATP6V0D1 genomic sequences acting as regulatory elements","pmids":["19285986"],"is_preprint":false},{"year":2020,"finding":"Affinity purification-mass spectrometry using PHF1-immunoreactive phosphorylated tau as bait from Alzheimer's disease brain tissue identified ATP6V0D1 as a protein that directly interacts with phosphorylated tau in neurofibrillary tangles.","method":"Affinity purification-mass spectrometry from microdissected neurofibrillary tangles in human Alzheimer's disease brain","journal":"Brain : a journal of neurology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single AP-MS study, no reciprocal validation or functional follow-up for ATP6V0D1 specifically","pmids":["32812023"],"is_preprint":false},{"year":2025,"finding":"ATP6V0D1 deletion in PDAC cells led to compensatory overactivation of STAT3-mediated lysosomal pH regulation and AKT signaling. Inhibition of STAT3 or AKT pathways in ATP6V0D1-deficient cells restored sensitivity to alkaliptosis, placing ATP6V0D1 upstream of STAT3/AKT as a suppressor of these oncogenic pathways.","method":"Gene knockdown/knockout, transcriptomic analysis, Western blotting, CCK-8/PI cell death assays, macropinocytosis assay, DepMap database analysis","journal":"Cancer drug resistance","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with transcriptomic and protein-level mechanistic follow-up, single lab","pmids":["41019981"],"is_preprint":false}],"current_model":"ATP6V0D1 encodes the d1 subunit of the vacuolar H⁺-ATPase (V-ATPase) V0 sector and functions as a direct regulator of lysosomal acidification; it is a druggable target of JTC801 (binding at Cys-335 allosterically activates the pump), interacts with STAT3 to maintain lysosomal pH homeostasis (restraining alkaliptosis), is trafficked to lysosome-related organelle membranes by HPS6/BLOC-2 for V-ATPase assembly, is required as a host factor for influenza and coronavirus replication, mediates chemoresistance in cancer cells via lysosomal drug sequestration (with ABCB1 regulation downstream), and in adipose tissue acts as a master regulator of adipogenesis whose loss causes lipodystrophy and secondary cardiomyopathy through a myocardin-IRS-1-FoxO1 axis."},"narrative":{"mechanistic_narrative":"ATP6V0D1 encodes the d1 subunit of the vacuolar H⁺-ATPase V0 sector and functions as a determinant of organellar acidification across diverse cellular contexts [PMID:33346633, PMID:36213534]. Loss of ATP6V0D1 raises lysosomal pH and impairs V-ATPase-dependent functions, including lysosome-dependent coronavirus entry/replication and a parallel host-factor requirement for influenza A virus [PMID:18615018, PMID:33346633]. The subunit is allosterically druggable: JTC801 stabilizes ATP6V0D1 and Schisandrol A binds cysteine-335 to drive a conformational change that activates V-ATPase-dependent lysosomal acidification [PMID:36640329, PMID:36213534]. Through this activity ATP6V0D1 restrains alkaliptosis, a pH-dependent cell death; mechanistically it acts upstream of STAT3 (with which it physically interacts) and AKT to sustain lysosomal pH homeostasis, such that STAT3/AKT inhibition restores alkaliptosis sensitivity in ATP6V0D1-deficient pancreatic cancer cells [PMID:36640329, PMID:41019981]. Its lysosomal acidifying role also underlies chemoresistance, sequestering drugs to limit cytotoxicity and modulating ABCB1 expression in a pH-dependent manner [PMID:40552114, PMID:38751020]. ATP6V0D1 is delivered to lysosome-related organelle membranes for V-ATPase assembly by the BLOC-2 subunit HPS6, supporting Weibel-Palade body biogenesis [PMID:35252216], and assembly is regulated by Tmem9 in microglia [PMID:39871402]. At the organismal level it is required for vertebrate eye development and melanosome biogenesis, and adipose-specific deletion in mice causes lipodystrophy and secondary cardiomyopathy through a myocardin–IRS-1–FoxO1 axis [PMID:18836173, PMID:38505620].","teleology":[{"year":2000,"claim":"Establishing the gene structure and conservation of ATP6V0D1 provided the molecular foundation for studying the d1 subunit and distinguished its regulation from its genomic neighbor.","evidence":"Gene cloning, exon-intron mapping, and forskolin expression assays in JEG3 cells","pmids":["11118322"],"confidence":"Medium","gaps":["Did not address subunit function within the V-ATPase complex","No protein-level activity characterized"]},{"year":2005,"claim":"Tissue localization raised the possibility that the d1 subunit has functions beyond canonical proton transport, by appearing at the apical membrane without other V-ATPase subunits.","evidence":"Immunostaining of V-ATPase subunit isoforms in rat epididymis and vas deferens","pmids":["16192400"],"confidence":"Low","gaps":["Single localization study with no functional rescue or knockout","Putative complex-independent role never validated mechanistically"]},{"year":2008,"claim":"Functional genetics defined ATP6V0D1 as a specific influenza host factor and, separately, as essential for vertebrate eye, melanosome, and RPE development, linking the subunit to acidification-dependent organelle function in vivo.","evidence":"Genome-wide RNAi screen with human siRNA viral assays; loss-of-function zebrafish mutant with histology, BrdU, TUNEL, and EM","pmids":["18615018","18836173"],"confidence":"Medium","gaps":["Molecular step in viral replication not pinpointed","Whether developmental phenotypes derive solely from acidification loss not isolated"]},{"year":2009,"claim":"Intronic sequences of ATP6V0D1 were shown to act as enhancers for the neighboring AgRP gene, a genomic-regulatory property distinct from the subunit's protein function.","evidence":"Enhancer screening and transgenic mouse reporter assays under dietary challenge","pmids":["19285986"],"confidence":"Medium","gaps":["Concerns the locus rather than the ATP6V0D1 protein","No link to V-ATPase activity"]},{"year":2020,"claim":"Knockdown tied ATP6V0D1 directly to lysosomal pH and coronavirus replication, mechanistically connecting its acidifying activity to viral entry, and AP-MS placed the protein in physical contact with phosphorylated tau in disease tissue.","evidence":"siRNA knockdown with coronavirus cytopathic-effect and lysosomal pH assays; AP-MS from Alzheimer neurofibrillary tangles","pmids":["33346633","32812023"],"confidence":"Medium","gaps":["Tau interaction lacks reciprocal validation or functional follow-up","Causal role of pH rise in blocking coronavirus not formally separated from off-target effects"]},{"year":2022,"claim":"ATP6V0D1 was established as a druggable, allosterically regulated acidification controller and a node restraining alkaliptosis, defining both a cysteine-335 ligand site and a STAT3-coupled pH-homeostasis mechanism.","evidence":"MS target ID, CETSA, cysteine-335 mutagenesis, Co-IP, knockout cell-death assays, xenografts; plus HPS6 Co-IP with parallel WPB knockdown phenotypes","pmids":["36640329","36213534","35252216"],"confidence":"High","gaps":["Structural basis of allosteric activation not resolved","How HPS6 selects ATP6V0D1 cargo for delivery unknown"]},{"year":2024,"claim":"In vivo deletion and pH-manipulation studies expanded ATP6V0D1 from a lysosomal subunit to a regulator of adipogenesis/cardiac metabolism and of the alkaliptosis-ABCB1 chemoresistance axis.","evidence":"Adipose-specific knockout mice with RNA-seq, ChIP-qPCR, luciferase, and AAV rescue; ATP6V0D1 overexpression and intracellular pH manipulation in ovarian cancer cells","pmids":["38505620","38751020"],"confidence":"High","gaps":["How an adipose lysosomal subunit controls myocardin transcriptionally is unresolved","Direct vs indirect control of ABCB1 by pH not fully separated"]},{"year":2025,"claim":"Recent work refined the downstream signaling and disease contexts, placing ATP6V0D1 upstream of STAT3/AKT in PDAC, implicating it in lysosomal drug sequestration in neuroblastoma, and connecting Tmem9-regulated V-ATPase assembly to microglial complement activity.","evidence":"Knockout transcriptomics/Western blot in PDAC; subunit-controlled siRNA with cell-cycle, migration, and lysosomal uptake assays; Tmem9 gain/loss in 5xFAD mice and BV2 cells","pmids":["41019981","40552114","39871402"],"confidence":"Medium","gaps":["ATP6V0D1's precise step in the Tmem9→complement pathway inferred not directly tested","Single-lab observations awaiting independent confirmation"]},{"year":null,"claim":"How a single V0 subunit integrates lysosomal acidification with such diverse outcomes—alkaliptosis, adipogenesis, viral entry, and chemoresistance—and whether these reflect one acidification mechanism or distinct complex-independent activities remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structure of ATP6V0D1 within the assembled V-ATPase","Complex-independent functions not mechanistically separated from canonical proton transport"]}],"mechanism_profile":{"molecular_activity":[],"localization":[{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[3,4,5,8]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[1]}],"pathway":[{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[3,5]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[4,14]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[0,3,8,11]}],"complexes":["vacuolar H+-ATPase (V-ATPase) V0 sector"],"partners":["STAT3","HPS6","TMEM9"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P61421","full_name":"V-type proton ATPase subunit d 1","aliases":["32 kDa accessory protein","V-ATPase 40 kDa accessory protein","V-ATPase AC39 subunit","p39","Vacuolar proton pump subunit d 1"],"length_aa":351,"mass_kda":40.3,"function":"Subunit of the V0 complex of vacuolar(H+)-ATPase (V-ATPase), a multisubunit enzyme composed of a peripheral complex (V1) that hydrolyzes ATP and a membrane integral complex (V0) that translocates protons (PubMed:28296633, PubMed:30374053, PubMed:33065002). V-ATPase is responsible for acidifying and maintaining the pH of intracellular compartments and in some cell types, is targeted to the plasma membrane, where it is responsible for acidifying the extracellular environment (PubMed:30374053). May play a role in coupling of proton transport and ATP hydrolysis (By similarity). In aerobic conditions, involved in intracellular iron homeostasis, thus triggering the activity of Fe(2+) prolyl hydroxylase (PHD) enzymes, and leading to HIF1A hydroxylation and subsequent proteasomal degradation (PubMed:28296633). May play a role in cilium biogenesis through regulation of the transport and the localization of proteins to the cilium (By similarity)","subcellular_location":"Membrane; Lysosome membrane; Cytoplasmic vesicle, clathrin-coated vesicle membrane","url":"https://www.uniprot.org/uniprotkb/P61421/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/ATP6V0D1","classification":"Common Essential","n_dependent_lines":1157,"n_total_lines":1208,"dependency_fraction":0.9577814569536424},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000159720","cell_line_id":"CID001643","localizations":[{"compartment":"vesicles","grade":3}],"interactors":[{"gene":"ATP6AP1","stoichiometry":10.0},{"gene":"ATP6AP2","stoichiometry":10.0},{"gene":"ATP6V0A1","stoichiometry":10.0},{"gene":"ATP6V0A2","stoichiometry":10.0},{"gene":"ATP6V1G1","stoichiometry":10.0},{"gene":"ATP6V1B2","stoichiometry":10.0},{"gene":"ATP6V1A","stoichiometry":4.0},{"gene":"STX12","stoichiometry":4.0},{"gene":"ATP6V1E1","stoichiometry":0.2},{"gene":"ATP6V0C","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID001643","total_profiled":1310},"omim":[{"mim_id":"616877","title":"TRANSMEMBRANE PROTEIN 9; TMEM9","url":"https://www.omim.org/entry/616877"},{"mim_id":"614232","title":"11-@BETA-HYDROXYSTEROID DEHYDROGENASE, TYPE II; HSD11B2","url":"https://www.omim.org/entry/614232"},{"mim_id":"607028","title":"ATPase, H+ TRANSPORTING, LYSOSOMAL, 38-KD, V0 SUBUNIT D, ISOFORM 1; ATP6V0D1","url":"https://www.omim.org/entry/607028"},{"mim_id":"300556","title":"ATPase, H+ TRANSPORTING, LYSOSOMAL, ACCESSORY PROTEIN 2; ATP6AP2","url":"https://www.omim.org/entry/300556"}],"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/ATP6V0D1"},"hgnc":{"alias_symbol":["ATP6DV","VATX","VPATPD","P39","Vma6"],"prev_symbol":["ATP6D"]},"alphafold":{"accession":"P61421","domains":[{"cath_id":"1.20.1690","chopping":"87-188","consensus_level":"medium","plddt":80.6393,"start":87,"end":188}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P61421","model_url":"https://alphafold.ebi.ac.uk/files/AF-P61421-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P61421-F1-predicted_aligned_error_v6.png","plddt_mean":85.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ATP6V0D1","jax_strain_url":"https://www.jax.org/strain/search?query=ATP6V0D1"},"sequence":{"accession":"P61421","fasta_url":"https://rest.uniprot.org/uniprotkb/P61421.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P61421/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P61421"}},"corpus_meta":[{"pmid":"18615016","id":"PMC_18615016","title":"Drosophila 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\"finding\": \"RNAi knockdown of ATP6V0D1 (the human homologue of the Drosophila gene identified in a genome-wide screen) in HEK 293 cells significantly inhibited replication of H5N1 and H1N1 influenza A viruses but not vesicular stomatitis virus or vaccinia virus, establishing a specific host-factor role for ATP6V0D1 in influenza virus replication.\",\n      \"method\": \"Genome-wide RNAi screen in Drosophila followed by siRNA knockdown of human ATP6V0D1 in HEK 293 cells with influenza virus infection assay\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean knockdown with specific viral phenotype, replicated across two influenza strains, single lab\",\n      \"pmids\": [\"18615018\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"ATP6V0D1 (d1 subunit) was localized by immunostaining to the apical membrane of principal cells in the rat epididymis and vas deferens in the apparent absence of other V-ATPase subunits, suggesting a physiological function distinct from its role in proton transport via the complete V-ATPase complex.\",\n      \"method\": \"Immunohistochemistry/immunofluorescence localization of V-ATPase subunit isoforms in rat epididymis and vas deferens\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single localization study, no functional rescue or KO validation\",\n      \"pmids\": [\"16192400\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Zebrafish carrying a loss-of-function mutation in atp6v0d1 exhibited microphthalmic eyes, defective retinoblast cell cycle exit, elevated retinal apoptosis, abnormal photoreceptor outer segment morphology, RPE malformation with undigested outer segment material in vacuoles, and oculocutaneous albinism with melanosome biogenesis defects, demonstrating that ATP6V0D1 is required for v-ATPase complex function during vertebrate eye development.\",\n      \"method\": \"Genetic loss-of-function zebrafish mutant analysis with histology, BrdU incorporation, TUNEL assay, in situ hybridization, and electron microscopy\",\n      \"journal\": \"Investigative ophthalmology & visual science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal readouts in a clean genetic model, single lab\",\n      \"pmids\": [\"18836173\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"siRNA knockdown of ATP6V0D1 in LLC-MK2 cells blocked the cytopathic effect of HCoV-NL63 coronavirus, and ATP6V0D1 knockdown was associated with increased lysosomal pH, implicating the subunit in lysosome-dependent coronavirus entry/replication.\",\n      \"method\": \"siRNA knockdown of ATP6V0D1 with coronavirus cytopathic effect assay and lysosomal pH measurement\",\n      \"journal\": \"ACS infectious diseases\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — clean knockdown with defined cytopathic phenotype, single lab, single method\",\n      \"pmids\": [\"33346633\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ATP6V0D1 was identified as a direct molecular target of JTC801 (the alkaliptosis-inducing drug) using mass-spectrometry-based target identification and cellular thermal shift assay. JTC801-mediated stabilization of ATP6V0D1 protein increases its interaction with STAT3, leading to increased STAT3 expression and activity that sustains lysosomal pH homeostasis. Genetic or pharmacological inhibition of STAT3 restored alkaliptosis sensitivity in ATP6V0D1-deficient pancreatic ductal adenocarcinoma cells.\",\n      \"method\": \"Mass-spectrometry-based drug target ID, cellular thermal shift assay, point mutation, co-immunoprecipitation, siRNA/gene knockout with cell death assays, mouse xenograft models\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (MS target ID, CETSA, mutagenesis, Co-IP, in vivo validation) in one study\",\n      \"pmids\": [\"36640329\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Schisandrol A (SolA) was shown to allosterically activate ATP6V0D1 by binding to cysteine 335, inducing a conformational change that activates V-ATPase-dependent lysosomal acidification. This lysosomal acidification selectively promoted mitochondrial BH3-only protein BIM degradation, preserving mitochondrial homeostasis and neuronal cell survival against AGEs-induced apoptosis.\",\n      \"method\": \"Drug target identification, cysteine-335 point mutation, lysosomal pH measurement, mitochondrial assays, cell survival assay\",\n      \"journal\": \"Acta pharmaceutica Sinica. B\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — site-directed mutagenesis identifying the binding residue, functional pH and cell survival readouts, multiple orthogonal methods in one study\",\n      \"pmids\": [\"36213534\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"HPS6 (a BLOC-2 complex subunit) was shown to interact with ATP6V0D1 by co-immunoprecipitation. Knockdown of either HPS6 or ATP6V0D1 in HUVECs produced similar Weibel-Palade body defects (misshaped WPBs, decreased WPB number, impaired vWF tubulation), indicating that HPS6 transports ATP6V0D1 to the WPB limiting membrane for V-ATPase assembly and maintenance of acidic luminal pH required for WPB biogenesis.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown of HPS6 and ATP6V0D1, fluorescence microscopy of WPB morphology\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP plus parallel KD phenotypes showing convergent defects, single lab\",\n      \"pmids\": [\"35252216\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Adipose-specific deletion of Atp6v0d1 in mice caused generalized lipodystrophy, identifying ATP6V0D1 as a master regulator of adipogenesis. The resulting Atp6v0d1AKO mice developed spontaneous cardiomyopathy with cardiac insulin resistance (decreased IRS-1/2 expression), lipid accumulation, and increased FoxO1. Myocardin was downregulated in these hearts and shown by RNAi, luciferase reporter, and ChIP-qPCR to directly regulate IRS-1 transcription; restoring cardiac myocardin expression reversed metabolic gene dysregulation and improved cardiac function.\",\n      \"method\": \"Adipose-specific Atp6v0d1 knockout mice, RNA-seq, RNAi, luciferase reporter assay, ChIP-qPCR, in vivo AAV-mediated gene delivery\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean conditional KO with defined metabolic phenotype, multiple orthogonal mechanistic assays (RNA-seq, ChIP, reporter, in vivo rescue)\",\n      \"pmids\": [\"38505620\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Knockdown of ATP6V0D1 (but not ATP6V1H) in neuroblastoma cells enhanced sensitivity to ellipticine, suppressed proliferation and migration, decreased lysosomal drug uptake, and induced G2/M arrest. ATP6V0D1 knockdown also suppressed ellipticine-induced cytoplasmic vacuolation (ER swelling), establishing a specific role for ATP6V0D1 in lysosomal drug sequestration-mediated chemoresistance.\",\n      \"method\": \"siRNA knockdown of ATP6V0D1 and ATP6V1H, cell viability assay, migration assay, flow cytometry (cell cycle), lysosomal uptake assay, EM for vacuolation characterization\",\n      \"journal\": \"Molecular & cellular oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — parallel KD of two V-ATPase subunits as controls with multiple orthogonal phenotypic readouts, single lab\",\n      \"pmids\": [\"40552114\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Microglial Tmem9 was shown to regulate V-ATPase assembly through ATP6V0D1. In an Alzheimer's disease mouse model (5xFAD), Tmem9 downregulation (by physical exercise) inhibited C1q complement activation and reduced microglial synapse engulfment; overexpression of Tmem9 promoted complement activation. The pathway was placed as Tmem9 → ATP6V0D1 (V-ATPase subunit) → V-ATPase assembly → complement activation.\",\n      \"method\": \"In vivo exercise intervention in 5xFAD mice, Tmem9 overexpression/knockdown in BV2 cells treated with oAβ, complement activation assay, synapse engulfment assay\",\n      \"journal\": \"Aging cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo and in vitro gain/loss-of-function with defined complement and synaptic phenotypes, single lab; ATP6V0D1's precise mechanistic role is inferred from the pathway rather than directly tested\",\n      \"pmids\": [\"39871402\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"The human VPATPD (ATP6V0D1) gene was characterized: it spans 19 kb, consists of 8 exons, and encodes a protein 99.5% identical to mouse subunit D at the amino acid level. The gene is located on chromosome 16q22, transcribed from the complementary strand relative to HSD11B2, with their 3' ends only 0.5 kb apart. Forskolin upregulates HSD11B2 but not VPATPD in JEG3 cells, showing distinct regulatory control despite genomic proximity.\",\n      \"method\": \"Gene cloning, sequencing, exon-intron mapping, RT-PCR, comparative sequence analysis, cell-based reporter/expression assay with forskolin\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct gene structure characterization with expression experiments; multiple methods in one study\",\n      \"pmids\": [\"11118322\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ATP6V0D1 overexpression or JTC801-mediated stabilization of ATP6V0D1 protein inhibited ABCB1 (multidrug resistance protein) upregulation in paclitaxel-resistant ovarian cancer cells, overcoming drug resistance via alkaliptosis. Increasing intracellular pH (pH 8.5 via NaOH) suppressed ABCB1 expression whereas acidification (pH 6.5 via HCl) amplified ABCB1, demonstrating that the ATP6V0D1-mediated alkaliptosis-ABCB1 axis mediates paclitaxel resistance.\",\n      \"method\": \"Gene transfection (ATP6V0D1 overexpression), JTC801 pharmacological treatment, intracellular pH manipulation, ABCB1 expression assay, cell growth inhibition assay\",\n      \"journal\": \"Molecular carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal interventions (genetic OE, pharmacological, pH manipulation) with defined resistance phenotype, single lab\",\n      \"pmids\": [\"38751020\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Control elements located within the first intron of ATP6V0D1 were shown experimentally to function as enhancers that modulate expression of the neighboring AgRP gene with spatiotemporal specificity, demonstrating that intronic sequences of ATP6V0D1 can act in trans on adjacent gene promoters.\",\n      \"method\": \"In vitro enhancer screening, transgenic mouse reporter assays with dietary/fasting challenges, comparative sequence analysis\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct functional demonstration in transgenic mice with multiple reporter readouts; finding is about ATP6V0D1 genomic sequences acting as regulatory elements\",\n      \"pmids\": [\"19285986\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Affinity purification-mass spectrometry using PHF1-immunoreactive phosphorylated tau as bait from Alzheimer's disease brain tissue identified ATP6V0D1 as a protein that directly interacts with phosphorylated tau in neurofibrillary tangles.\",\n      \"method\": \"Affinity purification-mass spectrometry from microdissected neurofibrillary tangles in human Alzheimer's disease brain\",\n      \"journal\": \"Brain : a journal of neurology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single AP-MS study, no reciprocal validation or functional follow-up for ATP6V0D1 specifically\",\n      \"pmids\": [\"32812023\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ATP6V0D1 deletion in PDAC cells led to compensatory overactivation of STAT3-mediated lysosomal pH regulation and AKT signaling. Inhibition of STAT3 or AKT pathways in ATP6V0D1-deficient cells restored sensitivity to alkaliptosis, placing ATP6V0D1 upstream of STAT3/AKT as a suppressor of these oncogenic pathways.\",\n      \"method\": \"Gene knockdown/knockout, transcriptomic analysis, Western blotting, CCK-8/PI cell death assays, macropinocytosis assay, DepMap database analysis\",\n      \"journal\": \"Cancer drug resistance\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with transcriptomic and protein-level mechanistic follow-up, single lab\",\n      \"pmids\": [\"41019981\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ATP6V0D1 encodes the d1 subunit of the vacuolar H⁺-ATPase (V-ATPase) V0 sector and functions as a direct regulator of lysosomal acidification; it is a druggable target of JTC801 (binding at Cys-335 allosterically activates the pump), interacts with STAT3 to maintain lysosomal pH homeostasis (restraining alkaliptosis), is trafficked to lysosome-related organelle membranes by HPS6/BLOC-2 for V-ATPase assembly, is required as a host factor for influenza and coronavirus replication, mediates chemoresistance in cancer cells via lysosomal drug sequestration (with ABCB1 regulation downstream), and in adipose tissue acts as a master regulator of adipogenesis whose loss causes lipodystrophy and secondary cardiomyopathy through a myocardin-IRS-1-FoxO1 axis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ATP6V0D1 encodes the d1 subunit of the vacuolar H\\u207a-ATPase V0 sector and functions as a determinant of organellar acidification across diverse cellular contexts [#3, #5]. Loss of ATP6V0D1 raises lysosomal pH and impairs V-ATPase-dependent functions, including lysosome-dependent coronavirus entry/replication and a parallel host-factor requirement for influenza A virus [#0, #3]. The subunit is allosterically druggable: JTC801 stabilizes ATP6V0D1 and Schisandrol A binds cysteine-335 to drive a conformational change that activates V-ATPase-dependent lysosomal acidification [#4, #5]. Through this activity ATP6V0D1 restrains alkaliptosis, a pH-dependent cell death; mechanistically it acts upstream of STAT3 (with which it physically interacts) and AKT to sustain lysosomal pH homeostasis, such that STAT3/AKT inhibition restores alkaliptosis sensitivity in ATP6V0D1-deficient pancreatic cancer cells [#4, #14]. Its lysosomal acidifying role also underlies chemoresistance, sequestering drugs to limit cytotoxicity and modulating ABCB1 expression in a pH-dependent manner [#8, #11]. ATP6V0D1 is delivered to lysosome-related organelle membranes for V-ATPase assembly by the BLOC-2 subunit HPS6, supporting Weibel-Palade body biogenesis [#6], and assembly is regulated by Tmem9 in microglia [#9]. At the organismal level it is required for vertebrate eye development and melanosome biogenesis, and adipose-specific deletion in mice causes lipodystrophy and secondary cardiomyopathy through a myocardin\\u2013IRS-1\\u2013FoxO1 axis [#2, #7].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Establishing the gene structure and conservation of ATP6V0D1 provided the molecular foundation for studying the d1 subunit and distinguished its regulation from its genomic neighbor.\",\n      \"evidence\": \"Gene cloning, exon-intron mapping, and forskolin expression assays in JEG3 cells\",\n      \"pmids\": [\"11118322\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not address subunit function within the V-ATPase complex\", \"No protein-level activity characterized\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Tissue localization raised the possibility that the d1 subunit has functions beyond canonical proton transport, by appearing at the apical membrane without other V-ATPase subunits.\",\n      \"evidence\": \"Immunostaining of V-ATPase subunit isoforms in rat epididymis and vas deferens\",\n      \"pmids\": [\"16192400\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single localization study with no functional rescue or knockout\", \"Putative complex-independent role never validated mechanistically\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Functional genetics defined ATP6V0D1 as a specific influenza host factor and, separately, as essential for vertebrate eye, melanosome, and RPE development, linking the subunit to acidification-dependent organelle function in vivo.\",\n      \"evidence\": \"Genome-wide RNAi screen with human siRNA viral assays; loss-of-function zebrafish mutant with histology, BrdU, TUNEL, and EM\",\n      \"pmids\": [\"18615018\", \"18836173\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular step in viral replication not pinpointed\", \"Whether developmental phenotypes derive solely from acidification loss not isolated\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Intronic sequences of ATP6V0D1 were shown to act as enhancers for the neighboring AgRP gene, a genomic-regulatory property distinct from the subunit's protein function.\",\n      \"evidence\": \"Enhancer screening and transgenic mouse reporter assays under dietary challenge\",\n      \"pmids\": [\"19285986\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Concerns the locus rather than the ATP6V0D1 protein\", \"No link to V-ATPase activity\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Knockdown tied ATP6V0D1 directly to lysosomal pH and coronavirus replication, mechanistically connecting its acidifying activity to viral entry, and AP-MS placed the protein in physical contact with phosphorylated tau in disease tissue.\",\n      \"evidence\": \"siRNA knockdown with coronavirus cytopathic-effect and lysosomal pH assays; AP-MS from Alzheimer neurofibrillary tangles\",\n      \"pmids\": [\"33346633\", \"32812023\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Tau interaction lacks reciprocal validation or functional follow-up\", \"Causal role of pH rise in blocking coronavirus not formally separated from off-target effects\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"ATP6V0D1 was established as a druggable, allosterically regulated acidification controller and a node restraining alkaliptosis, defining both a cysteine-335 ligand site and a STAT3-coupled pH-homeostasis mechanism.\",\n      \"evidence\": \"MS target ID, CETSA, cysteine-335 mutagenesis, Co-IP, knockout cell-death assays, xenografts; plus HPS6 Co-IP with parallel WPB knockdown phenotypes\",\n      \"pmids\": [\"36640329\", \"36213534\", \"35252216\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of allosteric activation not resolved\", \"How HPS6 selects ATP6V0D1 cargo for delivery unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"In vivo deletion and pH-manipulation studies expanded ATP6V0D1 from a lysosomal subunit to a regulator of adipogenesis/cardiac metabolism and of the alkaliptosis-ABCB1 chemoresistance axis.\",\n      \"evidence\": \"Adipose-specific knockout mice with RNA-seq, ChIP-qPCR, luciferase, and AAV rescue; ATP6V0D1 overexpression and intracellular pH manipulation in ovarian cancer cells\",\n      \"pmids\": [\"38505620\", \"38751020\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How an adipose lysosomal subunit controls myocardin transcriptionally is unresolved\", \"Direct vs indirect control of ABCB1 by pH not fully separated\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Recent work refined the downstream signaling and disease contexts, placing ATP6V0D1 upstream of STAT3/AKT in PDAC, implicating it in lysosomal drug sequestration in neuroblastoma, and connecting Tmem9-regulated V-ATPase assembly to microglial complement activity.\",\n      \"evidence\": \"Knockout transcriptomics/Western blot in PDAC; subunit-controlled siRNA with cell-cycle, migration, and lysosomal uptake assays; Tmem9 gain/loss in 5xFAD mice and BV2 cells\",\n      \"pmids\": [\"41019981\", \"40552114\", \"39871402\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"ATP6V0D1's precise step in the Tmem9\\u2192complement pathway inferred not directly tested\", \"Single-lab observations awaiting independent confirmation\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How a single V0 subunit integrates lysosomal acidification with such diverse outcomes\\u2014alkaliptosis, adipogenesis, viral entry, and chemoresistance\\u2014and whether these reflect one acidification mechanism or distinct complex-independent activities remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structure of ATP6V0D1 within the assembled V-ATPase\", \"Complex-independent functions not mechanistically separated from canonical proton transport\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0015078\", \"supporting_discovery_ids\": []}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [3, 4, 5, 8]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [3, 5]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [4, 14]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [0, 3, 8, 11]}\n    ],\n    \"complexes\": [\"vacuolar H+-ATPase (V-ATPase) V0 sector\"],\n    \"partners\": [\"STAT3\", \"HPS6\", \"TMEM9\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":{"gene":"ATP6V0D1","tier":"GROUNDING","verdict":"Evidence-grounding concern","subtype":"fabrication","uniprot_band":"medium","rules_fired":"R7","issue":"R7: fabricated (no corpus paper): 18615018"},"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}