{"gene":"ATP6AP1","run_date":"2026-06-09T22:02:44","timeline":{"discoveries":[{"year":2024,"finding":"ATP6AP1 directly binds Rheb through its last 12 amino acids and utilizes a tri-aspartate motif in its highly conserved C-tail to enhance Rheb GTP loading, functioning as an unconventional guanine nucleotide exchange factor (GEF) for Rheb; targeting the ATP6AP1 C-tail blocks Rheb activation and inhibits cancer cell proliferation and migration.","method":"Proximity labeling (PhastID), co-immunoprecipitation, in vitro GTP loading assay, domain deletion/mutagenesis, functional cell proliferation and migration assays","journal":"Cell research","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — multiple orthogonal methods including proximity labeling, direct binding assays, mutagenesis of functional domain, and functional readouts in a single rigorous study","pmids":["38448650"],"is_preprint":false},{"year":2022,"finding":"SARS-CoV-2 NSP6 directly interacts with ATP6AP1 (a V-ATPase proton pump component) and inhibits its cleavage-mediated activation, thereby impairing lysosome acidification and autophagic flux, which triggers NLRP3/ASC-dependent caspase-1 activation and pyroptosis in lung epithelial cells.","method":"Co-immunoprecipitation, overexpression, lysosomal acidification assays, autophagic flux assays, inflammasome/caspase-1 activation assays, pharmacological rescue experiments","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal interaction demonstrated, multiple orthogonal functional assays, supported by NSP6 variant (L37F) showing reduced binding and reduced pyroptosis induction","pmids":["34997207"],"is_preprint":false},{"year":2016,"finding":"ATP6AP1 (Ac45) is the functional human ortholog of yeast V-ATPase assembly factor Voa1; processed wild-type Ac45, but not disease-associated missense mutants, restores V-ATPase-dependent growth in Voa1 mutant yeast, establishing its role in V-ATPase assembly. Tissue-specific isoforms (40-kDa in brain, 62-kDa in liver, 50-kDa in B-cells) are present, linking V-ATPase assembly to immunoglobulin production and cognitive function.","method":"Yeast complementation assay, sequence profile homology detection, patient mutation analysis, Western blot of tissue-specific isoforms","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — yeast reconstitution complementation with wild-type vs. mutant forms, multiple orthogonal approaches, independently validated by patient phenotypes","pmids":["27231034"],"is_preprint":false},{"year":2012,"finding":"ATP6AP1 (Ac45) is required for RANKL-induced osteoclast differentiation, extracellular acidification, lysosomal trafficking to the ruffled border, and cathepsin K exocytosis; Ac45 knockdown severely impairs bone resorption and reduces osteoclast precursor proliferation and fusion, partially via downregulation of ERK phosphorylation and c-fos/NFATc1/Tm7sf4 expression. The effect on exocytosis is specific to Ac45 deficiency, distinct from general V-ATPase defects, and may involve interaction with the small GTPase Rab7.","method":"siRNA knockdown, bone resorption assays, extracellular acidification assay, lysosomal trafficking immunofluorescence, cathepsin K exocytosis assay, Western blot for signaling molecules","journal":"Journal of bone and mineral research","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean KD with multiple orthogonal functional readouts (acidification, trafficking, exocytosis, signaling), single lab","pmids":["22467241"],"is_preprint":false},{"year":2010,"finding":"Transgenic overexpression of Ac45 (ATP6AP1) specifically in Xenopus melanotrope cells increases granular acidification, reduces sensitivity to V-ATPase inhibitor, enhances early POMC processing by PC1, but reduces PC2 maturation; establishing Ac45 as the first identified regulator of V-ATPase-mediated granular acidification required for prohormone processing.","method":"Transgenic Xenopus melanotrope cell model, granular acidification assay, V-ATPase inhibitor sensitivity assay, prohormone processing Western blot analysis","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — transgenic gain-of-function with multiple orthogonal phenotypic readouts in a well-established in vivo neuroendocrine model","pmids":["20702583"],"is_preprint":false},{"year":2008,"finding":"Transgenic expression of Ac45 (ATP6AP1) in Xenopus melanotrope cells causes accumulation of V-ATPase at the plasma membrane, increased abundance of secretory granules, plasma membrane protrusions, and increased Ca2+-dependent secretion efficiency, demonstrating that Ac45 guides V-ATPase through the secretory pathway and regulates Ca2+-dependent peptide secretion.","method":"Transgenic Xenopus melanotrope cell model, immunofluorescence, electron microscopy, Ca2+-dependent secretion assay","journal":"Biochimica et biophysica acta","confidence":"High","confidence_rationale":"Tier 2 / Moderate — transgenic in vivo model with multiple orthogonal readouts (localization, secretion), consistent with prior and subsequent studies","pmids":["18657579"],"is_preprint":false},{"year":2008,"finding":"Furin cleaves Ac45 (ATP6AP1) at a defined site in pancreatic beta cells; furin knockout reduces granule acidification, and Ac45 knockdown similarly reduces regulated secretion and proinsulin II processing, establishing a furin–Ac45–V-ATPase axis controlling intragranular acidification in the regulated secretory pathway.","method":"Pdx1-Cre/loxP furin knockout mice, DAMP acidification assay, ex vivo cleavage assay to determine cleavage site, siRNA knockdown of furin and Ac45 in betaTC3 cells, insulin/proinsulin processing assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vivo KO combined with defined biochemical cleavage site mapping and in vitro functional validation with multiple readouts","pmids":["18713856"],"is_preprint":false},{"year":2012,"finding":"Ac45 (ATP6AP1) interacts with the V0 sector of the V-ATPase complex in neuroendocrine cells, regulating intragranular pH and Ca2+-dependent exocytotic membrane fusion.","method":"Biochemical co-purification/interaction studies, neuroendocrine cell models (review/synthesis of prior experimental work cited therein)","journal":"Current protein & peptide science","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — summarizes experimental findings from multiple studies but as a review; underlying data from prior primary studies support the interaction claim","pmids":["22044156"],"is_preprint":false},{"year":2012,"finding":"The N-terminal domain of Ac45 (ATP6AP1) controls its retention in the endoplasmic reticulum when intact; proteolytic cleavage releasing the N-terminal domain enables transport through the secretory pathway. The C-tail of cleaved Ac45 is required for V-ATPase recruitment into the secretory pathway and for Ca2+-dependent regulated exocytosis, but not for plasma membrane targeting.","method":"Deletion mutant analysis in transgenic Xenopus melanotrope cells, immunofluorescence, electron microscopy, secretion assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — structure-function deletion mutant analysis in in vivo transgenic model with multiple orthogonal readouts","pmids":["22736765"],"is_preprint":false},{"year":2002,"finding":"The cytoplasmic tail of Ac45 (ATP6AP1) contains autonomous internalization signal(s) in its membrane-distal region that mediate rapid retrieval from the cell surface and targeting to vacuolar compartments; multiple sites rather than a single short linear motif are responsible, and this routing is distinct from known tyrosine- or di-leucine-based sorting motifs.","method":"Transfection in CV-1 fibroblasts, antibody internalization experiments, chimeric protein (Ac45 tail fused to Tac), C-terminally truncated mutant expression, immunolocalization","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — domain mutagenesis combined with chimeric protein approach and direct trafficking assay, reproduced across multiple constructs","pmids":["9739073"],"is_preprint":false},{"year":2002,"finding":"Newly synthesized Ac45 (ATP6AP1) undergoes N-linked glycosylation (to ~62 kDa from ~46 kDa unglycosylated form), followed by slow proteolytic cleavage (half-life ~4–6 h) in the early secretory pathway producing an N-terminal (~22 kDa) and C-terminal (~42–44 kDa) fragment; N-linked glycosylation is required for proper folding and cleavage. The N-terminal fragment is rapidly degraded in a non-lysosomal, brefeldin A-sensitive manner.","method":"Pulse-chase radiolabeling, endoglycosidase H resistance assay, tunicamycin treatment, brefeldin A treatment, monensin treatment, deglycosylation, Western blot","journal":"European journal of biochemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — biochemical characterization with multiple inhibitors and enzymatic treatments providing mechanistic detail on processing pathway","pmids":["11952786"],"is_preprint":false},{"year":1999,"finding":"Xenopus Ac45 (ATP6AP1 ortholog) is synthesized as an N-glycosylated ~60 kDa precursor that is intracellularly cleaved to an ~40 kDa product; expression is predominantly in neuroendocrine tissues. The protein is associated with V-ATPase and localizes to biosynthetically active melanotrope cells in pituitary.","method":"cDNA cloning, Western blot, deglycosylation assay, biosynthetic pulse labeling, immunocytochemistry in Xenopus pituitary","journal":"European journal of biochemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — biochemical characterization with biosynthetic labeling and multiple complementary methods establishing processing and localization","pmids":["10336633"],"is_preprint":false},{"year":2018,"finding":"Loss-of-function mutations in ATP6AP1 impair vesicle acidification, cause redistribution of endosomal compartments, and lead to accumulation of intracytoplasmic granules in granular cell tumors; ATP6AP1 depletion in vitro recapitulates these phenotypes and results in acquisition of oncogenic properties.","method":"Whole-exome and targeted sequencing of patient tumors, in vitro siRNA silencing, vesicle acidification assay, endosomal compartment imaging, granule accumulation assay, oncogenic property assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — patient tumor genetics combined with in vitro KD recapitulating cardinal phenotypes using multiple orthogonal methods","pmids":["30166553"],"is_preprint":false},{"year":2015,"finding":"AAV-mediated Ac45 (ATP6AP1) knockdown in a mouse periodontitis model impairs osteoclast-mediated extracellular acidification and bone resorption in vivo, protects against bone erosion by >85%, and attenuates periodontal inflammation including reduced pro-inflammatory cytokine expression and immune cell infiltration.","method":"AAV-shRNA knockdown in vivo (mouse periodontitis model), histology, immunochemistry, ELISA, qRT-PCR","journal":"Journal of clinical periodontology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo knockdown with defined phenotypic readouts but single lab, and mechanism extends prior in vitro findings","pmids":["25952706"],"is_preprint":false},{"year":2002,"finding":"Targeted disruption of the Ac45 (ATP6AP1) gene in mouse embryonic stem cells generates null mutant (−/Y) ES cells; injection into blastocysts produced chimeric mice with severely compromised development, suggesting an essential role in early embryonic development consistent with other V-ATPase subunit knockouts.","method":"Gene targeting/homologous recombination in mouse ES cells, blastocyst injection, developmental phenotype assessment","journal":"Molecular membrane biology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — gene targeting establishes essentiality but developmental lethality prevents detailed mechanistic characterization; single study","pmids":["11989824"],"is_preprint":false},{"year":2020,"finding":"A novel hemizygous ATP6AP1 mutation (c.221T>C, p.L74P) causes increased reactive oxygen species in patient fibroblasts and striking hepatic copper accumulation, with tissue-specific differences in ATP6AP1 protein levels and post-translational modification pattern, demonstrating that ATP6AP1 deficiency leads to peroxisomal dysfunction and disrupted copper homeostasis.","method":"Patient fibroblast analysis, ROS assay, hepatic copper quantification, Western blot of tissue-specific protein pattern","journal":"Journal of inherited metabolic disease","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — patient-derived cells with direct biochemical measurements, single case report with two siblings, single lab","pmids":["32216104"],"is_preprint":false}],"current_model":"ATP6AP1 (Ac45) is a type I transmembrane accessory subunit of the V-ATPase that associates with the V0 sector of the proton pump, undergoes furin-mediated proteolytic cleavage after N-linked glycosylation in the early secretory pathway, and functions as both a V-ATPase assembly factor (orthologous to yeast Voa1) and a regulator of organellar acidification, lysosomal trafficking, and Ca2+-dependent exocytosis in neuroendocrine and osteoclast cells; additionally, its C-terminal tail acts as an unconventional GEF for the small GTPase Rheb to activate mTORC1, and its inhibition by SARS-CoV-2 NSP6 blocks lysosomal acidification to trigger NLRP3-dependent pyroptosis."},"narrative":{"mechanistic_narrative":"ATP6AP1 (Ac45) is a type I transmembrane accessory subunit of the vacuolar H+-ATPase (V-ATPase) that functions as a V-ATPase assembly factor and regulator of organellar acidification across the secretory and endolysosomal pathways [PMID:27231034, PMID:22044156]. It is the functional human ortholog of the yeast V-ATPase assembly factor Voa1: processed wild-type Ac45, but not disease-associated missense mutants, restores V-ATPase-dependent growth in Voa1-deficient yeast [PMID:27231034]. The newly synthesized protein is N-glycosylated and then proteolytically cleaved in the early secretory pathway — cleavage by furin separates an N-terminal fragment from a C-terminal fragment, and glycosylation is required for proper folding and cleavage [PMID:11952786, PMID:18713856]; the intact N-terminal domain confers ER retention, while the cleaved C-tail is required to recruit V-ATPase into the secretory pathway and to support Ca2+-dependent regulated exocytosis, and the cytoplasmic tail also carries autonomous signals for retrieval from the cell surface to vacuolar compartments [PMID:22736765, PMID:9739073]. Through these activities Ac45 controls intragranular pH and prohormone processing in neuroendocrine cells [PMID:20702583, PMID:18657579] and drives RANKL-induced osteoclast differentiation, extracellular acidification, lysosomal trafficking to the ruffled border, and cathepsin K exocytosis required for bone resorption [PMID:22467241, PMID:25952706]. Loss-of-function mutations impair vesicle acidification, cause endosomal redistribution and intracytoplasmic granule accumulation, and confer oncogenic properties [PMID:30166553]. Independent of its V-ATPase role, the conserved C-tail binds Rheb through its last 12 amino acids and uses a tri-aspartate motif to enhance Rheb GTP loading, acting as an unconventional GEF that promotes cancer cell proliferation and migration [PMID:38448650]. ATP6AP1 is a target of viral subversion: SARS-CoV-2 NSP6 binds ATP6AP1 and blocks its cleavage-mediated activation, impairing lysosomal acidification and autophagic flux to trigger NLRP3/ASC-dependent caspase-1 activation and pyroptosis [PMID:34997207].","teleology":[{"year":1999,"claim":"Established that ATP6AP1/Ac45 is a neuroendocrine-enriched V-ATPase-associated protein synthesized as a glycosylated precursor that is intracellularly cleaved, defining its basic biosynthetic identity.","evidence":"cDNA cloning, biosynthetic pulse labeling, deglycosylation, and immunocytochemistry in Xenopus pituitary","pmids":["10336633"],"confidence":"High","gaps":["Cleaving protease not identified","Functional consequence of cleavage unresolved"]},{"year":2002,"claim":"Defined the post-translational processing pathway, showing N-glycosylation precedes slow proteolytic cleavage in the early secretory pathway and is required for folding and cleavage.","evidence":"Pulse-chase radiolabeling with endoglycosidase H, tunicamycin, brefeldin A, and monensin treatments","pmids":["11952786"],"confidence":"High","gaps":["Protease responsible not yet identified","Fate/function of cleavage fragments not assigned"]},{"year":2002,"claim":"Mapped autonomous endocytic sorting determinants in the cytoplasmic tail, explaining how Ac45 is retrieved from the cell surface to vacuolar compartments.","evidence":"Chimeric Tac-tail constructs and truncation mutants with antibody internalization assays in CV-1 fibroblasts","pmids":["9739073"],"confidence":"High","gaps":["Trafficking adaptors mediating retrieval unknown","Distinct from canonical tyrosine/di-leucine motifs but motif identity undefined"]},{"year":2002,"claim":"Tested organismal requirement by gene disruption, indicating an essential role in early development consistent with V-ATPase biology.","evidence":"Homologous recombination in mouse ES cells and blastocyst injection with developmental assessment","pmids":["11989824"],"confidence":"Medium","gaps":["Developmental lethality prevents mechanistic dissection","Tissue-specific roles not addressed"]},{"year":2008,"claim":"Identified furin as the protease cleaving Ac45 and placed the furin–Ac45–V-ATPase axis in control of granule acidification and regulated secretion.","evidence":"Pdx1-Cre furin knockout mice, ex vivo cleavage-site mapping, DAMP acidification, and siRNA in betaTC3 cells","pmids":["18713856"],"confidence":"High","gaps":["Whether furin is the sole protease in all tissues unclear","Structural basis of cleavage-dependent activation unresolved"]},{"year":2008,"claim":"Demonstrated through gain-of-function that Ac45 guides V-ATPase trafficking and enhances Ca2+-dependent secretion, linking acidification machinery to exocytosis.","evidence":"Transgenic Xenopus melanotrope overexpression with immunofluorescence, electron microscopy, and secretion assays","pmids":["18657579"],"confidence":"High","gaps":["Direct molecular partners in exocytosis not identified","Mechanism coupling V-ATPase to fusion machinery unknown"]},{"year":2010,"claim":"Showed Ac45 regulates granular acidification required for prohormone processing, establishing it as the first identified regulator of V-ATPase-mediated granular pH.","evidence":"Transgenic Xenopus melanotrope model with granular acidification, inhibitor sensitivity, and POMC processing assays","pmids":["20702583"],"confidence":"High","gaps":["Differential effect on PC1 vs PC2 maturation mechanistically unexplained"]},{"year":2012,"claim":"Resolved the domain logic of Ac45 trafficking, showing intact N-terminal domain confers ER retention while the cleaved C-tail recruits V-ATPase into the secretory pathway for regulated exocytosis.","evidence":"Deletion-mutant structure-function analysis in transgenic Xenopus melanotrope cells with imaging and secretion readouts","pmids":["22736765"],"confidence":"High","gaps":["Receptor/adaptor reading the C-tail signal unidentified","C-tail interaction partners not defined"]},{"year":2012,"claim":"Demonstrated a cell-type-specific requirement for Ac45 in osteoclast differentiation, acidification, lysosomal trafficking, and cathepsin K exocytosis beyond generic V-ATPase function.","evidence":"siRNA knockdown with bone resorption, acidification, trafficking, exocytosis, and signaling Western blots","pmids":["22467241"],"confidence":"High","gaps":["Proposed Rab7 interaction not biochemically confirmed","Link from acidification to ERK/c-fos/NFATc1 signaling indirect"]},{"year":2012,"claim":"Consolidated the V0-association model, framing Ac45 as a V0-sector partner governing intragranular pH and exocytotic membrane fusion.","evidence":"Review synthesis of biochemical co-purification and neuroendocrine cell studies","pmids":["22044156"],"confidence":"Medium","gaps":["Review-level synthesis, not new primary data","Stoichiometry and structural arrangement within V0 not defined"]},{"year":2015,"claim":"Validated the osteoclast role in vivo, showing Ac45 knockdown protects against bone erosion and attenuates inflammation in periodontitis.","evidence":"AAV-shRNA knockdown in a mouse periodontitis model with histology, ELISA, and qRT-PCR","pmids":["25952706"],"confidence":"Medium","gaps":["Single lab in vivo knockdown","Mechanism of anti-inflammatory effect not dissected"]},{"year":2016,"claim":"Established ATP6AP1 as the human functional ortholog of yeast V-ATPase assembly factor Voa1 and tied disease mutations to failed assembly.","evidence":"Yeast complementation with wild-type vs patient missense mutants, homology detection, and tissue isoform Western blots","pmids":["27231034"],"confidence":"High","gaps":["Molecular step of assembly that Ac45 chaperones not defined","Basis of tissue-specific isoform pattern unknown"]},{"year":2018,"claim":"Linked ATP6AP1 loss-of-function to vesicle de-acidification, endosomal disruption, granule accumulation, and oncogenic transformation in granular cell tumors.","evidence":"Tumor exome/targeted sequencing plus in vitro siRNA recapitulation with acidification and oncogenic assays","pmids":["30166553"],"confidence":"High","gaps":["Mechanism converting acidification loss to oncogenic properties unresolved"]},{"year":2020,"claim":"Extended the phenotypic spectrum of ATP6AP1 deficiency to peroxisomal dysfunction and disrupted copper homeostasis via a new hemizygous mutation.","evidence":"Patient fibroblast ROS assay, hepatic copper quantification, and tissue-specific Western blot in two siblings","pmids":["32216104"],"confidence":"Medium","gaps":["Single family case report","Mechanistic link between V-ATPase defect and copper accumulation unestablished"]},{"year":2022,"claim":"Identified ATP6AP1 as a target of SARS-CoV-2 NSP6, defining a viral mechanism that blocks Ac45 activation to impair lysosomal acidification and trigger pyroptosis.","evidence":"Reciprocal co-IP, lysosomal acidification and autophagic flux assays, inflammasome/caspase-1 readouts, and an NSP6 L37F binding-deficient variant","pmids":["34997207"],"confidence":"High","gaps":["Structural interface of NSP6–ATP6AP1 binding not resolved","Step of cleavage-activation blocked by NSP6 undefined"]},{"year":2024,"claim":"Uncovered a non-canonical, V-ATPase-independent function of the ATP6AP1 C-tail as an unconventional GEF for Rheb that activates mTORC1 signaling and drives cancer phenotypes.","evidence":"Proximity labeling, co-IP, in vitro GTP loading, tri-aspartate motif mutagenesis, and proliferation/migration assays","pmids":["38448650"],"confidence":"High","gaps":["Whether GEF and V-ATPase functions are spatially/temporally separated unclear","In vivo contribution to mTORC1 regulation not established"]},{"year":null,"claim":"How ATP6AP1 partitions between its V-ATPase assembly/acidification role and its Rheb-GEF/mTORC1 role, and how cleavage state governs that switch, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of full-length or cleaved Ac45 within V0","Determinants directing the C-tail to V-ATPase versus Rheb unknown","Tissue-specific consequences of dual function not mapped"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,2]},{"term_id":"GO:0005215","term_label":"transporter activity","supporting_discovery_ids":[4,7]}],"localization":[{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[8,10]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[5,8]},{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[1,3]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[9,12]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[5,9]}],"pathway":[{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[2,3,4]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[5,8]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[1]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[6,10]}],"complexes":["V-ATPase (V0 sector)"],"partners":["RHEB","FURIN","NSP6 (SARS-COV-2)"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q15904","full_name":"V-type proton ATPase subunit S1","aliases":["Protein XAP-3","V-ATPase Ac45 subunit","V-ATPase S1 accessory protein","Vacuolar proton pump subunit S1"],"length_aa":470,"mass_kda":52.0,"function":"Accessory subunit of the proton-transporting vacuolar (V)-ATPase protein pump, which is required for luminal acidification of secretory vesicles (PubMed:33065002). Guides the V-type ATPase into specialized subcellular compartments, such as neuroendocrine regulated secretory vesicles or the ruffled border of the osteoclast, thereby regulating its activity (PubMed:27231034). Involved in membrane trafficking and Ca(2+)-dependent membrane fusion (PubMed:27231034). May play a role in the assembly of the V-type ATPase complex (Probable). 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). 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virology","url":"https://pubmed.ncbi.nlm.nih.gov/27283114","citation_count":17,"is_preprint":false},{"pmid":"15351483","id":"PMC_15351483","title":"Heparin binding activity of orf virus F1L protein.","date":"2004","source":"Virus research","url":"https://pubmed.ncbi.nlm.nih.gov/15351483","citation_count":17,"is_preprint":false},{"pmid":"9335578","id":"PMC_9335578","title":"Roles for lambda Orf and Escherichia coli RecO, RecR and RecF in lambda recombination.","date":"1997","source":"Genetics","url":"https://pubmed.ncbi.nlm.nih.gov/9335578","citation_count":17,"is_preprint":false},{"pmid":"25952706","id":"PMC_25952706","title":"Ac45 silencing mediated by AAV-sh-Ac45-RNAi prevents both bone loss and inflammation caused by periodontitis.","date":"2015","source":"Journal of clinical periodontology","url":"https://pubmed.ncbi.nlm.nih.gov/25952706","citation_count":15,"is_preprint":false},{"pmid":"20702583","id":"PMC_20702583","title":"V-ATPase-mediated granular acidification is regulated by the V-ATPase accessory subunit Ac45 in POMC-producing cells.","date":"2010","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/20702583","citation_count":15,"is_preprint":false},{"pmid":"26645035","id":"PMC_26645035","title":"Isolation and phylogenetic analysis of caprine Orf virus in Malaysia.","date":"2015","source":"Virusdisease","url":"https://pubmed.ncbi.nlm.nih.gov/26645035","citation_count":15,"is_preprint":false},{"pmid":"36574498","id":"PMC_36574498","title":"Natural Compound Allicin Containing Thiosulfinate Moieties as Transmembrane Protein 16A (TMEM16A) Ion Channel Inhibitor for Food Adjuvant Therapy of Lung Cancer.","date":"2022","source":"Journal of agricultural and food chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/36574498","citation_count":15,"is_preprint":false},{"pmid":"27795413","id":"PMC_27795413","title":"Ankyrin Repeat Proteins of Orf Virus Influence the Cellular Hypoxia Response Pathway.","date":"2016","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/27795413","citation_count":14,"is_preprint":false},{"pmid":"11962638","id":"PMC_11962638","title":"RAPD isolation of a Y chromosome specific ORF in a dioecious plant, Silene latifolia.","date":"2002","source":"Genome","url":"https://pubmed.ncbi.nlm.nih.gov/11962638","citation_count":14,"is_preprint":false},{"pmid":"31510805","id":"PMC_31510805","title":"iNK-CD64/16A cells: a promising approach for ADCC?","date":"2019","source":"Expert opinion on biological therapy","url":"https://pubmed.ncbi.nlm.nih.gov/31510805","citation_count":13,"is_preprint":false},{"pmid":"33506118","id":"PMC_33506118","title":"Biochemical and molecular study on serum miRNA-16a and miRNA- 451 as neonatal sepsis biomarkers.","date":"2021","source":"Biochemistry and biophysics reports","url":"https://pubmed.ncbi.nlm.nih.gov/33506118","citation_count":13,"is_preprint":false},{"pmid":"22736765","id":"PMC_22736765","title":"Identification of domains within the V-ATPase accessory subunit Ac45 involved in V-ATPase transport and Ca2+-dependent exocytosis.","date":"2012","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/22736765","citation_count":13,"is_preprint":false},{"pmid":"29896166","id":"PMC_29896166","title":"Orf Virus Encoded Protein ORFV119 Induces Cell Apoptosis Through the Extrinsic and Intrinsic Pathways.","date":"2018","source":"Frontiers in microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/29896166","citation_count":13,"is_preprint":false},{"pmid":"11952786","id":"PMC_11952786","title":"The fate of newly synthesized V-ATPase accessory subunit Ac45 in the secretory pathway.","date":"2002","source":"European journal of biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11952786","citation_count":13,"is_preprint":false},{"pmid":"9705253","id":"PMC_9705253","title":"Repetitive DNA insertion in a protein kinase ORF of a latent FSV (Feldmannia sp. virus) genome.","date":"1998","source":"Virology","url":"https://pubmed.ncbi.nlm.nih.gov/9705253","citation_count":13,"is_preprint":false},{"pmid":"32058063","id":"PMC_32058063","title":"Prenatal and postnatal phenotype of a pathologic variant in the ATP6AP1 gene.","date":"2020","source":"European journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/32058063","citation_count":12,"is_preprint":false},{"pmid":"8972571","id":"PMC_8972571","title":"Gene homology between orf virus and smallpox variola virus.","date":"1996","source":"Virus genes","url":"https://pubmed.ncbi.nlm.nih.gov/8972571","citation_count":12,"is_preprint":false},{"pmid":"37217030","id":"PMC_37217030","title":"Host range, severity and trans boundary transmission of Orf virus (ORFV).","date":"2023","source":"Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases","url":"https://pubmed.ncbi.nlm.nih.gov/37217030","citation_count":12,"is_preprint":false},{"pmid":"28338219","id":"PMC_28338219","title":"Human orf complicated by epidermolysis bullosa acquisita.","date":"2017","source":"The British journal of dermatology","url":"https://pubmed.ncbi.nlm.nih.gov/28338219","citation_count":12,"is_preprint":false},{"pmid":"11520578","id":"PMC_11520578","title":"Expression and functional properties of proteins encoded in the x-II ORF of HTLV-I.","date":"2001","source":"Virus research","url":"https://pubmed.ncbi.nlm.nih.gov/11520578","citation_count":11,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":47673,"output_tokens":4218,"usd":0.103145,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12235,"output_tokens":4910,"usd":0.091963,"stage2_stop_reason":"end_turn"},"total_usd":0.195108,"stage1_batch_id":"msgbatch_0179Z2pxRYBsXi2xADneafRw","stage2_batch_id":"msgbatch_01LaY9ZaVdgjWArcZo9eqQE5","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2024,\n      \"finding\": \"ATP6AP1 directly binds Rheb through its last 12 amino acids and utilizes a tri-aspartate motif in its highly conserved C-tail to enhance Rheb GTP loading, functioning as an unconventional guanine nucleotide exchange factor (GEF) for Rheb; targeting the ATP6AP1 C-tail blocks Rheb activation and inhibits cancer cell proliferation and migration.\",\n      \"method\": \"Proximity labeling (PhastID), co-immunoprecipitation, in vitro GTP loading assay, domain deletion/mutagenesis, functional cell proliferation and migration assays\",\n      \"journal\": \"Cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — multiple orthogonal methods including proximity labeling, direct binding assays, mutagenesis of functional domain, and functional readouts in a single rigorous study\",\n      \"pmids\": [\"38448650\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SARS-CoV-2 NSP6 directly interacts with ATP6AP1 (a V-ATPase proton pump component) and inhibits its cleavage-mediated activation, thereby impairing lysosome acidification and autophagic flux, which triggers NLRP3/ASC-dependent caspase-1 activation and pyroptosis in lung epithelial cells.\",\n      \"method\": \"Co-immunoprecipitation, overexpression, lysosomal acidification assays, autophagic flux assays, inflammasome/caspase-1 activation assays, pharmacological rescue experiments\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal interaction demonstrated, multiple orthogonal functional assays, supported by NSP6 variant (L37F) showing reduced binding and reduced pyroptosis induction\",\n      \"pmids\": [\"34997207\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ATP6AP1 (Ac45) is the functional human ortholog of yeast V-ATPase assembly factor Voa1; processed wild-type Ac45, but not disease-associated missense mutants, restores V-ATPase-dependent growth in Voa1 mutant yeast, establishing its role in V-ATPase assembly. Tissue-specific isoforms (40-kDa in brain, 62-kDa in liver, 50-kDa in B-cells) are present, linking V-ATPase assembly to immunoglobulin production and cognitive function.\",\n      \"method\": \"Yeast complementation assay, sequence profile homology detection, patient mutation analysis, Western blot of tissue-specific isoforms\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — yeast reconstitution complementation with wild-type vs. mutant forms, multiple orthogonal approaches, independently validated by patient phenotypes\",\n      \"pmids\": [\"27231034\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ATP6AP1 (Ac45) is required for RANKL-induced osteoclast differentiation, extracellular acidification, lysosomal trafficking to the ruffled border, and cathepsin K exocytosis; Ac45 knockdown severely impairs bone resorption and reduces osteoclast precursor proliferation and fusion, partially via downregulation of ERK phosphorylation and c-fos/NFATc1/Tm7sf4 expression. The effect on exocytosis is specific to Ac45 deficiency, distinct from general V-ATPase defects, and may involve interaction with the small GTPase Rab7.\",\n      \"method\": \"siRNA knockdown, bone resorption assays, extracellular acidification assay, lysosomal trafficking immunofluorescence, cathepsin K exocytosis assay, Western blot for signaling molecules\",\n      \"journal\": \"Journal of bone and mineral research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KD with multiple orthogonal functional readouts (acidification, trafficking, exocytosis, signaling), single lab\",\n      \"pmids\": [\"22467241\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Transgenic overexpression of Ac45 (ATP6AP1) specifically in Xenopus melanotrope cells increases granular acidification, reduces sensitivity to V-ATPase inhibitor, enhances early POMC processing by PC1, but reduces PC2 maturation; establishing Ac45 as the first identified regulator of V-ATPase-mediated granular acidification required for prohormone processing.\",\n      \"method\": \"Transgenic Xenopus melanotrope cell model, granular acidification assay, V-ATPase inhibitor sensitivity assay, prohormone processing Western blot analysis\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — transgenic gain-of-function with multiple orthogonal phenotypic readouts in a well-established in vivo neuroendocrine model\",\n      \"pmids\": [\"20702583\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Transgenic expression of Ac45 (ATP6AP1) in Xenopus melanotrope cells causes accumulation of V-ATPase at the plasma membrane, increased abundance of secretory granules, plasma membrane protrusions, and increased Ca2+-dependent secretion efficiency, demonstrating that Ac45 guides V-ATPase through the secretory pathway and regulates Ca2+-dependent peptide secretion.\",\n      \"method\": \"Transgenic Xenopus melanotrope cell model, immunofluorescence, electron microscopy, Ca2+-dependent secretion assay\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — transgenic in vivo model with multiple orthogonal readouts (localization, secretion), consistent with prior and subsequent studies\",\n      \"pmids\": [\"18657579\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Furin cleaves Ac45 (ATP6AP1) at a defined site in pancreatic beta cells; furin knockout reduces granule acidification, and Ac45 knockdown similarly reduces regulated secretion and proinsulin II processing, establishing a furin–Ac45–V-ATPase axis controlling intragranular acidification in the regulated secretory pathway.\",\n      \"method\": \"Pdx1-Cre/loxP furin knockout mice, DAMP acidification assay, ex vivo cleavage assay to determine cleavage site, siRNA knockdown of furin and Ac45 in betaTC3 cells, insulin/proinsulin processing assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vivo KO combined with defined biochemical cleavage site mapping and in vitro functional validation with multiple readouts\",\n      \"pmids\": [\"18713856\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Ac45 (ATP6AP1) interacts with the V0 sector of the V-ATPase complex in neuroendocrine cells, regulating intragranular pH and Ca2+-dependent exocytotic membrane fusion.\",\n      \"method\": \"Biochemical co-purification/interaction studies, neuroendocrine cell models (review/synthesis of prior experimental work cited therein)\",\n      \"journal\": \"Current protein & peptide science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — summarizes experimental findings from multiple studies but as a review; underlying data from prior primary studies support the interaction claim\",\n      \"pmids\": [\"22044156\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The N-terminal domain of Ac45 (ATP6AP1) controls its retention in the endoplasmic reticulum when intact; proteolytic cleavage releasing the N-terminal domain enables transport through the secretory pathway. The C-tail of cleaved Ac45 is required for V-ATPase recruitment into the secretory pathway and for Ca2+-dependent regulated exocytosis, but not for plasma membrane targeting.\",\n      \"method\": \"Deletion mutant analysis in transgenic Xenopus melanotrope cells, immunofluorescence, electron microscopy, secretion assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — structure-function deletion mutant analysis in in vivo transgenic model with multiple orthogonal readouts\",\n      \"pmids\": [\"22736765\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"The cytoplasmic tail of Ac45 (ATP6AP1) contains autonomous internalization signal(s) in its membrane-distal region that mediate rapid retrieval from the cell surface and targeting to vacuolar compartments; multiple sites rather than a single short linear motif are responsible, and this routing is distinct from known tyrosine- or di-leucine-based sorting motifs.\",\n      \"method\": \"Transfection in CV-1 fibroblasts, antibody internalization experiments, chimeric protein (Ac45 tail fused to Tac), C-terminally truncated mutant expression, immunolocalization\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — domain mutagenesis combined with chimeric protein approach and direct trafficking assay, reproduced across multiple constructs\",\n      \"pmids\": [\"9739073\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Newly synthesized Ac45 (ATP6AP1) undergoes N-linked glycosylation (to ~62 kDa from ~46 kDa unglycosylated form), followed by slow proteolytic cleavage (half-life ~4–6 h) in the early secretory pathway producing an N-terminal (~22 kDa) and C-terminal (~42–44 kDa) fragment; N-linked glycosylation is required for proper folding and cleavage. The N-terminal fragment is rapidly degraded in a non-lysosomal, brefeldin A-sensitive manner.\",\n      \"method\": \"Pulse-chase radiolabeling, endoglycosidase H resistance assay, tunicamycin treatment, brefeldin A treatment, monensin treatment, deglycosylation, Western blot\",\n      \"journal\": \"European journal of biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — biochemical characterization with multiple inhibitors and enzymatic treatments providing mechanistic detail on processing pathway\",\n      \"pmids\": [\"11952786\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Xenopus Ac45 (ATP6AP1 ortholog) is synthesized as an N-glycosylated ~60 kDa precursor that is intracellularly cleaved to an ~40 kDa product; expression is predominantly in neuroendocrine tissues. The protein is associated with V-ATPase and localizes to biosynthetically active melanotrope cells in pituitary.\",\n      \"method\": \"cDNA cloning, Western blot, deglycosylation assay, biosynthetic pulse labeling, immunocytochemistry in Xenopus pituitary\",\n      \"journal\": \"European journal of biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical characterization with biosynthetic labeling and multiple complementary methods establishing processing and localization\",\n      \"pmids\": [\"10336633\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Loss-of-function mutations in ATP6AP1 impair vesicle acidification, cause redistribution of endosomal compartments, and lead to accumulation of intracytoplasmic granules in granular cell tumors; ATP6AP1 depletion in vitro recapitulates these phenotypes and results in acquisition of oncogenic properties.\",\n      \"method\": \"Whole-exome and targeted sequencing of patient tumors, in vitro siRNA silencing, vesicle acidification assay, endosomal compartment imaging, granule accumulation assay, oncogenic property assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — patient tumor genetics combined with in vitro KD recapitulating cardinal phenotypes using multiple orthogonal methods\",\n      \"pmids\": [\"30166553\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"AAV-mediated Ac45 (ATP6AP1) knockdown in a mouse periodontitis model impairs osteoclast-mediated extracellular acidification and bone resorption in vivo, protects against bone erosion by >85%, and attenuates periodontal inflammation including reduced pro-inflammatory cytokine expression and immune cell infiltration.\",\n      \"method\": \"AAV-shRNA knockdown in vivo (mouse periodontitis model), histology, immunochemistry, ELISA, qRT-PCR\",\n      \"journal\": \"Journal of clinical periodontology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo knockdown with defined phenotypic readouts but single lab, and mechanism extends prior in vitro findings\",\n      \"pmids\": [\"25952706\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Targeted disruption of the Ac45 (ATP6AP1) gene in mouse embryonic stem cells generates null mutant (−/Y) ES cells; injection into blastocysts produced chimeric mice with severely compromised development, suggesting an essential role in early embryonic development consistent with other V-ATPase subunit knockouts.\",\n      \"method\": \"Gene targeting/homologous recombination in mouse ES cells, blastocyst injection, developmental phenotype assessment\",\n      \"journal\": \"Molecular membrane biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — gene targeting establishes essentiality but developmental lethality prevents detailed mechanistic characterization; single study\",\n      \"pmids\": [\"11989824\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"A novel hemizygous ATP6AP1 mutation (c.221T>C, p.L74P) causes increased reactive oxygen species in patient fibroblasts and striking hepatic copper accumulation, with tissue-specific differences in ATP6AP1 protein levels and post-translational modification pattern, demonstrating that ATP6AP1 deficiency leads to peroxisomal dysfunction and disrupted copper homeostasis.\",\n      \"method\": \"Patient fibroblast analysis, ROS assay, hepatic copper quantification, Western blot of tissue-specific protein pattern\",\n      \"journal\": \"Journal of inherited metabolic disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — patient-derived cells with direct biochemical measurements, single case report with two siblings, single lab\",\n      \"pmids\": [\"32216104\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ATP6AP1 (Ac45) is a type I transmembrane accessory subunit of the V-ATPase that associates with the V0 sector of the proton pump, undergoes furin-mediated proteolytic cleavage after N-linked glycosylation in the early secretory pathway, and functions as both a V-ATPase assembly factor (orthologous to yeast Voa1) and a regulator of organellar acidification, lysosomal trafficking, and Ca2+-dependent exocytosis in neuroendocrine and osteoclast cells; additionally, its C-terminal tail acts as an unconventional GEF for the small GTPase Rheb to activate mTORC1, and its inhibition by SARS-CoV-2 NSP6 blocks lysosomal acidification to trigger NLRP3-dependent pyroptosis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ATP6AP1 (Ac45) is a type I transmembrane accessory subunit of the vacuolar H+-ATPase (V-ATPase) that functions as a V-ATPase assembly factor and regulator of organellar acidification across the secretory and endolysosomal pathways [#2, #7]. It is the functional human ortholog of the yeast V-ATPase assembly factor Voa1: processed wild-type Ac45, but not disease-associated missense mutants, restores V-ATPase-dependent growth in Voa1-deficient yeast [#2]. The newly synthesized protein is N-glycosylated and then proteolytically cleaved in the early secretory pathway — cleavage by furin separates an N-terminal fragment from a C-terminal fragment, and glycosylation is required for proper folding and cleavage [#10, #6]; the intact N-terminal domain confers ER retention, while the cleaved C-tail is required to recruit V-ATPase into the secretory pathway and to support Ca2+-dependent regulated exocytosis, and the cytoplasmic tail also carries autonomous signals for retrieval from the cell surface to vacuolar compartments [#8, #9]. Through these activities Ac45 controls intragranular pH and prohormone processing in neuroendocrine cells [#4, #5] and drives RANKL-induced osteoclast differentiation, extracellular acidification, lysosomal trafficking to the ruffled border, and cathepsin K exocytosis required for bone resorption [#3, #13]. Loss-of-function mutations impair vesicle acidification, cause endosomal redistribution and intracytoplasmic granule accumulation, and confer oncogenic properties [#12]. Independent of its V-ATPase role, the conserved C-tail binds Rheb through its last 12 amino acids and uses a tri-aspartate motif to enhance Rheb GTP loading, acting as an unconventional GEF that promotes cancer cell proliferation and migration [#0]. ATP6AP1 is a target of viral subversion: SARS-CoV-2 NSP6 binds ATP6AP1 and blocks its cleavage-mediated activation, impairing lysosomal acidification and autophagic flux to trigger NLRP3/ASC-dependent caspase-1 activation and pyroptosis [#1].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Established that ATP6AP1/Ac45 is a neuroendocrine-enriched V-ATPase-associated protein synthesized as a glycosylated precursor that is intracellularly cleaved, defining its basic biosynthetic identity.\",\n      \"evidence\": \"cDNA cloning, biosynthetic pulse labeling, deglycosylation, and immunocytochemistry in Xenopus pituitary\",\n      \"pmids\": [\"10336633\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cleaving protease not identified\", \"Functional consequence of cleavage unresolved\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Defined the post-translational processing pathway, showing N-glycosylation precedes slow proteolytic cleavage in the early secretory pathway and is required for folding and cleavage.\",\n      \"evidence\": \"Pulse-chase radiolabeling with endoglycosidase H, tunicamycin, brefeldin A, and monensin treatments\",\n      \"pmids\": [\"11952786\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Protease responsible not yet identified\", \"Fate/function of cleavage fragments not assigned\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Mapped autonomous endocytic sorting determinants in the cytoplasmic tail, explaining how Ac45 is retrieved from the cell surface to vacuolar compartments.\",\n      \"evidence\": \"Chimeric Tac-tail constructs and truncation mutants with antibody internalization assays in CV-1 fibroblasts\",\n      \"pmids\": [\"9739073\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Trafficking adaptors mediating retrieval unknown\", \"Distinct from canonical tyrosine/di-leucine motifs but motif identity undefined\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Tested organismal requirement by gene disruption, indicating an essential role in early development consistent with V-ATPase biology.\",\n      \"evidence\": \"Homologous recombination in mouse ES cells and blastocyst injection with developmental assessment\",\n      \"pmids\": [\"11989824\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Developmental lethality prevents mechanistic dissection\", \"Tissue-specific roles not addressed\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identified furin as the protease cleaving Ac45 and placed the furin–Ac45–V-ATPase axis in control of granule acidification and regulated secretion.\",\n      \"evidence\": \"Pdx1-Cre furin knockout mice, ex vivo cleavage-site mapping, DAMP acidification, and siRNA in betaTC3 cells\",\n      \"pmids\": [\"18713856\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether furin is the sole protease in all tissues unclear\", \"Structural basis of cleavage-dependent activation unresolved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Demonstrated through gain-of-function that Ac45 guides V-ATPase trafficking and enhances Ca2+-dependent secretion, linking acidification machinery to exocytosis.\",\n      \"evidence\": \"Transgenic Xenopus melanotrope overexpression with immunofluorescence, electron microscopy, and secretion assays\",\n      \"pmids\": [\"18657579\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct molecular partners in exocytosis not identified\", \"Mechanism coupling V-ATPase to fusion machinery unknown\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Showed Ac45 regulates granular acidification required for prohormone processing, establishing it as the first identified regulator of V-ATPase-mediated granular pH.\",\n      \"evidence\": \"Transgenic Xenopus melanotrope model with granular acidification, inhibitor sensitivity, and POMC processing assays\",\n      \"pmids\": [\"20702583\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Differential effect on PC1 vs PC2 maturation mechanistically unexplained\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Resolved the domain logic of Ac45 trafficking, showing intact N-terminal domain confers ER retention while the cleaved C-tail recruits V-ATPase into the secretory pathway for regulated exocytosis.\",\n      \"evidence\": \"Deletion-mutant structure-function analysis in transgenic Xenopus melanotrope cells with imaging and secretion readouts\",\n      \"pmids\": [\"22736765\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Receptor/adaptor reading the C-tail signal unidentified\", \"C-tail interaction partners not defined\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Demonstrated a cell-type-specific requirement for Ac45 in osteoclast differentiation, acidification, lysosomal trafficking, and cathepsin K exocytosis beyond generic V-ATPase function.\",\n      \"evidence\": \"siRNA knockdown with bone resorption, acidification, trafficking, exocytosis, and signaling Western blots\",\n      \"pmids\": [\"22467241\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Proposed Rab7 interaction not biochemically confirmed\", \"Link from acidification to ERK/c-fos/NFATc1 signaling indirect\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Consolidated the V0-association model, framing Ac45 as a V0-sector partner governing intragranular pH and exocytotic membrane fusion.\",\n      \"evidence\": \"Review synthesis of biochemical co-purification and neuroendocrine cell studies\",\n      \"pmids\": [\"22044156\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Review-level synthesis, not new primary data\", \"Stoichiometry and structural arrangement within V0 not defined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Validated the osteoclast role in vivo, showing Ac45 knockdown protects against bone erosion and attenuates inflammation in periodontitis.\",\n      \"evidence\": \"AAV-shRNA knockdown in a mouse periodontitis model with histology, ELISA, and qRT-PCR\",\n      \"pmids\": [\"25952706\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab in vivo knockdown\", \"Mechanism of anti-inflammatory effect not dissected\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Established ATP6AP1 as the human functional ortholog of yeast V-ATPase assembly factor Voa1 and tied disease mutations to failed assembly.\",\n      \"evidence\": \"Yeast complementation with wild-type vs patient missense mutants, homology detection, and tissue isoform Western blots\",\n      \"pmids\": [\"27231034\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular step of assembly that Ac45 chaperones not defined\", \"Basis of tissue-specific isoform pattern unknown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Linked ATP6AP1 loss-of-function to vesicle de-acidification, endosomal disruption, granule accumulation, and oncogenic transformation in granular cell tumors.\",\n      \"evidence\": \"Tumor exome/targeted sequencing plus in vitro siRNA recapitulation with acidification and oncogenic assays\",\n      \"pmids\": [\"30166553\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism converting acidification loss to oncogenic properties unresolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Extended the phenotypic spectrum of ATP6AP1 deficiency to peroxisomal dysfunction and disrupted copper homeostasis via a new hemizygous mutation.\",\n      \"evidence\": \"Patient fibroblast ROS assay, hepatic copper quantification, and tissue-specific Western blot in two siblings\",\n      \"pmids\": [\"32216104\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single family case report\", \"Mechanistic link between V-ATPase defect and copper accumulation unestablished\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified ATP6AP1 as a target of SARS-CoV-2 NSP6, defining a viral mechanism that blocks Ac45 activation to impair lysosomal acidification and trigger pyroptosis.\",\n      \"evidence\": \"Reciprocal co-IP, lysosomal acidification and autophagic flux assays, inflammasome/caspase-1 readouts, and an NSP6 L37F binding-deficient variant\",\n      \"pmids\": [\"34997207\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural interface of NSP6–ATP6AP1 binding not resolved\", \"Step of cleavage-activation blocked by NSP6 undefined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Uncovered a non-canonical, V-ATPase-independent function of the ATP6AP1 C-tail as an unconventional GEF for Rheb that activates mTORC1 signaling and drives cancer phenotypes.\",\n      \"evidence\": \"Proximity labeling, co-IP, in vitro GTP loading, tri-aspartate motif mutagenesis, and proliferation/migration assays\",\n      \"pmids\": [\"38448650\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether GEF and V-ATPase functions are spatially/temporally separated unclear\", \"In vivo contribution to mTORC1 regulation not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How ATP6AP1 partitions between its V-ATPase assembly/acidification role and its Rheb-GEF/mTORC1 role, and how cleavage state governs that switch, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of full-length or cleaved Ac45 within V0\", \"Determinants directing the C-tail to V-ATPase versus Rheb unknown\", \"Tissue-specific consequences of dual function not mapped\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 2]},\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [4, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [8, 10]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [5, 8]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [1, 3]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [9, 12]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [5, 9]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [2, 3, 4]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [5, 8]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [6, 10]}\n    ],\n    \"complexes\": [\"V-ATPase (V0 sector)\"],\n    \"partners\": [\"RHEB\", \"FURIN\", \"NSP6 (SARS-CoV-2)\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}