{"gene":"ATP5PO","run_date":"2026-06-09T22:02:44","timeline":{"discoveries":[{"year":1995,"finding":"ATP5PO (OSCP subunit) was cloned and identified as a key structural component of the stalk of the mitochondrial F1F0-ATP synthase, encoded on chromosome 21q22.1-q22.2, with >80% amino acid identity to bovine and murine OSCP subunits, and expressed in all human tissues, most strongly in muscle and heart.","method":"Exon trapping, cDNA cloning, sequence analysis, chromosomal mapping to YAC contigs","journal":"Genomics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct cloning and sequence characterization with chromosomal mapping, single lab, multiple methods but no functional reconstitution","pmids":["7490082"],"is_preprint":false},{"year":2022,"finding":"A homozygous splice variant (c.87+3A>G) in ATP5PO causing skipping of exon 2 results in decreased ATP5PO protein, defective complex V assembly with markedly reduced peripheral stalk proteins, and reduced complex V hydrolytic activity in patient fibroblasts; expression of the exon-2-deleted human ATP5PO in yeast deleted for its homolog (yATP5) failed to rescue oxidative phosphorylation-dependent growth, demonstrating that exon 2 is required for protein function.","method":"cDNA splicing analysis, fibroblast biochemical assays (complex V assembly, hydrolytic activity), yeast complementation assay","journal":"Journal of inherited metabolic disease","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods (patient fibroblast biochemistry, yeast complementation), two independent families, functional mutagenesis-equivalent via splice variant","pmids":["35621276"],"is_preprint":false},{"year":2022,"finding":"CLDN10 overexpression upregulates acetylation and expression levels of ATP5PO (ATP5O), leading to mitochondrial dysfunction with increased ROS, increased NDUFS2/SDHB/Cleaved-Caspase 3/E-cadherin, and decreased mitochondrial membrane potential; knockdown of ATP5O in CLDN10-overexpressing cells reverses these effects, placing ATP5O downstream of CLDN10 in regulating mitochondrial function and suppressing ccRCC growth and metastasis.","method":"Gain-of-function (CLDN10 overexpression), loss-of-function (ATP5O knockdown), Western blotting, ROS measurement, mitochondrial membrane potential assay, orthotopic mouse model","journal":"International journal of biological sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis via knockdown rescue in cell line and in vivo, single lab, multiple readouts","pmids":["35414767"],"is_preprint":false},{"year":2022,"finding":"Under chronic stress, ATP5O K51 crotonylation is decreased due to HDAC2 hyperphosphorylation at S424 (which enhances HDAC2 decrotonylation activity); this reduction in ATP5O crotonylation causes decreased gross ATP5O protein levels and downregulated phospholipid metabolism. Correcting HDAC2 hyperphosphorylation restored ATP5O levels and partially rescued phospholipid metabolism.","method":"Quantitative PTM-omics, phospho-omics, metabolomics, site-specific PTM analysis, genetic correction of HDAC2 phosphorylation in mouse model","journal":"Research (Washington, D.C.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multi-omic approach with site-specific PTM identification and in vivo rescue, single lab","pmids":["38645677"],"is_preprint":false},{"year":2023,"finding":"ATP5PO overexpression in zebrafish caused a significant reduction in enteric nervous system (ENS) cells; in vitro, overexpression paradoxically reduced ATP5PO protein levels, impaired neuronal differentiation, and reduced mitochondrial ATP production in a neuroblastoma cell line. Genetic epistasis was demonstrated between ATP5PO and ret (the principal Hirschsprung disease gene), establishing ATP5PO as a regulator of ENS development acting in the same pathway as RET.","method":"Zebrafish overexpression screen (ENS cell quantification), in vitro overexpression in neuroblastoma cells (ATP production assay, differentiation assay), genetic epistasis analysis with ret","journal":"Biochimica et biophysica acta. Molecular basis of disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional overexpression in vivo and in vitro with epistasis, single lab, multiple orthogonal readouts","pmids":["38128843"],"is_preprint":false},{"year":2025,"finding":"SIRT3 deacetylates ATP5O at the K162 site in primary mouse cardiomyocytes; deacetylation mimics decreased mitochondrial damage while acetylation mimics promoted mitochondrial damage. CVB-D upregulates SIRT3 expression, which increases ATP5O acetylation (at K162) and enhances mitochondrial function. ATP5O knockout inhibited the protective effects of SIRT3 overexpression in DCM, establishing SIRT3 as a writer and K162 as a functional acetylation site on ATP5O.","method":"Co-immunoprecipitation, LC-MS/MS acetylation site mapping, acetylation/deacetylation mimic plasmid transfection, MST/SPR/ITC binding assays, AAV9-mediated SIRT3 overexpression and ATP5O knockout mouse models","journal":"Chinese medicine","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — site-specific PTM identified by LC-MS/MS, functional validation with mimic mutants, Co-IP, binding assays, and in vivo AAV knockout/overexpression, multiple orthogonal methods in one study","pmids":["41233904"],"is_preprint":false},{"year":2025,"finding":"A homozygous splice variant (c.87+3A>G) in ATP5PO in a patient caused approximately 35% of normal ATPase enzyme activity in fibroblasts (relative to citrate synthase), providing functional evidence that ATP5PO is required for complex V assembly and function.","method":"Fibroblast mitochondrial respiratory chain enzyme activity assay, whole-exome sequencing","journal":"American journal of medical genetics. Part A","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct enzymatic assay in patient-derived fibroblasts, single case, consistent with prior report (PMID 35621276)","pmids":["40913360"],"is_preprint":false}],"current_model":"ATP5PO encodes the oligomycin sensitivity-conferring protein (OSCP), a structural component of the stalk linking the F1 and F0 domains of mitochondrial complex V (ATP synthase); loss-of-function splice variants impair complex V assembly and hydrolytic/synthetic activity causing Leigh syndrome, while post-translational modifications—specifically SIRT3-mediated deacetylation at K162 and HDAC2-regulated crotonylation at K51—modulate ATP5PO stability and mitochondrial function, and balanced ATP5PO expression is required for enteric nervous system development acting in a pathway with RET."},"narrative":{"mechanistic_narrative":"ATP5PO encodes the oligomycin sensitivity-conferring protein (OSCP), a structural component of the peripheral stalk that links the F1 and F0 domains of the mitochondrial F1F0-ATP synthase (complex V), expressed ubiquitously but most strongly in muscle and heart [PMID:7490082]. Its function is required for proper complex V assembly and activity: a homozygous splice variant (c.87+3A>G) causing skipping of exon 2 reduces ATP5PO protein, destabilizes the peripheral stalk, and lowers complex V hydrolytic activity, and the exon-2-deleted protein fails to complement a yeast OSCP-null strain, establishing exon 2 as essential for function [PMID:35621276, PMID:40913360]. This loss of function underlies a mitochondrial disorder presenting as Leigh-like disease in affected patients [PMID:35621276, PMID:40913360]. ATP5PO activity is tuned by post-translational modification: SIRT3 binds and deacetylates ATP5PO at K162 in cardiomyocytes to limit mitochondrial damage [PMID:41233904], and HDAC2-regulated crotonylation at K51 controls ATP5PO protein stability and downstream phospholipid metabolism under chronic stress [PMID:38645677]. Balanced ATP5PO expression is also required for enteric nervous system development, where it acts genetically in the RET pathway, with both loss and overexpression compromising neuronal differentiation and mitochondrial ATP production [PMID:38128843].","teleology":[{"year":1995,"claim":"Established the molecular identity of ATP5PO as the OSCP stalk subunit of mitochondrial ATP synthase, defining its place in complex V.","evidence":"Exon trapping, cDNA cloning, sequence analysis and chromosomal mapping of the human gene","pmids":["7490082"],"confidence":"Medium","gaps":["No functional reconstitution of the protein in the synthase","Tissue expression described but not linked to phenotype","No structural placement within the assembled complex shown directly"]},{"year":2022,"claim":"Demonstrated that ATP5PO loss of function impairs complex V assembly and activity, linking the gene to human mitochondrial disease.","evidence":"Patient fibroblast biochemistry (assembly, hydrolytic activity) and yeast complementation of an OSCP-null strain across two families","pmids":["35621276"],"confidence":"High","gaps":["Mechanism by which stalk loss destabilizes the whole complex not resolved at structural level","Tissue-specific disease manifestation not fully explained"]},{"year":2022,"claim":"Placed ATP5PO downstream of CLDN10 in a pathway regulating mitochondrial function and tumor suppression, implicating its acetylation state.","evidence":"CLDN10 gain-of-function with ATP5O knockdown rescue, mitochondrial readouts, and orthotopic mouse model in ccRCC","pmids":["35414767"],"confidence":"Medium","gaps":["Acetylation sites and writers/erasers not identified here","Direct CLDN10–ATP5PO molecular relationship unestablished","Single lab, single cancer context"]},{"year":2022,"claim":"Identified K51 crotonylation as a stress-responsive modification controlling ATP5PO stability and phospholipid metabolism via HDAC2.","evidence":"Quantitative PTM/phospho/metabolomics with site-specific PTM mapping and genetic correction of HDAC2 phosphorylation in mouse","pmids":["38645677"],"confidence":"Medium","gaps":["Direct enzymatic decrotonylation of ATP5PO by HDAC2 not reconstituted","Mechanism linking crotonylation to phospholipid metabolism unclear","Single lab"]},{"year":2023,"claim":"Revealed a non-canonical role for ATP5PO in enteric nervous system development within the RET pathway, where dosage is critical.","evidence":"Zebrafish overexpression ENS quantification, neuroblastoma differentiation/ATP assays, and genetic epistasis with ret","pmids":["38128843"],"confidence":"Medium","gaps":["Molecular basis of the paradoxical protein decrease upon overexpression unknown","Direct biochemical link between ATP5PO and RET signaling not defined","Human ENS relevance inferred from model systems"]},{"year":2025,"claim":"Established SIRT3 as a writer and K162 as a functional acetylation site governing ATP5PO-dependent mitochondrial protection in the heart.","evidence":"Co-IP, LC-MS/MS site mapping, acetylation mimic mutants, MST/SPR/ITC binding, and AAV9 SIRT3 overexpression / ATP5O knockout mouse models in DCM","pmids":["41233904"],"confidence":"High","gaps":["How K162 acetylation alters complex V function structurally not resolved","Interplay between K162 acetylation and K51 crotonylation not examined","Cardiac-focused; generality to other tissues untested"]},{"year":null,"claim":"How the multiple PTMs (K162 acetylation, K51 crotonylation) integrate to control complex V assembly and the non-OSCP role of ATP5PO in RET-dependent ENS development remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified structural model of how stalk modifications affect synthase activity","Mechanism linking mitochondrial ATP5PO to RET signaling unknown","Cross-talk among PTM sites uncharacterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,1]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[0,1,5]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[1,6]}],"complexes":["mitochondrial ATP synthase (complex V)"],"partners":["SIRT3","HDAC2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P48047","full_name":"ATP synthase peripheral stalk subunit OSCP, mitochondrial","aliases":["ATP synthase subunit O","Oligomycin sensitivity conferral protein","OSCP"],"length_aa":213,"mass_kda":23.3,"function":"Subunit OSCP, of the mitochondrial membrane ATP synthase complex (F(1)F(0) ATP synthase or Complex V) that produces ATP from ADP in the presence of a proton gradient across the membrane which is generated by electron transport complexes of the respiratory chain (PubMed:37244256). ATP synthase complex consist of a soluble F(1) head domain - the catalytic core - and a membrane F(1) domain - the membrane proton channel (PubMed:37244256). These two domains are linked by a central stalk rotating inside the F(1) region and a stationary peripheral stalk (PubMed:37244256). During catalysis, ATP synthesis in the catalytic domain of F(1) is coupled via a rotary mechanism of the central stalk subunits to proton translocation (Probable). In vivo, can only synthesize ATP although its ATP hydrolase activity can be activated artificially in vitro (By similarity). Part of the complex F(0) domain (PubMed:37244256). Part of the complex F(0) domain and the peripheric stalk, which acts as a stator to hold the catalytic alpha(3)beta(3) subcomplex and subunit a/ATP6 static relative to the rotary elements (By similarity)","subcellular_location":"Mitochondrion; Mitochondrion inner membrane","url":"https://www.uniprot.org/uniprotkb/P48047/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ATP5PO","classification":"Not Classified","n_dependent_lines":427,"n_total_lines":1208,"dependency_fraction":0.353476821192053},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CPSF6","stoichiometry":0.2},{"gene":"HEATR3","stoichiometry":0.2},{"gene":"MYH9","stoichiometry":0.2},{"gene":"RER1","stoichiometry":0.2},{"gene":"RTN4","stoichiometry":0.2},{"gene":"SAR1B","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/ATP5PO","total_profiled":1310},"omim":[{"mim_id":"620359","title":"MITOCHONDRIAL COMPLEX V (ATP SYNTHASE) DEFICIENCY, NUCLEAR TYPE 7; MC5DN7","url":"https://www.omim.org/entry/620359"},{"mim_id":"617228","title":"COMBINED OXIDATIVE PHOSPHORYLATION DEFICIENCY 31; COXPD31","url":"https://www.omim.org/entry/617228"},{"mim_id":"604273","title":"MITOCHONDRIAL COMPLEX V (ATP SYNTHASE) DEFICIENCY, NUCLEAR TYPE 1; MC5DN1","url":"https://www.omim.org/entry/604273"},{"mim_id":"602241","title":"MITOCHONDRIAL INTERMEDIATE PEPTIDASE; MIPEP","url":"https://www.omim.org/entry/602241"},{"mim_id":"600828","title":"ATP SYNTHASE PERIPHERAL STALK, SUBUNIT OSCP; ATP5PO","url":"https://www.omim.org/entry/600828"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Mitochondria","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"tongue","ntpm":1494.1}],"url":"https://www.proteinatlas.org/search/ATP5PO"},"hgnc":{"alias_symbol":["OSCP","ATPO"],"prev_symbol":["ATP5O"]},"alphafold":{"accession":"P48047","domains":[{"cath_id":"1.10.520.20","chopping":"36-133","consensus_level":"high","plddt":88.3756,"start":36,"end":133},{"cath_id":"3.30.429","chopping":"137-211","consensus_level":"high","plddt":87.694,"start":137,"end":211}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P48047","model_url":"https://alphafold.ebi.ac.uk/files/AF-P48047-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P48047-F1-predicted_aligned_error_v6.png","plddt_mean":84.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ATP5PO","jax_strain_url":"https://www.jax.org/strain/search?query=ATP5PO"},"sequence":{"accession":"P48047","fasta_url":"https://rest.uniprot.org/uniprotkb/P48047.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P48047/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P48047"}},"corpus_meta":[{"pmid":"12519060","id":"PMC_12519060","title":"Competitive antagonism of AMPA receptors by ligands of different classes: crystal structure of ATPO bound to the GluR2 ligand-binding core, in comparison with DNQX.","date":"2003","source":"Journal of medicinal chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12519060","citation_count":84,"is_preprint":false},{"pmid":"9799528","id":"PMC_9799528","title":"Mitochondrial ATP synthesis in permeabilized cells: assessment of the ATP/O values in situ.","date":"1998","source":"Analytical biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/9799528","citation_count":62,"is_preprint":false},{"pmid":"7540110","id":"PMC_7540110","title":"ATPo but not cAMPi activates a chloride conductance in mouse ventricular myocytes.","date":"1995","source":"Cardiovascular research","url":"https://pubmed.ncbi.nlm.nih.gov/7540110","citation_count":34,"is_preprint":false},{"pmid":"35414767","id":"PMC_35414767","title":"Claudin-10 overexpression suppresses human clear cell renal cell carcinoma growth and metastasis by regulating ATP5O and causing mitochondrial dysfunction.","date":"2022","source":"International journal of biological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/35414767","citation_count":22,"is_preprint":false},{"pmid":"19274082","id":"PMC_19274082","title":"Genetic variation in ATP5O is associated with skeletal muscle ATP50 mRNA expression and glucose uptake in young twins.","date":"2009","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/19274082","citation_count":22,"is_preprint":false},{"pmid":"7490082","id":"PMC_7490082","title":"Cloning of the cDNA for the human ATP synthase OSCP subunit (ATP5O) by exon trapping and mapping to chromosome 21q22.1-q22.2.","date":"1995","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/7490082","citation_count":22,"is_preprint":false},{"pmid":"35621276","id":"PMC_35621276","title":"A homozygous splice variant in ATP5PO, disrupts mitochondrial complex V function and causes Leigh syndrome in two unrelated families.","date":"2022","source":"Journal of inherited metabolic disease","url":"https://pubmed.ncbi.nlm.nih.gov/35621276","citation_count":12,"is_preprint":false},{"pmid":"38645677","id":"PMC_38645677","title":"ATP5O Hypo-crotonylation Caused by HDAC2 Hyper-Phosphorylation Is a Primary Detrimental Factor for Downregulated Phospholipid Metabolism under Chronic Stress.","date":"2022","source":"Research (Washington, D.C.)","url":"https://pubmed.ncbi.nlm.nih.gov/38645677","citation_count":7,"is_preprint":false},{"pmid":"34885151","id":"PMC_34885151","title":"Proteomic Analysis Identifies NDUFS1 and ATP5O as Novel Markers for Survival Outcome in Prostate Cancer.","date":"2021","source":"Cancers","url":"https://pubmed.ncbi.nlm.nih.gov/34885151","citation_count":5,"is_preprint":false},{"pmid":"38128843","id":"PMC_38128843","title":"ATP5PO levels regulate enteric nervous system development in zebrafish, linking Hirschsprung disease to Down Syndrome.","date":"2023","source":"Biochimica et biophysica acta. Molecular basis of disease","url":"https://pubmed.ncbi.nlm.nih.gov/38128843","citation_count":4,"is_preprint":false},{"pmid":"9884081","id":"PMC_9884081","title":"Studies of the antagonist actions of (RS)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl] propionic acid (ATPO) on non-NMDA receptors in cultured rat neurones.","date":"1998","source":"British journal of pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/9884081","citation_count":3,"is_preprint":false},{"pmid":"34754791","id":"PMC_34754791","title":"Dynamic calculation of ATP/O ratios measured using Magnesium Green (MgGr)™.","date":"2021","source":"MethodsX","url":"https://pubmed.ncbi.nlm.nih.gov/34754791","citation_count":3,"is_preprint":false},{"pmid":"41233904","id":"PMC_41233904","title":"Cyclovirobuxine D ameliorates cardiomyocyte senescence in diabetic cardiomyopathy mice by enhancing mitochondrial function via sirtuin 3-ATP5O signal axis.","date":"2025","source":"Chinese medicine","url":"https://pubmed.ncbi.nlm.nih.gov/41233904","citation_count":1,"is_preprint":false},{"pmid":"39124785","id":"PMC_39124785","title":"Evaluation of Anti-Thyroperoxidase (A-TPO) and Anti-Thyroglobulin (A-Tg) Antibodies in Women with Previous Hashimoto's Thyroiditis during and after Pregnancy.","date":"2024","source":"Journal of clinical medicine","url":"https://pubmed.ncbi.nlm.nih.gov/39124785","citation_count":1,"is_preprint":false},{"pmid":"40913360","id":"PMC_40913360","title":"Mitochondrial Complex V Deficiency Caused by a Homozygous Splice Variant in ATP5PO.","date":"2025","source":"American journal of medical genetics. Part A","url":"https://pubmed.ncbi.nlm.nih.gov/40913360","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.12.09.627532","title":"Genetic basis of immunity in Indian cattle as revealed by comparative analysis of  <i>Bos</i>  genome","date":"2024-12-10","source":"bioRxiv","url":"https://doi.org/10.1101/2024.12.09.627532","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11827,"output_tokens":2040,"usd":0.033041,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9067,"output_tokens":2322,"usd":0.051693,"stage2_stop_reason":"end_turn"},"total_usd":0.084734,"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\": 1995,\n      \"finding\": \"ATP5PO (OSCP subunit) was cloned and identified as a key structural component of the stalk of the mitochondrial F1F0-ATP synthase, encoded on chromosome 21q22.1-q22.2, with >80% amino acid identity to bovine and murine OSCP subunits, and expressed in all human tissues, most strongly in muscle and heart.\",\n      \"method\": \"Exon trapping, cDNA cloning, sequence analysis, chromosomal mapping to YAC contigs\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct cloning and sequence characterization with chromosomal mapping, single lab, multiple methods but no functional reconstitution\",\n      \"pmids\": [\"7490082\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"A homozygous splice variant (c.87+3A>G) in ATP5PO causing skipping of exon 2 results in decreased ATP5PO protein, defective complex V assembly with markedly reduced peripheral stalk proteins, and reduced complex V hydrolytic activity in patient fibroblasts; expression of the exon-2-deleted human ATP5PO in yeast deleted for its homolog (yATP5) failed to rescue oxidative phosphorylation-dependent growth, demonstrating that exon 2 is required for protein function.\",\n      \"method\": \"cDNA splicing analysis, fibroblast biochemical assays (complex V assembly, hydrolytic activity), yeast complementation assay\",\n      \"journal\": \"Journal of inherited metabolic disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods (patient fibroblast biochemistry, yeast complementation), two independent families, functional mutagenesis-equivalent via splice variant\",\n      \"pmids\": [\"35621276\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CLDN10 overexpression upregulates acetylation and expression levels of ATP5PO (ATP5O), leading to mitochondrial dysfunction with increased ROS, increased NDUFS2/SDHB/Cleaved-Caspase 3/E-cadherin, and decreased mitochondrial membrane potential; knockdown of ATP5O in CLDN10-overexpressing cells reverses these effects, placing ATP5O downstream of CLDN10 in regulating mitochondrial function and suppressing ccRCC growth and metastasis.\",\n      \"method\": \"Gain-of-function (CLDN10 overexpression), loss-of-function (ATP5O knockdown), Western blotting, ROS measurement, mitochondrial membrane potential assay, orthotopic mouse model\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis via knockdown rescue in cell line and in vivo, single lab, multiple readouts\",\n      \"pmids\": [\"35414767\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Under chronic stress, ATP5O K51 crotonylation is decreased due to HDAC2 hyperphosphorylation at S424 (which enhances HDAC2 decrotonylation activity); this reduction in ATP5O crotonylation causes decreased gross ATP5O protein levels and downregulated phospholipid metabolism. Correcting HDAC2 hyperphosphorylation restored ATP5O levels and partially rescued phospholipid metabolism.\",\n      \"method\": \"Quantitative PTM-omics, phospho-omics, metabolomics, site-specific PTM analysis, genetic correction of HDAC2 phosphorylation in mouse model\",\n      \"journal\": \"Research (Washington, D.C.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multi-omic approach with site-specific PTM identification and in vivo rescue, single lab\",\n      \"pmids\": [\"38645677\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ATP5PO overexpression in zebrafish caused a significant reduction in enteric nervous system (ENS) cells; in vitro, overexpression paradoxically reduced ATP5PO protein levels, impaired neuronal differentiation, and reduced mitochondrial ATP production in a neuroblastoma cell line. Genetic epistasis was demonstrated between ATP5PO and ret (the principal Hirschsprung disease gene), establishing ATP5PO as a regulator of ENS development acting in the same pathway as RET.\",\n      \"method\": \"Zebrafish overexpression screen (ENS cell quantification), in vitro overexpression in neuroblastoma cells (ATP production assay, differentiation assay), genetic epistasis analysis with ret\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular basis of disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional overexpression in vivo and in vitro with epistasis, single lab, multiple orthogonal readouts\",\n      \"pmids\": [\"38128843\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SIRT3 deacetylates ATP5O at the K162 site in primary mouse cardiomyocytes; deacetylation mimics decreased mitochondrial damage while acetylation mimics promoted mitochondrial damage. CVB-D upregulates SIRT3 expression, which increases ATP5O acetylation (at K162) and enhances mitochondrial function. ATP5O knockout inhibited the protective effects of SIRT3 overexpression in DCM, establishing SIRT3 as a writer and K162 as a functional acetylation site on ATP5O.\",\n      \"method\": \"Co-immunoprecipitation, LC-MS/MS acetylation site mapping, acetylation/deacetylation mimic plasmid transfection, MST/SPR/ITC binding assays, AAV9-mediated SIRT3 overexpression and ATP5O knockout mouse models\",\n      \"journal\": \"Chinese medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — site-specific PTM identified by LC-MS/MS, functional validation with mimic mutants, Co-IP, binding assays, and in vivo AAV knockout/overexpression, multiple orthogonal methods in one study\",\n      \"pmids\": [\"41233904\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"A homozygous splice variant (c.87+3A>G) in ATP5PO in a patient caused approximately 35% of normal ATPase enzyme activity in fibroblasts (relative to citrate synthase), providing functional evidence that ATP5PO is required for complex V assembly and function.\",\n      \"method\": \"Fibroblast mitochondrial respiratory chain enzyme activity assay, whole-exome sequencing\",\n      \"journal\": \"American journal of medical genetics. Part A\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct enzymatic assay in patient-derived fibroblasts, single case, consistent with prior report (PMID 35621276)\",\n      \"pmids\": [\"40913360\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ATP5PO encodes the oligomycin sensitivity-conferring protein (OSCP), a structural component of the stalk linking the F1 and F0 domains of mitochondrial complex V (ATP synthase); loss-of-function splice variants impair complex V assembly and hydrolytic/synthetic activity causing Leigh syndrome, while post-translational modifications—specifically SIRT3-mediated deacetylation at K162 and HDAC2-regulated crotonylation at K51—modulate ATP5PO stability and mitochondrial function, and balanced ATP5PO expression is required for enteric nervous system development acting in a pathway with RET.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ATP5PO encodes the oligomycin sensitivity-conferring protein (OSCP), a structural component of the peripheral stalk that links the F1 and F0 domains of the mitochondrial F1F0-ATP synthase (complex V), expressed ubiquitously but most strongly in muscle and heart [#0]. Its function is required for proper complex V assembly and activity: a homozygous splice variant (c.87+3A>G) causing skipping of exon 2 reduces ATP5PO protein, destabilizes the peripheral stalk, and lowers complex V hydrolytic activity, and the exon-2-deleted protein fails to complement a yeast OSCP-null strain, establishing exon 2 as essential for function [#1, #6]. This loss of function underlies a mitochondrial disorder presenting as Leigh-like disease in affected patients [#1, #6]. ATP5PO activity is tuned by post-translational modification: SIRT3 binds and deacetylates ATP5PO at K162 in cardiomyocytes to limit mitochondrial damage [#5], and HDAC2-regulated crotonylation at K51 controls ATP5PO protein stability and downstream phospholipid metabolism under chronic stress [#3]. Balanced ATP5PO expression is also required for enteric nervous system development, where it acts genetically in the RET pathway, with both loss and overexpression compromising neuronal differentiation and mitochondrial ATP production [#4].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"Established the molecular identity of ATP5PO as the OSCP stalk subunit of mitochondrial ATP synthase, defining its place in complex V.\",\n      \"evidence\": \"Exon trapping, cDNA cloning, sequence analysis and chromosomal mapping of the human gene\",\n      \"pmids\": [\"7490082\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No functional reconstitution of the protein in the synthase\", \"Tissue expression described but not linked to phenotype\", \"No structural placement within the assembled complex shown directly\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Demonstrated that ATP5PO loss of function impairs complex V assembly and activity, linking the gene to human mitochondrial disease.\",\n      \"evidence\": \"Patient fibroblast biochemistry (assembly, hydrolytic activity) and yeast complementation of an OSCP-null strain across two families\",\n      \"pmids\": [\"35621276\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which stalk loss destabilizes the whole complex not resolved at structural level\", \"Tissue-specific disease manifestation not fully explained\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Placed ATP5PO downstream of CLDN10 in a pathway regulating mitochondrial function and tumor suppression, implicating its acetylation state.\",\n      \"evidence\": \"CLDN10 gain-of-function with ATP5O knockdown rescue, mitochondrial readouts, and orthotopic mouse model in ccRCC\",\n      \"pmids\": [\"35414767\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Acetylation sites and writers/erasers not identified here\", \"Direct CLDN10–ATP5PO molecular relationship unestablished\", \"Single lab, single cancer context\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified K51 crotonylation as a stress-responsive modification controlling ATP5PO stability and phospholipid metabolism via HDAC2.\",\n      \"evidence\": \"Quantitative PTM/phospho/metabolomics with site-specific PTM mapping and genetic correction of HDAC2 phosphorylation in mouse\",\n      \"pmids\": [\"38645677\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct enzymatic decrotonylation of ATP5PO by HDAC2 not reconstituted\", \"Mechanism linking crotonylation to phospholipid metabolism unclear\", \"Single lab\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Revealed a non-canonical role for ATP5PO in enteric nervous system development within the RET pathway, where dosage is critical.\",\n      \"evidence\": \"Zebrafish overexpression ENS quantification, neuroblastoma differentiation/ATP assays, and genetic epistasis with ret\",\n      \"pmids\": [\"38128843\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis of the paradoxical protein decrease upon overexpression unknown\", \"Direct biochemical link between ATP5PO and RET signaling not defined\", \"Human ENS relevance inferred from model systems\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Established SIRT3 as a writer and K162 as a functional acetylation site governing ATP5PO-dependent mitochondrial protection in the heart.\",\n      \"evidence\": \"Co-IP, LC-MS/MS site mapping, acetylation mimic mutants, MST/SPR/ITC binding, and AAV9 SIRT3 overexpression / ATP5O knockout mouse models in DCM\",\n      \"pmids\": [\"41233904\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How K162 acetylation alters complex V function structurally not resolved\", \"Interplay between K162 acetylation and K51 crotonylation not examined\", \"Cardiac-focused; generality to other tissues untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the multiple PTMs (K162 acetylation, K51 crotonylation) integrate to control complex V assembly and the non-OSCP role of ATP5PO in RET-dependent ENS development remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified structural model of how stalk modifications affect synthase activity\", \"Mechanism linking mitochondrial ATP5PO to RET signaling unknown\", \"Cross-talk among PTM sites uncharacterized\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [0, 1, 5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [1, 6]}\n    ],\n    \"complexes\": [\"mitochondrial ATP synthase (complex V)\"],\n    \"partners\": [\"SIRT3\", \"HDAC2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}