{"gene":"ACAA2","run_date":"2026-06-09T22:02:37","timeline":{"discoveries":[{"year":2021,"finding":"ACAA2, a mitochondrial thiolase enzyme, was identified as a novel interacting protein of thyroid hormone receptor β1 (TRβ1) via GST pull-down on cardiac tissue followed by LC-MS/MS. ACAA2 was confirmed to localize to the nucleus and acts as a thyroid hormone (TH)-dependent coactivator for TRβ1 in a luciferase reporter assay. ACAA2 can bind TR recognition sequences but does not alter TRβ1 DNA-binding ability.","method":"GST pull-down on cardiac tissue, LC-MS/MS identification, luciferase reporter assay, nuclear localization confirmation","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal pull-down with MS identification and functional reporter assay, single lab, two orthogonal methods","pmids":["34474245"],"is_preprint":false},{"year":2023,"finding":"CAND1 mitigates NAFLD by preventing ubiquitin-mediated degradation of ACAA2. Mechanistically, CAND1 deficiency enhances assembly of a Cullin1/FBXO42/ACAA2 E3 ubiquitin ligase complex, promoting ubiquitinated degradation of ACAA2. ACAA2 overexpression abolishes the exacerbating effects of CAND1 deficiency on NAFLD.","method":"Co-immunoprecipitation of Cullin1/FBXO42/ACAA2 complex, hepatocyte-specific CAND1 knockout and knockin mouse models, ACAA2 overexpression rescue experiment","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP defining the E3 ligase complex, genetic KO/KI mouse models, and rescue experiment with ACAA2 overexpression in a single rigorous study","pmids":["37528093"],"is_preprint":false},{"year":2022,"finding":"Egr1 transcriptionally upregulates Acaa2, a key fatty acid β-oxidation (FAO) gene, in hepatocytes. Liver-specific Egr1 knockout inhibited mitochondrial respiratory function and FAO activity, while Egr1 overexpression promoted these functions. Knockdown of Acaa2 abolished the protective effect of Egr1 in APAP-induced liver injury, establishing Acaa2 as the downstream mediator of Egr1-dependent FAO.","method":"Chromatin immunoprecipitation-sequencing (ChIP-seq), RNA-seq, liver-specific Egr1 knockout and adenoviral Egr1 overexpression in vivo, Acaa2 knockdown epistasis, Seahorse XF analysis, targeted fatty acid analysis","journal":"International journal of biological sciences","confidence":"High","confidence_rationale":"Tier 2 / Strong — ChIP-seq for transcriptional regulation, genetic KO and OE mouse models, epistasis knockdown of Acaa2, and functional metabolic readouts across multiple orthogonal methods","pmids":["35813467"],"is_preprint":false},{"year":2018,"finding":"ACAA2 is a direct target of miR-152 in mammary epithelial cells (MECs), validated by dual-luciferase reporter assay. Overexpression of ACAA2 inhibits triglyceride production and cell proliferation while inducing apoptosis in MECs; shRNA-mediated knockdown of ACAA2 reverses these effects.","method":"Dual-luciferase reporter assay, miR-152 transfection, ACAA2 overexpression and shRNA knockdown in MECs, qPCR, western blot","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — luciferase validation of miRNA targeting and functional OE/KD experiments, single lab, multiple cellular readouts","pmids":["29323178"],"is_preprint":false},{"year":2024,"finding":"p46Shc (mitochondrial Shc isoform) represses ACAA2 thiolase activity in vitro. In vivo induction of p46Shc in mice reduced ACAA2-dependent mitochondrial β-oxidation, suppressed β-hydroxybutyrate production, increased reactive oxygen species, and caused mitochondrial structural damage. Expression of dominant-negative p46Shc reduced ACAA2 thiolase activity, improved β-oxidation, and reduced lipid peroxidation.","method":"In vitro thiolase activity assay, p46Shc-inducible transgenic mouse model, mitochondrial oxygen consumption by Oroboros, dominant-negative p46Shc expression, electron microscopy","journal":"The American journal of pathology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro enzymatic assay combined with in vivo genetic model, single lab, multiple orthogonal methods","pmids":["39733992"],"is_preprint":false},{"year":2024,"finding":"OGT (O-GlcNAc transferase) induces O-GlcNAc glycosylation of ACAA2 and regulates the nucleocytoplasmic (karyoplasmic) distribution of ACAA2 in ovarian cancer cells. ACAA2 overexpression promoted ovarian cancer growth, proliferation, migration, and invasion; ACAA2 knockdown inhibited these processes and reduced subcutaneous tumor formation in nude mice. RNA-seq revealed ACAA2 regulates DIXDC1 expression, likely through the WNT/β-Catenin signaling pathway.","method":"OGT overexpression and immunofluorescence/fractionation for karyoplasmic distribution, gain/loss of function (OE and KD), nude mouse xenograft, RNA-seq","journal":"Molecular carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — OGT-mediated glycosylation and localization change established by OE experiments, functional KD/OE with xenograft, RNA-seq pathway inference, single lab","pmids":["38656551"],"is_preprint":false},{"year":2025,"finding":"In phenylephrine (PE)-induced cardiomyocyte hypertrophy, ACAA2 undergoes lactylation (confirmed by immunoprecipitation) which is reduced upon PE stimulation. Knockdown of ACAA2 exacerbated PE-induced hypertrophy in NRVMs, accompanied by accumulation of free fatty acids, decreased lactate and ATP, and impaired mitochondrial oxidative respiration (measured by Seahorse). Sodium lactate treatment partially rescued these effects.","method":"Immunoprecipitation for lactylation detection, RNA interference knockdown of ACAA2, Seahorse extracellular flux analysis, ELISA-based substrate utilization, RNA-seq of TAC mouse cardiac tissue","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — lactylation confirmed by IP, functional KD with multiple metabolic and mitochondrial readouts, rescue experiment, single lab","pmids":["40858063"],"is_preprint":false},{"year":2025,"finding":"ACAA2 knockdown in cardiomyocytes led to accumulation of lipid droplets and exacerbation of oxidative stress, while ACAA2 overexpression reversed these effects. The transcription factor FOXO4 was found to regulate ACAA2 expression: FOXO4 knockdown partially restored ACAA2 expression and reduced oxidative stress in cardiomyocytes in a renal insufficiency model.","method":"ACAA2 knockdown and overexpression in cardiomyocytes, FOXO4 knockdown, Oil Red O staining for lipid accumulation, oxidative stress assays","journal":"Biomolecules","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — KD/OE with defined cellular phenotype and epistasis between FOXO4 and ACAA2, single lab, multiple readouts","pmids":["40149900"],"is_preprint":false},{"year":2022,"finding":"RPL34-AS1 acts as a competing endogenous RNA (ceRNA) sponging miR-575 to relieve repression of its target ACAA2 in esophageal squamous cell carcinoma cells. This was validated by luciferase reporter assay, RNA immunoprecipitation (RIP), and western blot, establishing a RPL34-AS1/miR-575/ACAA2 regulatory axis suppressing tumor progression.","method":"Luciferase reporter assay, RNA immunoprecipitation (RIP), FISH, western blot, in vitro and in vivo functional assays","journal":"BMC cancer","confidence":"Low","confidence_rationale":"Tier 3 / Weak — miRNA target validation by luciferase and RIP is mechanistically informative for ACAA2 regulation, but the primary subject is the lncRNA; single lab, single study","pmids":["36162992"],"is_preprint":false}],"current_model":"ACAA2 is a mitochondrial thiolase that catalyzes the final step of fatty acid β-oxidation (FAO); it is subject to ubiquitin-mediated degradation via the Cullin1/FBXO42 E3 ligase complex (counteracted by CAND1), transcriptional upregulation by Egr1, repression by p46Shc (which inhibits its thiolase activity), O-GlcNAc glycosylation by OGT (affecting its nucleocytoplasmic distribution), and lactylation that modulates its activity during cardiac stress; additionally, ACAA2 localizes to the nucleus where it functions as a ligand-dependent coactivator for thyroid hormone receptor TRβ1, linking metabolic status to transcriptional regulation."},"narrative":{"mechanistic_narrative":"ACAA2 is a mitochondrial thiolase that catalyzes the final step of fatty acid β-oxidation (FAO), and its abundance and activity serve as a regulatory node coupling lipid catabolism to cellular stress responses [PMID:35813467, PMID:39733992]. In hepatocytes, Egr1 transcriptionally upregulates Acaa2 to drive mitochondrial respiration and FAO, and Acaa2 is the downstream effector through which Egr1 protects against acetaminophen-induced liver injury [PMID:35813467]. ACAA2 protein stability is controlled by the ubiquitin–proteasome system: assembly of a Cullin1/FBXO42 E3 ligase complex promotes its ubiquitin-mediated degradation, which is restrained by CAND1, and preserving ACAA2 limits non-alcoholic fatty liver disease [PMID:37528093]. ACAA2 thiolase activity is further tuned by post-translational and protein–protein inputs — the mitochondrial Shc isoform p46Shc represses its activity, reducing β-oxidation and β-hydroxybutyrate output while raising reactive oxygen species, and lactylation modulates its activity during cardiac hypertrophic stress [PMID:39733992, PMID:40858063]. Consistent with a protective metabolic role, loss of ACAA2 in cardiomyocytes causes lipid droplet accumulation, impaired oxidative respiration, and heightened oxidative stress [PMID:40858063, PMID:40149900]. Beyond its mitochondrial role, ACAA2 also localizes to the nucleus, where it binds thyroid hormone receptor β1 (TRβ1) and acts as a ligand-dependent transcriptional coactivator without altering TR DNA binding, and OGT-mediated O-GlcNAc glycosylation governs its nucleocytoplasmic distribution [PMID:34474245, PMID:38656551]. ACAA2 expression is additionally set by miRNA regulatory axes, and gain or loss of ACAA2 modulates proliferation, apoptosis, and tumor behavior in epithelial and cancer cells [PMID:29323178, PMID:38656551].","teleology":[{"year":2018,"claim":"Established that ACAA2 levels are set by miRNA control and that its level dictates lipid synthesis and cell fate, framing it as more than a constitutive housekeeping enzyme.","evidence":"Dual-luciferase miR-152 target validation plus ACAA2 overexpression/knockdown in mammary epithelial cells","pmids":["29323178"],"confidence":"Medium","gaps":["Does not address thiolase mechanism or whether phenotypes depend on FAO flux","Single cell type, no in vivo confirmation"]},{"year":2021,"claim":"Revealed a non-canonical nuclear function for this mitochondrial enzyme by identifying it as a ligand-dependent coactivator of TRβ1, linking metabolic enzyme identity to transcriptional regulation.","evidence":"GST pull-down on cardiac tissue with LC-MS/MS, luciferase reporter assay, nuclear localization confirmation","pmids":["34474245"],"confidence":"Medium","gaps":["Mechanism of nuclear import not defined","Direct target genes of the ACAA2/TRβ1 complex not identified","Whether thiolase catalytic activity is required for coactivation unknown"]},{"year":2022,"claim":"Defined an upstream transcriptional driver, showing Egr1 directly upregulates Acaa2 to sustain FAO and that ACAA2 is the obligate downstream mediator of Egr1 hepatoprotection.","evidence":"ChIP-seq, RNA-seq, liver-specific Egr1 KO and adenoviral OE, Acaa2 knockdown epistasis, Seahorse and fatty acid analysis","pmids":["35813467"],"confidence":"High","gaps":["Does not address post-transcriptional control of ACAA2","Cardiac/other tissue generalization untested in this study"]},{"year":2022,"claim":"Added a ceRNA layer to ACAA2 regulation, showing a lncRNA/miRNA axis relieves repression of ACAA2 to suppress tumor progression.","evidence":"Luciferase, RNA immunoprecipitation, FISH, western blot, and functional assays in esophageal squamous cell carcinoma (lncRNA-centric study)","pmids":["36162992"],"confidence":"Low","gaps":["Primary subject is the lncRNA, not ACAA2; direct enzymatic relevance not tested","Single lab, single study","ACAA2-specific causality not isolated from the broader axis"]},{"year":2023,"claim":"Identified the degradation machinery controlling ACAA2 stability, defining a Cullin1/FBXO42 E3 ligase complex opposed by CAND1 and linking ACAA2 preservation to protection against NAFLD.","evidence":"Reciprocal Co-IP of the Cullin1/FBXO42/ACAA2 complex, hepatocyte-specific CAND1 KO/KI mice, ACAA2 overexpression rescue","pmids":["37528093"],"confidence":"High","gaps":["Ubiquitination site(s) on ACAA2 not mapped","Substrate-recognition determinant of FBXO42 for ACAA2 unknown"]},{"year":2024,"claim":"Showed that ACAA2 thiolase activity is directly repressible, with p46Shc inhibiting activity to reduce β-oxidation and ketogenesis while promoting ROS and mitochondrial damage.","evidence":"In vitro thiolase activity assay, p46Shc-inducible and dominant-negative transgenic mice, Oroboros respirometry, electron microscopy","pmids":["39733992"],"confidence":"Medium","gaps":["Whether p46Shc physically binds ACAA2 directly not established","Molecular basis of activity inhibition unresolved"]},{"year":2024,"claim":"Demonstrated that O-GlcNAcylation by OGT controls ACAA2 nucleocytoplasmic distribution and that ACAA2 abundance drives ovarian cancer growth, connecting a post-translational modification to its dual localization and oncogenic output.","evidence":"OGT overexpression with immunofluorescence/fractionation, ACAA2 gain/loss of function, nude mouse xenograft, RNA-seq implicating DIXDC1/WNT-β-Catenin","pmids":["38656551"],"confidence":"Medium","gaps":["O-GlcNAc site(s) not mapped","Direct link between glycosylation, localization, and the WNT axis remains correlative","Whether nuclear ACAA2 here is the TRβ1 coactivator pool untested"]},{"year":2025,"claim":"Extended modification-based control to the heart, showing ACAA2 lactylation changes during hypertrophic stress and that ACAA2 loss worsens lipid accumulation, oxidative stress, and respiratory impairment, with transcriptional input from FOXO4.","evidence":"Immunoprecipitation for lactylation, RNAi knockdown and overexpression in cardiomyocytes, Seahorse, ELISA substrate assays, FOXO4 knockdown epistasis, Oil Red O staining","pmids":["40858063","40149900"],"confidence":"Medium","gaps":["Lactylation site(s) and effect on catalytic mechanism not mapped","Causal contribution of lactylation vs. abundance to phenotype not separated","FOXO4 regulation of ACAA2 not shown to be direct"]},{"year":null,"claim":"How ACAA2's mitochondrial thiolase function and its nuclear TRβ1 coactivator role are coordinated — and whether the array of modifications (ubiquitination, O-GlcNAc, lactylation) constitute a unified switch governing localization and activity — remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model integrating catalytic and coactivator functions","Modification crosstalk not dissected","Tissue-specific hierarchy of regulators unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[4,6]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,4]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[0,4,6]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,5]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[2,4,6]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,2]}],"complexes":["Cullin1/FBXO42 E3 ubiquitin ligase complex (as substrate)"],"partners":["THRB","CUL1","FBXO42","CAND1","OGT","SHC1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P42765","full_name":"3-ketoacyl-CoA thiolase, mitochondrial","aliases":["Acetyl-CoA acetyltransferase","Acetyl-CoA acyltransferase","Acyl-CoA hydrolase, mitochondrial","Beta-ketothiolase","Mitochondrial 3-oxoacyl-CoA thiolase","T1"],"length_aa":397,"mass_kda":41.9,"function":"In the production of energy from fats, this is one of the enzymes that catalyzes the last step of the mitochondrial beta-oxidation pathway, an aerobic process breaking down fatty acids into acetyl-CoA (Probable). Using free coenzyme A/CoA, catalyzes the thiolytic cleavage of medium- to long-chain unbranched 3-oxoacyl-CoAs into acetyl-CoA and a fatty acyl-CoA shortened by two carbon atoms (Probable). Also catalyzes the condensation of two acetyl-CoA molecules into acetoacetyl-CoA and could be involved in the production of ketone bodies (Probable). Also displays hydrolase activity on various fatty acyl-CoAs (PubMed:25478839). Thereby, could be responsible for the production of acetate in a side reaction to beta-oxidation (Probable). Abolishes BNIP3-mediated apoptosis and mitochondrial damage (PubMed:18371312)","subcellular_location":"Mitochondrion","url":"https://www.uniprot.org/uniprotkb/P42765/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ACAA2","classification":"Not Classified","n_dependent_lines":8,"n_total_lines":1208,"dependency_fraction":0.006622516556291391},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CLIP1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/ACAA2","total_profiled":1310},"omim":[{"mim_id":"604770","title":"ACETYL-CoA ACYLTRANSFERASE 2; ACAA2","url":"https://www.omim.org/entry/604770"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Mitochondria","reliability":"Supported"},{"location":"Basal body","reliability":"Additional"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"liver","ntpm":945.6}],"url":"https://www.proteinatlas.org/search/ACAA2"},"hgnc":{"alias_symbol":["DSAEC"],"prev_symbol":[]},"alphafold":{"accession":"P42765","domains":[{"cath_id":"3.40.47.10","chopping":"8-126_253-396","consensus_level":"medium","plddt":98.2451,"start":8,"end":396}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P42765","model_url":"https://alphafold.ebi.ac.uk/files/AF-P42765-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P42765-F1-predicted_aligned_error_v6.png","plddt_mean":97.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ACAA2","jax_strain_url":"https://www.jax.org/strain/search?query=ACAA2"},"sequence":{"accession":"P42765","fasta_url":"https://rest.uniprot.org/uniprotkb/P42765.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P42765/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P42765"}},"corpus_meta":[{"pmid":"29323178","id":"PMC_29323178","title":"MiR-152 Regulates Apoptosis and Triglyceride Production in MECs via Targeting ACAA2 and HSD17B12 Genes.","date":"2018","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/29323178","citation_count":35,"is_preprint":false},{"pmid":"37528093","id":"PMC_37528093","title":"Cullin-associated and neddylation-dissociated protein 1 (CAND1) alleviates NAFLD by reducing ubiquitinated degradation of ACAA2.","date":"2023","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/37528093","citation_count":24,"is_preprint":false},{"pmid":"35813467","id":"PMC_35813467","title":"Egr1 confers protection against acetaminophen‑induced hepatotoxicity via transcriptional upregulating of Acaa2.","date":"2022","source":"International journal of biological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/35813467","citation_count":24,"is_preprint":false},{"pmid":"32008581","id":"PMC_32008581","title":"ACAA2 and FASN polymorphisms affect the fatty acid profile of Chios sheep milk.","date":"2020","source":"The Journal of dairy research","url":"https://pubmed.ncbi.nlm.nih.gov/32008581","citation_count":12,"is_preprint":false},{"pmid":"22612976","id":"PMC_22612976","title":"A single nucleotide polymorphism in the acetyl-coenzyme A acyltransferase 2 (ACAA2) gene is associated with milk yield in Chios sheep.","date":"2012","source":"Journal of dairy science","url":"https://pubmed.ncbi.nlm.nih.gov/22612976","citation_count":12,"is_preprint":false},{"pmid":"36162992","id":"PMC_36162992","title":"LncRNA RPL34-AS1 suppresses the proliferation, migration and invasion of esophageal squamous cell carcinoma via targeting miR-575/ACAA2 axis.","date":"2022","source":"BMC cancer","url":"https://pubmed.ncbi.nlm.nih.gov/36162992","citation_count":10,"is_preprint":false},{"pmid":"38656551","id":"PMC_38656551","title":"The oncogenic role and regulatory mechanism of ACAA2 in human ovarian cancer.","date":"2024","source":"Molecular carcinogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/38656551","citation_count":7,"is_preprint":false},{"pmid":"34474245","id":"PMC_34474245","title":"ACAA2 is a ligand-dependent coactivator for thyroid hormone receptor β1.","date":"2021","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/34474245","citation_count":7,"is_preprint":false},{"pmid":"37798372","id":"PMC_37798372","title":"ACAA2 is a novel molecular indicator for cancers with neuroendocrine phenotype.","date":"2023","source":"British journal of cancer","url":"https://pubmed.ncbi.nlm.nih.gov/37798372","citation_count":6,"is_preprint":false},{"pmid":"40149900","id":"PMC_40149900","title":"ACAA2 Protects Against Cardiac Dysfunction and Lipid Peroxidation in Renal Insufficiency with the Treatment of S-Nitroso-L-Cysteine.","date":"2025","source":"Biomolecules","url":"https://pubmed.ncbi.nlm.nih.gov/40149900","citation_count":4,"is_preprint":false},{"pmid":"39733992","id":"PMC_39733992","title":"P46Shc Inhibits Mitochondrial ACAA2 Thiolase, Exacerbating Mitochondrial Injury and Inflammation in Aging Livers.","date":"2024","source":"The American journal of pathology","url":"https://pubmed.ncbi.nlm.nih.gov/39733992","citation_count":2,"is_preprint":false},{"pmid":"40858063","id":"PMC_40858063","title":"ACAA2 lactylation and expression mediate mitochondrial dysfunction in phenylephrine-induced cardiomyocyte hypertrophy.","date":"2025","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/40858063","citation_count":1,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.05.29.656916","title":"Reverse remodelling of the mitochondria and cytoskeleton after respiratory heart rate variability pacing of the failing sheep heart","date":"2025-05-30","source":"bioRxiv","url":"https://doi.org/10.1101/2025.05.29.656916","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9286,"output_tokens":2731,"usd":0.034411,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10045,"output_tokens":3414,"usd":0.067788,"stage2_stop_reason":"end_turn"},"total_usd":0.102199,"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\": 2021,\n      \"finding\": \"ACAA2, a mitochondrial thiolase enzyme, was identified as a novel interacting protein of thyroid hormone receptor β1 (TRβ1) via GST pull-down on cardiac tissue followed by LC-MS/MS. ACAA2 was confirmed to localize to the nucleus and acts as a thyroid hormone (TH)-dependent coactivator for TRβ1 in a luciferase reporter assay. ACAA2 can bind TR recognition sequences but does not alter TRβ1 DNA-binding ability.\",\n      \"method\": \"GST pull-down on cardiac tissue, LC-MS/MS identification, luciferase reporter assay, nuclear localization confirmation\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal pull-down with MS identification and functional reporter assay, single lab, two orthogonal methods\",\n      \"pmids\": [\"34474245\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CAND1 mitigates NAFLD by preventing ubiquitin-mediated degradation of ACAA2. Mechanistically, CAND1 deficiency enhances assembly of a Cullin1/FBXO42/ACAA2 E3 ubiquitin ligase complex, promoting ubiquitinated degradation of ACAA2. ACAA2 overexpression abolishes the exacerbating effects of CAND1 deficiency on NAFLD.\",\n      \"method\": \"Co-immunoprecipitation of Cullin1/FBXO42/ACAA2 complex, hepatocyte-specific CAND1 knockout and knockin mouse models, ACAA2 overexpression rescue experiment\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP defining the E3 ligase complex, genetic KO/KI mouse models, and rescue experiment with ACAA2 overexpression in a single rigorous study\",\n      \"pmids\": [\"37528093\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Egr1 transcriptionally upregulates Acaa2, a key fatty acid β-oxidation (FAO) gene, in hepatocytes. Liver-specific Egr1 knockout inhibited mitochondrial respiratory function and FAO activity, while Egr1 overexpression promoted these functions. Knockdown of Acaa2 abolished the protective effect of Egr1 in APAP-induced liver injury, establishing Acaa2 as the downstream mediator of Egr1-dependent FAO.\",\n      \"method\": \"Chromatin immunoprecipitation-sequencing (ChIP-seq), RNA-seq, liver-specific Egr1 knockout and adenoviral Egr1 overexpression in vivo, Acaa2 knockdown epistasis, Seahorse XF analysis, targeted fatty acid analysis\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — ChIP-seq for transcriptional regulation, genetic KO and OE mouse models, epistasis knockdown of Acaa2, and functional metabolic readouts across multiple orthogonal methods\",\n      \"pmids\": [\"35813467\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"ACAA2 is a direct target of miR-152 in mammary epithelial cells (MECs), validated by dual-luciferase reporter assay. Overexpression of ACAA2 inhibits triglyceride production and cell proliferation while inducing apoptosis in MECs; shRNA-mediated knockdown of ACAA2 reverses these effects.\",\n      \"method\": \"Dual-luciferase reporter assay, miR-152 transfection, ACAA2 overexpression and shRNA knockdown in MECs, qPCR, western blot\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — luciferase validation of miRNA targeting and functional OE/KD experiments, single lab, multiple cellular readouts\",\n      \"pmids\": [\"29323178\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"p46Shc (mitochondrial Shc isoform) represses ACAA2 thiolase activity in vitro. In vivo induction of p46Shc in mice reduced ACAA2-dependent mitochondrial β-oxidation, suppressed β-hydroxybutyrate production, increased reactive oxygen species, and caused mitochondrial structural damage. Expression of dominant-negative p46Shc reduced ACAA2 thiolase activity, improved β-oxidation, and reduced lipid peroxidation.\",\n      \"method\": \"In vitro thiolase activity assay, p46Shc-inducible transgenic mouse model, mitochondrial oxygen consumption by Oroboros, dominant-negative p46Shc expression, electron microscopy\",\n      \"journal\": \"The American journal of pathology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro enzymatic assay combined with in vivo genetic model, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"39733992\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"OGT (O-GlcNAc transferase) induces O-GlcNAc glycosylation of ACAA2 and regulates the nucleocytoplasmic (karyoplasmic) distribution of ACAA2 in ovarian cancer cells. ACAA2 overexpression promoted ovarian cancer growth, proliferation, migration, and invasion; ACAA2 knockdown inhibited these processes and reduced subcutaneous tumor formation in nude mice. RNA-seq revealed ACAA2 regulates DIXDC1 expression, likely through the WNT/β-Catenin signaling pathway.\",\n      \"method\": \"OGT overexpression and immunofluorescence/fractionation for karyoplasmic distribution, gain/loss of function (OE and KD), nude mouse xenograft, RNA-seq\",\n      \"journal\": \"Molecular carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — OGT-mediated glycosylation and localization change established by OE experiments, functional KD/OE with xenograft, RNA-seq pathway inference, single lab\",\n      \"pmids\": [\"38656551\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In phenylephrine (PE)-induced cardiomyocyte hypertrophy, ACAA2 undergoes lactylation (confirmed by immunoprecipitation) which is reduced upon PE stimulation. Knockdown of ACAA2 exacerbated PE-induced hypertrophy in NRVMs, accompanied by accumulation of free fatty acids, decreased lactate and ATP, and impaired mitochondrial oxidative respiration (measured by Seahorse). Sodium lactate treatment partially rescued these effects.\",\n      \"method\": \"Immunoprecipitation for lactylation detection, RNA interference knockdown of ACAA2, Seahorse extracellular flux analysis, ELISA-based substrate utilization, RNA-seq of TAC mouse cardiac tissue\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — lactylation confirmed by IP, functional KD with multiple metabolic and mitochondrial readouts, rescue experiment, single lab\",\n      \"pmids\": [\"40858063\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ACAA2 knockdown in cardiomyocytes led to accumulation of lipid droplets and exacerbation of oxidative stress, while ACAA2 overexpression reversed these effects. The transcription factor FOXO4 was found to regulate ACAA2 expression: FOXO4 knockdown partially restored ACAA2 expression and reduced oxidative stress in cardiomyocytes in a renal insufficiency model.\",\n      \"method\": \"ACAA2 knockdown and overexpression in cardiomyocytes, FOXO4 knockdown, Oil Red O staining for lipid accumulation, oxidative stress assays\",\n      \"journal\": \"Biomolecules\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — KD/OE with defined cellular phenotype and epistasis between FOXO4 and ACAA2, single lab, multiple readouts\",\n      \"pmids\": [\"40149900\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RPL34-AS1 acts as a competing endogenous RNA (ceRNA) sponging miR-575 to relieve repression of its target ACAA2 in esophageal squamous cell carcinoma cells. This was validated by luciferase reporter assay, RNA immunoprecipitation (RIP), and western blot, establishing a RPL34-AS1/miR-575/ACAA2 regulatory axis suppressing tumor progression.\",\n      \"method\": \"Luciferase reporter assay, RNA immunoprecipitation (RIP), FISH, western blot, in vitro and in vivo functional assays\",\n      \"journal\": \"BMC cancer\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — miRNA target validation by luciferase and RIP is mechanistically informative for ACAA2 regulation, but the primary subject is the lncRNA; single lab, single study\",\n      \"pmids\": [\"36162992\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ACAA2 is a mitochondrial thiolase that catalyzes the final step of fatty acid β-oxidation (FAO); it is subject to ubiquitin-mediated degradation via the Cullin1/FBXO42 E3 ligase complex (counteracted by CAND1), transcriptional upregulation by Egr1, repression by p46Shc (which inhibits its thiolase activity), O-GlcNAc glycosylation by OGT (affecting its nucleocytoplasmic distribution), and lactylation that modulates its activity during cardiac stress; additionally, ACAA2 localizes to the nucleus where it functions as a ligand-dependent coactivator for thyroid hormone receptor TRβ1, linking metabolic status to transcriptional regulation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ACAA2 is a mitochondrial thiolase that catalyzes the final step of fatty acid β-oxidation (FAO), and its abundance and activity serve as a regulatory node coupling lipid catabolism to cellular stress responses [#2, #4]. In hepatocytes, Egr1 transcriptionally upregulates Acaa2 to drive mitochondrial respiration and FAO, and Acaa2 is the downstream effector through which Egr1 protects against acetaminophen-induced liver injury [#2]. ACAA2 protein stability is controlled by the ubiquitin–proteasome system: assembly of a Cullin1/FBXO42 E3 ligase complex promotes its ubiquitin-mediated degradation, which is restrained by CAND1, and preserving ACAA2 limits non-alcoholic fatty liver disease [#1]. ACAA2 thiolase activity is further tuned by post-translational and protein–protein inputs — the mitochondrial Shc isoform p46Shc represses its activity, reducing β-oxidation and β-hydroxybutyrate output while raising reactive oxygen species, and lactylation modulates its activity during cardiac hypertrophic stress [#4, #6]. Consistent with a protective metabolic role, loss of ACAA2 in cardiomyocytes causes lipid droplet accumulation, impaired oxidative respiration, and heightened oxidative stress [#6, #7]. Beyond its mitochondrial role, ACAA2 also localizes to the nucleus, where it binds thyroid hormone receptor β1 (TRβ1) and acts as a ligand-dependent transcriptional coactivator without altering TR DNA binding, and OGT-mediated O-GlcNAc glycosylation governs its nucleocytoplasmic distribution [#0, #5]. ACAA2 expression is additionally set by miRNA regulatory axes, and gain or loss of ACAA2 modulates proliferation, apoptosis, and tumor behavior in epithelial and cancer cells [#3, #5].\",\n  \"teleology\": [\n    {\n      \"year\": 2018,\n      \"claim\": \"Established that ACAA2 levels are set by miRNA control and that its level dictates lipid synthesis and cell fate, framing it as more than a constitutive housekeeping enzyme.\",\n      \"evidence\": \"Dual-luciferase miR-152 target validation plus ACAA2 overexpression/knockdown in mammary epithelial cells\",\n      \"pmids\": [\"29323178\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not address thiolase mechanism or whether phenotypes depend on FAO flux\", \"Single cell type, no in vivo confirmation\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Revealed a non-canonical nuclear function for this mitochondrial enzyme by identifying it as a ligand-dependent coactivator of TRβ1, linking metabolic enzyme identity to transcriptional regulation.\",\n      \"evidence\": \"GST pull-down on cardiac tissue with LC-MS/MS, luciferase reporter assay, nuclear localization confirmation\",\n      \"pmids\": [\"34474245\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of nuclear import not defined\", \"Direct target genes of the ACAA2/TRβ1 complex not identified\", \"Whether thiolase catalytic activity is required for coactivation unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined an upstream transcriptional driver, showing Egr1 directly upregulates Acaa2 to sustain FAO and that ACAA2 is the obligate downstream mediator of Egr1 hepatoprotection.\",\n      \"evidence\": \"ChIP-seq, RNA-seq, liver-specific Egr1 KO and adenoviral OE, Acaa2 knockdown epistasis, Seahorse and fatty acid analysis\",\n      \"pmids\": [\"35813467\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not address post-transcriptional control of ACAA2\", \"Cardiac/other tissue generalization untested in this study\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Added a ceRNA layer to ACAA2 regulation, showing a lncRNA/miRNA axis relieves repression of ACAA2 to suppress tumor progression.\",\n      \"evidence\": \"Luciferase, RNA immunoprecipitation, FISH, western blot, and functional assays in esophageal squamous cell carcinoma (lncRNA-centric study)\",\n      \"pmids\": [\"36162992\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Primary subject is the lncRNA, not ACAA2; direct enzymatic relevance not tested\", \"Single lab, single study\", \"ACAA2-specific causality not isolated from the broader axis\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identified the degradation machinery controlling ACAA2 stability, defining a Cullin1/FBXO42 E3 ligase complex opposed by CAND1 and linking ACAA2 preservation to protection against NAFLD.\",\n      \"evidence\": \"Reciprocal Co-IP of the Cullin1/FBXO42/ACAA2 complex, hepatocyte-specific CAND1 KO/KI mice, ACAA2 overexpression rescue\",\n      \"pmids\": [\"37528093\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitination site(s) on ACAA2 not mapped\", \"Substrate-recognition determinant of FBXO42 for ACAA2 unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Showed that ACAA2 thiolase activity is directly repressible, with p46Shc inhibiting activity to reduce β-oxidation and ketogenesis while promoting ROS and mitochondrial damage.\",\n      \"evidence\": \"In vitro thiolase activity assay, p46Shc-inducible and dominant-negative transgenic mice, Oroboros respirometry, electron microscopy\",\n      \"pmids\": [\"39733992\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether p46Shc physically binds ACAA2 directly not established\", \"Molecular basis of activity inhibition unresolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstrated that O-GlcNAcylation by OGT controls ACAA2 nucleocytoplasmic distribution and that ACAA2 abundance drives ovarian cancer growth, connecting a post-translational modification to its dual localization and oncogenic output.\",\n      \"evidence\": \"OGT overexpression with immunofluorescence/fractionation, ACAA2 gain/loss of function, nude mouse xenograft, RNA-seq implicating DIXDC1/WNT-β-Catenin\",\n      \"pmids\": [\"38656551\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"O-GlcNAc site(s) not mapped\", \"Direct link between glycosylation, localization, and the WNT axis remains correlative\", \"Whether nuclear ACAA2 here is the TRβ1 coactivator pool untested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Extended modification-based control to the heart, showing ACAA2 lactylation changes during hypertrophic stress and that ACAA2 loss worsens lipid accumulation, oxidative stress, and respiratory impairment, with transcriptional input from FOXO4.\",\n      \"evidence\": \"Immunoprecipitation for lactylation, RNAi knockdown and overexpression in cardiomyocytes, Seahorse, ELISA substrate assays, FOXO4 knockdown epistasis, Oil Red O staining\",\n      \"pmids\": [\"40858063\", \"40149900\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Lactylation site(s) and effect on catalytic mechanism not mapped\", \"Causal contribution of lactylation vs. abundance to phenotype not separated\", \"FOXO4 regulation of ACAA2 not shown to be direct\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How ACAA2's mitochondrial thiolase function and its nuclear TRβ1 coactivator role are coordinated — and whether the array of modifications (ubiquitination, O-GlcNAc, lactylation) constitute a unified switch governing localization and activity — remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model integrating catalytic and coactivator functions\", \"Modification crosstalk not dissected\", \"Tissue-specific hierarchy of regulators unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [4, 6]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 4]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [0, 4, 6]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [2, 4, 6]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 2]}\n    ],\n    \"complexes\": [\"Cullin1/FBXO42 E3 ubiquitin ligase complex (as substrate)\"],\n    \"partners\": [\"THRB\", \"CUL1\", \"FBXO42\", \"CAND1\", \"OGT\", \"SHC1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}