Existing Annotations Review

GO Term	Evidence	Action	Reason
GO:0003985 acetyl-CoA C-acetyltransferase activity	IBA GO_REF:0000033	ACCEPT	Summary: Phylogenetic (IBA) assignment of the acetyl-CoA C-acetyltransferase (acetoacetyl-CoA thiolase, EC 2.3.1.9) activity. This is the well-established core catalytic function of ACAT1/T2 and is concordant with direct biochemical and structural evidence. Reason: Core molecular function, supported across IBA, EXP, IDA, and IMP evidence and by crystal structures of human T2. Supporting Evidence: PMID:17371050 Mitochondrial acetoacetyl-coenzyme A (CoA) thiolase (T2) is important in the pathways for the synthesis and degradation of ketone bodies as well as for the degradation of 2-methylacetoacetyl-CoA.
GO:0003985 acetyl-CoA C-acetyltransferase activity	IEA GO_REF:0000120	ACCEPT	Summary: Electronic assignment (RHEA:21036 / EC 2.3.1.9 mapping) of the core acetyl-CoA C-acetyltransferase activity. Consistent with the IBA and experimental annotations. Reason: Redundant with experimental evidence but correctly captures the core catalytic function.
GO:0003985 acetyl-CoA C-acetyltransferase activity	EXP PMID:1715688 Evidence for a structural mutation (347Ala to Thr) in a Germ...	ACCEPT	Summary: Experimental annotation from characterization of a 3KTD disease allele (A380T / "347Ala to Thr"), where expression analysis showed the mutation destabilizes T2 and impairs the thiolase enzyme. This links the EC 2.3.1.9 thiolase activity directly to ACAT1. Reason: Core molecular function with experimental (disease-variant expression) support. The curator read the full text; the cached record is abstract-only. Supporting Evidence: PMID:1715688 mitochondrial acetoacetyl-CoA thiolase (T2) biosynthesized in the patient's fibroblasts (GK06) was unstable
GO:0003985 acetyl-CoA C-acetyltransferase activity	EXP PMID:7728148 Molecular, biochemical, and clinical characterization of mit...	ACCEPT	Summary: Experimental annotation from characterization of 3KTD alleles (N158D, T297M, A301P); only T297M retained detectable residual thiolase activity, confirming that the assayed activity is the acetoacetyl-CoA thiolase function of ACAT1. Reason: Core molecular function supported by mutant-enzyme expression assays. Supporting Evidence: PMID:7728148 only the mutant T2 polypeptide with T297M appeared to have a detectable residual activity, in spite of its instability
GO:0003985 acetyl-CoA C-acetyltransferase activity	EXP PMID:9744475 Characterization of N93S, I312T, and A333P missense mutation...	ACCEPT	Summary: Experimental annotation from characterization of 3KTD missense alleles (N93S, I312T, A333P) with reduced residual thiolase activity, again establishing the acetoacetyl-CoA thiolase activity of ACAT1. Reason: Core molecular function; the paper explicitly frames T2 deficiency as an error of "ketone body and isoleucine catabolisms". Supporting Evidence: PMID:9744475 Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency is an inborn error of ketone body and isoleucine catabolisms.
GO:0003985 acetyl-CoA C-acetyltransferase activity	IDA PMID:17371050 Crystallographic and kinetic studies of human mitochondrial ...	ACCEPT	Summary: Direct assay annotation from the crystallographic/kinetic study of human T2, which measured degradation of acetoacetyl-CoA (and 2-methylacetoacetyl-CoA) and the reverse condensation of two acetyl-CoA molecules. Reason: Core molecular function with direct biochemical and structural evidence (homotetramer, active-site residues, K+ activation). Supporting Evidence: PMID:17371050 The kinetic measurements show that T2 can degrade acetoacetyl-CoA and 2-methylacetoacetyl-CoA with similar catalytic efficiencies.
GO:0003985 acetyl-CoA C-acetyltransferase activity	IMP PMID:1979337 Molecular cloning and sequence of the complementary DNA enco...	ACCEPT	Summary: IMP annotation from the original cDNA cloning paper, which analyzed T2 in fibroblasts from four 3-ketothiolase-deficiency patients (loss/reduction of the thiolase). Supports the core enzymatic function. Reason: Core molecular function inferred from patient phenotype/enzyme deficiency. Supporting Evidence: PMID:1979337 Complementary DNAs encoding the precursor of human hepatic mitochondrial acetoacetyl-CoA thiolase (T2) (EC 2.3.1.9) were cloned and sequenced.
GO:0003985 acetyl-CoA C-acetyltransferase activity	IMP PMID:8103405 Molecular studies of mitochondrial acetoacetyl-coenzyme A th...	ACCEPT	Summary: IMP annotation from molecular study of the two original 3KTD probands ("the K(+)-activated enzyme, mitochondrial acetoacetyl-coenzyme A thiolase (T2)"), whose mutant alleles impair the thiolase. Supports the core enzymatic function. Reason: Core molecular function; redundant with other EXP/IMP annotations but correctly assigned. Supporting Evidence: PMID:8103405 shown later to have a deficiency of the K(+)-activated enzyme, mitochondrial acetoacetyl-coenzyme A thiolase (T2)
GO:0003985 acetyl-CoA C-acetyltransferase activity	TAS PMID:1979337 Molecular cloning and sequence of the complementary DNA enco...	ACCEPT	Summary: TAS restatement of the core acetyl-CoA C-acetyltransferase activity from the cloning paper. Reason: Core molecular function; consistent with all other lines of evidence.
GO:0016453 C-acetyltransferase activity	IDA PMID:17371050 Crystallographic and kinetic studies of human mitochondrial ...	KEEP AS NON CORE	Summary: Direct-assay annotation to the more general parent term C-acetyltransferase activity. Correct but less informative than the specific child GO:0003985. Reason: Correct parent term; the specific acetyl-CoA C-acetyltransferase activity (GO:0003985) is the preferred core annotation.
GO:0016746 acyltransferase activity	IEA GO_REF:0000002	KEEP AS NON CORE	Summary: InterPro-based assignment of the broad acyltransferase activity parent term (thiolase domain). Reason: Technically correct but uninformative; superseded by the specific thiolase term.
GO:0016747 acyltransferase activity, transferring groups other than amino-acyl groups	IEA GO_REF:0000002	KEEP AS NON CORE	Summary: InterPro-based assignment of an intermediate acyltransferase parent term based on the thiolase InterPro signatures. Reason: Correct parent term; the specific acetyl-CoA C-acetyltransferase activity better captures the molecular function.
GO:0030955 potassium ion binding	IDA PMID:17371050 Crystallographic and kinetic studies of human mitochondrial ...	ACCEPT	Summary: Direct annotation supported by the crystal structures, which resolve a bound potassium ion near the CoA-binding and catalytic sites; T2 is uniquely activated by K+ (not Na+). Reason: Genuine, well-evidenced activator/cofactor binding that is a distinctive functional feature of T2, though it is supporting rather than the catalytic core function itself. Supporting Evidence: PMID:17371050 The potassium ion is bound near the CoA binding site and the catalytic site.
GO:0120225 coenzyme A binding	IEA GO_REF:0000107	KEEP AS NON CORE	Summary: Electronic (ortholog-transfer) annotation for coenzyme A binding. CoA is a substrate/cosubstrate of the thiolase reaction and the crystal structures define a CoA-binding site, so this is correct. Reason: Correct substrate-binding annotation, but it is an intrinsic part of the catalytic mechanism already captured by the thiolase MF.
GO:0019899 enzyme binding	IEA GO_REF:0000107	MARK AS OVER ANNOTATED	Summary: Generic "enzyme binding" transferred electronically from a rodent ortholog. Uninformative and not tied to any specific, verified interaction for human ACAT1. Reason: Vague binding term with only ortholog-based electronic support; conveys no specific functional information.
GO:0042802 identical protein binding	IEA GO_REF:0000107	KEEP AS NON CORE	Summary: Electronic annotation for self-association. ACAT1/T2 is a homotetramer, so self-interaction is real, but the generic term is uninformative and the catalytically relevant fact (homotetramer) is better captured by subunit/complex description. Reason: Consistent with the homotetrameric quaternary structure but uninformative as a function term.
GO:0034736 cholesterol O-acyltransferase activity	IDA PMID:32944968 Cholesterol 25-Hydroxylase inhibits SARS-CoV-2 and other cor...	REMOVE	Summary: This annotation mis-attributes a cholesterol-esterifying activity to the mitochondrial thiolase P24752. PMID:32944968 studies the ER acyl-CoA:cholesterol acyltransferase ("ACAT", EC 2.3.1.26), which is sterol O-acyltransferase SOAT1/SOAT2 - distinct genes that merely share the historical nickname "ACAT1/ACAT2". The paper's functional perturbation used "shRNAs targeting ACAT1 and ACAT2" (i.e., SOAT1/SOAT2), and describes the enzyme as ER-localized. The mitochondrial T2 (EC 2.3.1.9) has no cholesterol O-acyltransferase activity. Reason: Wrong-gene mis-annotation arising from the ACAT1/SOAT1 name collision. Cholesterol esterification is an EC 2.3.1.26 activity of the ER enzyme SOAT1/SOAT2, not of the mitochondrial acetoacetyl-CoA thiolase P24752. This contradicts the established mitochondrial-matrix thiolase function and should not be propagated. Proposed replacements: acetyl-CoA C-acetyltransferase activity Supporting Evidence: PMID:32944968 25HC inhibits viral membrane fusion by activating the ER-localized acyl-CoA:cholesterol acyltransferase (ACAT) which leads to the depletion of accessible cholesterol from the plasma membrane.
GO:0005783 endoplasmic reticulum	IDA PMID:32944968 Cholesterol 25-Hydroxylase inhibits SARS-CoV-2 and other cor...	REMOVE	Summary: ER localization derived from the same SOAT/cholesterol-acyltransferase study (PMID:32944968). The ER-localized enzyme in that paper is the cholesterol-esterifying SOAT1/SOAT2, not the mitochondrial matrix thiolase P24752. ACAT1/T2 is a soluble mitochondrial matrix protein. Reason: Wrong-gene mis-annotation (ACAT1/SOAT1 collision). The "is_active_in ER" claim belongs to the cholesterol acyltransferase SOAT1/SOAT2; the mitochondrial T2 acts in the mitochondrial matrix. Proposed replacements: mitochondrial matrix Supporting Evidence: PMID:32944968 25HC inhibits viral membrane fusion by activating the ER-localized acyl-CoA:cholesterol acyltransferase (ACAT) which leads to the depletion of accessible cholesterol from the plasma membrane.
GO:0005739 mitochondrion	IBA GO_REF:0000033	KEEP AS NON CORE	Summary: Phylogenetic localization to the mitochondrion. Correct; the more specific matrix term is preferred for the core localization. Reason: Correct but less specific than mitochondrial matrix (GO:0005759).
GO:0005739 mitochondrion	IEA GO_REF:0000120	KEEP AS NON CORE	Summary: Electronic mitochondrial localization consistent with experimental evidence. Reason: Correct but less specific than the matrix annotation.
GO:0005739 mitochondrion	IDA GO_REF:0000052	KEEP AS NON CORE	Summary: Immunofluorescence (HPA) mitochondrial localization. Correct localization. Reason: Correct mitochondrial localization; matrix term is more specific.
GO:0005739 mitochondrion	IDA PMID:1979337 Molecular cloning and sequence of the complementary DNA enco...	KEEP AS NON CORE	Summary: Direct localization from subcellular fractionation/pulse labeling in the original cloning paper, showing T2 protein resides in mitochondria. The more specific matrix term is preferred for the core localization, so this is kept as non-core for consistency with the other mitochondrion annotations. Reason: Correct, experimentally supported mitochondrial localization, but less specific than the mitochondrial-matrix core annotation. Supporting Evidence: PMID:1979337 Pulse labeling followed by subcellular fractionation revealed that the T2 proteins in the fibroblasts from these patients are present in the mitochondria.
GO:0005739 mitochondrion	HTP PMID:34800366 Quantitative high-confidence human mitochondrial proteome an...	KEEP AS NON CORE	Summary: High-throughput quantitative mitochondrial-proteome localization. Consistent with the established mitochondrial localization of ACAT1. Reason: Correct mitochondrial localization from proteomics; less specific than matrix.
GO:0005759 mitochondrial matrix	IEA GO_REF:0000120	ACCEPT	Summary: Electronic assignment to the mitochondrial matrix, the correct compartment for this soluble homotetrameric thiolase. Reason: Core cellular localization; concordant with Reactome curation and the soluble crystal structures.
GO:0005759 mitochondrial matrix	TAS Reactome:R-HSA-70844	ACCEPT	Summary: Reactome localization of ACAT1 to the mitochondrial matrix in the isoleucine-catabolism reaction (2-methylacetoacetyl-CoA thiolysis). Reason: Correct core localization from pathway curation.
GO:0005759 mitochondrial matrix	TAS Reactome:R-HSA-73916	ACCEPT	Summary: Reactome localization to the matrix in the ketone-body condensation reaction (2 acetyl-CoA <=> acetoacetyl-CoA + CoA). Reason: Correct core localization.
GO:0005759 mitochondrial matrix	TAS Reactome:R-HSA-74181	ACCEPT	Summary: Reactome localization to the matrix in the ketolysis reaction (acetoacetyl-CoA + CoA <=> 2 acetyl-CoA). Reason: Correct core localization.
GO:0005759 mitochondrial matrix	TAS Reactome:R-HSA-9838035	ACCEPT	Summary: Reactome matrix localization in the context of mitochondrial protein quality control (CLPXP substrate binding). Reason: Correct localization; the matrix compartment is right even though the cited reaction concerns protease handling of matrix proteins.
GO:0005759 mitochondrial matrix	TAS Reactome:R-HSA-9838081	ACCEPT	Summary: Reactome matrix localization in a LONP1-mediated matrix-protein degradation reaction. Reason: Correct matrix localization.
GO:0005759 mitochondrial matrix	TAS Reactome:R-HSA-9838093	ACCEPT	Summary: Reactome matrix localization in a LONP1 matrix-protein-binding reaction. Reason: Correct matrix localization.
GO:0005759 mitochondrial matrix	TAS Reactome:R-HSA-9838289	ACCEPT	Summary: Reactome matrix localization in a CLPXP matrix-protein-degradation reaction. Reason: Correct matrix localization.
GO:0005759 mitochondrial matrix	TAS Reactome:R-HSA-9854415	ACCEPT	Summary: Reactome matrix localization in the reaction "ACAT1 tetramer acetylates IDH2 dimer". This reflects a genuine moonlighting protein-lysine-acetyltransferase activity of the ACAT1 homotetramer reported in cancer metabolism (acetylation of IDH2 at K413 / PDH components); here it is used only as a matrix-localization annotation. Reason: Correct matrix localization. The associated acetyltransferase moonlighting activity is noted in proposed_new_terms/suggested_questions but is not part of the curated GOA MF set under review.
GO:0070062 extracellular exosome	HDA PMID:23533145 In-depth proteomic analyses of exosomes isolated from expres...	KEEP AS NON CORE	Summary: High-throughput detection of ACAT1 in prostatic-secretion urinary exosome proteomics. This is a common finding for abundant mitochondrial/metabolic proteins in vesicle proteomes and does not reflect the functional site of the enzyme. Reason: Proteomic vesicle-detection localization, not the functional mitochondrial-matrix site.
GO:0070062 extracellular exosome	HDA PMID:19056867 Large-scale proteomics and phosphoproteomics of urinary exos...	KEEP AS NON CORE	Summary: High-throughput detection of ACAT1 in urinary exosome proteomics. As above, not a functional localization. Reason: Proteomic detection in secreted vesicles; non-core.
GO:0046952 ketone body catabolic process	IMP PMID:1979337 Molecular cloning and sequence of the complementary DNA enco...	ACCEPT	Summary: IMP annotation linking ACAT1 to ketone body catabolism (ketolysis), via patient fibroblast analysis in 3-ketothiolase deficiency. T2 catalyzes acetoacetyl-CoA + CoA <=> 2 acetyl-CoA, the key ketolytic thiolase step. Reason: Core biological process; ketone body metabolism is a principal physiological role of T2. Supporting Evidence: PMID:17371050 important in the pathways for the synthesis and degradation of ketone bodies
GO:0006085 acetyl-CoA biosynthetic process	IDA PMID:17371050 Crystallographic and kinetic studies of human mitochondrial ...	KEEP AS NON CORE	Summary: Curator (BHF-UCL) IDA annotation reflecting that thiolytic cleavage of acetoacetyl-CoA yields acetyl-CoA. This is a reaction-level restatement of the thiolase activity rather than the gene's physiological process. Reason: Chemically defensible product of the reaction, but the physiological process core is ketone body metabolism / isoleucine catabolism, not generic acetyl-CoA biosynthesis.
GO:0046356 acetyl-CoA catabolic process	IDA PMID:17371050 Crystallographic and kinetic studies of human mitochondrial ...	KEEP AS NON CORE	Summary: BHF-UCL IDA annotation reflecting the condensation direction (consumption of acetyl-CoA to form acetoacetyl-CoA). Reaction-level restatement of the reversible thiolase chemistry. Reason: Defensible at the reaction level; the physiological BP core is ketone body metabolism.
GO:0006550 L-isoleucine catabolic process	TAS PMID:17371050 Crystallographic and kinetic studies of human mitochondrial ...	ACCEPT	Summary: TAS annotation placing ACAT1 in isoleucine catabolism, via its unique ability to cleave the 2-methyl-branched intermediate 2-methylacetoacetyl-CoA - a defining property of T2. Reason: Core biological process. The 2-methyl-branched substrate specificity is a distinctive feature of T2 and the basis of beta-ketothiolase deficiency as an isoleucine-catabolism disorder. Supporting Evidence: PMID:17371050 A unique property of T2 is its ability to use 2-methyl-branched acetoacetyl-CoA as a substrate
GO:0006550 L-isoleucine catabolic process	IMP PMID:1979337 Molecular cloning and sequence of the complementary DNA enco...	ACCEPT	Summary: IMP annotation from patient-fibroblast analysis in 3-ketothiolase deficiency, an inborn error of isoleucine catabolism. Supports the isoleucine-catabolism role. Reason: Core biological process supported by the disease phenotype.
GO:0006550 L-isoleucine catabolic process	IMP PMID:8103405 Molecular studies of mitochondrial acetoacetyl-coenzyme A th...	ACCEPT	Summary: IMP annotation from molecular study of the two original 2-methylacetoacetic-aciduria families with T2 deficiency, consistent with the isoleucine-catabolism role. Reason: Core biological process; redundant but correctly assigned. Supporting Evidence: PMID:8103405 first reported with 2-methylacetoacetic aciduria, and shown later to have a deficiency of the K(+)-activated enzyme, mitochondrial acetoacetyl-coenzyme A thiolase (T2)
GO:0015936 coenzyme A metabolic process	IDA PMID:17371050 Crystallographic and kinetic studies of human mitochondrial ...	KEEP AS NON CORE	Summary: BHF-UCL IDA annotation reflecting that CoA is consumed/released in the thiolase reaction. Reaction-level rather than a distinct physiological process. Reason: CoA is intrinsic to the catalytic mechanism; not an independent core process.
GO:0015937 coenzyme A biosynthetic process	IDA PMID:17371050 Crystallographic and kinetic studies of human mitochondrial ...	MARK AS OVER ANNOTATED	Summary: BHF-UCL IDA annotation reflecting CoA release during thiolytic cleavage. This is not genuine CoA biosynthesis (the de novo CoA biosynthetic pathway is unrelated); it is a reaction-level artifact. Reason: ACAT1 does not participate in coenzyme A biosynthesis; releasing free CoA as a thiolase product is not the biosynthetic pathway. Over-annotation.
GO:1902860 propionyl-CoA biosynthetic process	IDA PMID:17371050 Crystallographic and kinetic studies of human mitochondrial ...	KEEP AS NON CORE	Summary: BHF-UCL IDA annotation reflecting that thiolysis of 2-methylacetoacetyl-CoA in the isoleucine pathway yields propionyl-CoA (plus acetyl-CoA). This is a product of the isoleucine-catabolism step rather than a separate core process. Reason: Defensible as a product of the isoleucine-catabolism reaction; the physiological BP is better captured by L-isoleucine catabolic process.
GO:0006635 fatty acid beta-oxidation	IEA GO_REF:0000041	KEEP AS NON CORE	Summary: UniPathway-derived electronic annotation to fatty acid beta-oxidation. UniProt lists T2 under this pathway as a thiolase, but the unbranched long-/medium-chain 3-oxoacyl-CoA thiolysis of classic beta-oxidation is principally performed by ACAA2 and the MTP (HADHA/HADHB); ACAT1/T2's dominant roles are the acetoacetyl-CoA (ketone body) node and the 2-methyl-branched isoleucine intermediate. Reason: Defensible at the pathway-parent level but not the core physiological role of this thiolase.
GO:0001889 liver development	IEA GO_REF:0000107	MARK AS OVER ANNOTATED	Summary: Electronic ortholog-transfer (Ensembl Compara) annotation. ACAT1 is liver-enhanced in expression, but there is no evidence it has a developmental role in the liver; this reflects expression context rather than function. Reason: Ortholog-based IEA with no mechanistic link to liver development for a metabolic thiolase.
GO:0009725 response to hormone	IEA GO_REF:0000107	MARK AS OVER ANNOTATED	Summary: Electronic ortholog-transfer annotation. Generic stimulus-response term without specific evidence for human ACAT1. Reason: Vague, ortholog-based; not a core function.
GO:0042594 response to starvation	IEA GO_REF:0000107	KEEP AS NON CORE	Summary: Electronic ortholog-transfer annotation. Plausible in spirit (ketogenesis is induced by fasting and T2 contributes to ketone body metabolism), but the specific "response to starvation" process is an indirect ortholog inference rather than a demonstrated role of ACAT1. Reason: Biologically plausible via ketogenesis but only electronically inferred from orthologs; non-core.
GO:0060612 adipose tissue development	IEA GO_REF:0000107	MARK AS OVER ANNOTATED	Summary: Electronic ortholog-transfer annotation. No evidence for a developmental role of ACAT1 in adipose tissue; reflects expression/physiology context. Reason: Ortholog-based IEA with no mechanistic support for a developmental function.
GO:0072229 metanephric proximal convoluted tubule development	IEA GO_REF:0000107	MARK AS OVER ANNOTATED	Summary: Highly specific developmental term transferred electronically from a rodent ortholog. ACAT1 is kidney-enhanced in expression, but there is no evidence for a direct role in nephron development. Reason: Over-specific ortholog-based IEA with no mechanistic basis for this metabolic enzyme.

Core Functions

Potassium-activated mitochondrial-matrix acetoacetyl-CoA thiolase (EC 2.3.1.9) that catalyzes the reversible thiolytic cleavage/condensation at the acetoacetyl-CoA node (acetoacetyl-CoA + CoA <-> 2 acetyl-CoA), central to ketone body metabolism (ketogenesis and ketolysis).

Molecular Function:

acetyl-CoA C-acetyltransferase activity

Directly Involved In:

ketone body catabolic process

Cellular Locations:

mitochondrial matrix

Supporting Evidence:

PMID:17371050
important in the pathways for the synthesis and degradation of ketone bodies
PMID:9744475
Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency is an inborn error of ketone body and isoleucine catabolisms.

Catalyzes the final thiolytic step of L-isoleucine catabolism, cleaving the 2-methyl-branched intermediate 2-methylacetoacetyl-CoA into propionyl-CoA and acetyl-CoA - a substrate specificity unique among the structurally characterized thiolases. Loss of this activity causes beta-ketothiolase (3-ketothiolase) deficiency.

Molecular Function:

acetyl-CoA C-acetyltransferase activity

Directly Involved In:

L-isoleucine catabolic process

Cellular Locations:

mitochondrial matrix

Supporting Evidence:

PMID:17371050
A unique property of T2 is its ability to use 2-methyl-branched acetoacetyl-CoA as a substrate
PMID:9744475
Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency is an inborn error of ketone body and isoleucine catabolisms.

References

GO_REF:0000002

Gene Ontology annotation through association of InterPro records with GO terms

GO_REF:0000033

Annotation inferences using phylogenetic trees

GO_REF:0000041

Gene Ontology annotation based on UniPathway vocabulary mapping

GO_REF:0000052

Gene Ontology annotation based on curation of immunofluorescence data

GO_REF:0000107

Automatic transfer of experimentally verified manual GO annotation data to orthologs using Ensembl Compara

GO_REF:0000120

Combined Automated Annotation using Multiple IEA Methods

PMID:1715688

Evidence for a structural mutation (347Ala to Thr) in a German family with 3-ketothiolase deficiency.

Characterized a 3KTD missense allele (A380T / "347Ala to Thr" in the mature subunit) shown by expression analysis to destabilize the T2 protein, linking EC 2.3.1.9 thiolase activity to ACAT1.

PMID:17371050

Crystallographic and kinetic studies of human mitochondrial acetoacetyl-CoA thiolase: the importance of potassium and chloride ions for its structure and function.

High-resolution crystal structures of human T2 (apo and CoA complex, with/without K+). Defines the homotetramer, the CoA- and K+-binding sites near the catalytic site, and K+ activation.
T2 is important for synthesis and degradation of ketone bodies and for degradation of 2-methylacetoacetyl-CoA; it uniquely accepts 2-methyl-branched acetoacetyl-CoA and degrades acetoacetyl-CoA and 2-methylacetoacetyl-CoA with similar catalytic efficiencies.

PMID:19056867

Large-scale proteomics and phosphoproteomics of urinary exosomes.

Proteomic profiling of human urinary exosomes; basis for the extracellular-exosome localization annotation. Not a functional localization for the mitochondrial thiolase.

PMID:1979337

Molecular cloning and sequence of the complementary DNA encoding human mitochondrial acetoacetyl-coenzyme A thiolase and study of the variant enzymes in cultured fibroblasts from patients with 3-ketothiolase deficiency.

Cloned/sequenced the 427-aa human T2 precursor (33-residue leader + mature subunit) and showed by subcellular fractionation that T2 protein localizes to mitochondria in patient fibroblasts.

PMID:23533145

In-depth proteomic analyses of exosomes isolated from expressed prostatic secretions in urine.

Proteomic analysis of prostatic-secretion urinary exosomes; basis for an extracellular-exosome localization annotation. Incidental, not functional.

PMID:32944968

Cholesterol 25-Hydroxylase inhibits SARS-CoV-2 and other coronaviruses by depleting membrane cholesterol.

Studies CH25H/25HC activating the ER acyl-CoA:cholesterol acyltransferase ("ACAT", EC 2.3.1.26) - i.e. sterol O-acyltransferase SOAT1/SOAT2 - to deplete plasma-membrane cholesterol and block coronavirus fusion. Functional perturbation used shRNAs targeting "ACAT1 and ACAT2" (SOAT1/SOAT2).

PMID:34800366

Quantitative high-confidence human mitochondrial proteome and its dynamics in cellular context.

High-throughput quantitative mitochondrial proteome; supports mitochondrial localization of ACAT1.

PMID:7728148

Molecular, biochemical, and clinical characterization of mitochondrial acetoacetyl-coenzyme A thiolase deficiency in two further patients.

Characterized 3KTD alleles (N158D, T297M, A301P); expression analyses showed reduced/abolished T2 activity, with only T297M retaining detectable residual activity.

PMID:8103405

Molecular studies of mitochondrial acetoacetyl-coenzyme A thiolase deficiency in the two original families.

Molecular characterization of the two original 2-methylacetoacetic-aciduria probands, confirmed as deficient in the K+-activated mitochondrial acetoacetyl-CoA thiolase (T2); identified inactivating alleles.

PMID:9744475

Characterization of N93S, I312T, and A333P missense mutations in two Japanese families with mitochondrial acetoacetyl-CoA thiolase deficiency.

Characterized 3KTD missense alleles (N93S, I312T, A333P) with reduced residual thiolase activity; states explicitly that T2 deficiency is an inborn error of ketone body and isoleucine catabolisms.

PMID:27867011

Tetrameric Acetyl-CoA Acetyltransferase 1 Is Important for Tumor Growth.

Reports that the tetrameric form of mitochondrial ACAT1 acts as a protein-lysine acetyltransferase, acetylating and inhibiting pyruvate dehydrogenase (PDHA1) and PDH phosphatase (PDP1), and that tetrameric ACAT1 promotes tumor growth. Basis for the moonlighting acetyltransferase activity noted in proposed_new_terms.

"We identified mitochondrial acetyl-CoA acetyltransferase 1 (ACAT1) and SIRT3 as upstream acetyltransferase and deacetylase, respectively, of PDHA1 and PDP1"

Reactome:R-HSA-70844

alpha-methylacetoacetyl-CoA + CoA => propionyl-CoA + acetyl-CoA

Reactome reaction for the final thiolytic step of isoleucine catabolism catalyzed by ACAT1 in the mitochondrial matrix; supports the matrix localization and isoleucine role.

Reactome:R-HSA-73916

2 acetyl-CoA <=> acetoacetyl-CoA + CoA

Reactome reaction for the ketone-body condensation direction catalyzed by ACAT1; supports matrix localization and ketone body metabolism.

Reactome:R-HSA-74181

acetoacetyl-CoA + CoA <=> 2 acetyl-CoA

Reactome reaction for the ketolytic thiolysis direction catalyzed by ACAT1; supports matrix localization and ketone body catabolism.

Reactome:R-HSA-9838035

CLPXP binds mitochondrial matrix proteins

Reactome quality-control reaction in which ACAT1, as a matrix protein, is a CLPXP substrate; supports mitochondrial-matrix localization.

Reactome:R-HSA-9838081

LONP1 degrades mitochondrial matrix proteins

Reactome quality-control reaction; ACAT1 is annotated as a matrix protein substrate, supporting matrix localization.

Reactome:R-HSA-9838093

LONP1 binds mitochondrial matrix proteins

Reactome quality-control reaction; supports ACAT1 matrix localization.

Reactome:R-HSA-9838289

CLPXP degrades mitochondrial matrix proteins

Reactome quality-control reaction; supports ACAT1 matrix localization.

Reactome:R-HSA-9854415

ACAT1 tetramer acetylates IDH2 dimer

Reactome reaction capturing the moonlighting protein-lysine-acetyltransferase activity of the ACAT1 homotetramer (acetylation of IDH2 at K413), localized to the mitochondrial matrix. Used here as a matrix-localization annotation; the acetyltransferase activity itself is not in the curated GOA MF set under review.

Suggested Experiments

Experiment: Express recombinant human ACAT1/T2 and directly assay for cholesterol O-acyltransferase (sterol esterification, EC 2.3.1.26) activity alongside SOAT1 as a positive control.

Hypothesis: Recombinant mitochondrial ACAT1/T2 will show no cholesterol-esterifying activity, confirming that GO:0034736 is a wrong-gene mis-annotation belonging to SOAT1/SOAT2.

Experiment: Quantify ketone body and isoleucine-pathway flux (acetoacetyl-CoA turnover, 2-methylacetoacetyl-CoA cleavage, urinary 2-methyl-3-hydroxybutyrate/tiglylglycine) in ACAT1-knockout versus wild-type cells or patient fibroblasts.

Hypothesis: ACAT1 loss will impair both ketolysis and the final thiolytic step of isoleucine catabolism, producing the characteristic 2-methyl-branched metabolite accumulation of beta-ketothiolase deficiency.

Experiment: Reconstitute the ACAT1-tetramer-mediated acetylation of IDH2 in vitro and in cells, and test whether K+ activation and tetramer formation are required for the acetyltransferase (versus thiolase) activity.

Hypothesis: The protein-acetyltransferase moonlighting activity depends on the tetrameric assembly of ACAT1 and is mechanistically separable from its acetoacetyl-CoA thiolase activity.

📚 Additional Documentation

Notes

(ACAT1-notes.md)

ACAT1 (P24752) research notes

Gene: ACAT1 (HGNC:93); UniProt P24752 (THIL_HUMAN); EC 2.3.1.9.
Protein: Acetyl-CoA acetyltransferase, mitochondrial; AltNames: Acetoacetyl-CoA thiolase; "T2".
427 aa precursor (1-33 mitochondrial transit peptide; mature chain 34-427). Homotetramer.

CRITICAL nomenclature warning: ACAT1 (P24752) is NOT the cholesterol-esterifying enzyme

There is a long-standing naming collision. "ACAT" / "ACAT1" is used in two unrelated senses:
- This gene/protein (HGNC:93, P24752) = mitochondrial acetoacetyl-CoA thiolase / T2,
EC 2.3.1.9, a matrix homotetramer in ketone body and isoleucine metabolism.
- SOAT1 (HGNC:11177) = sterol O-acyltransferase 1 / acyl-CoA:cholesterol acyltransferase,
EC 2.3.1.26, an ER integral-membrane enzyme that esterifies cholesterol. SOAT1's old/alias
name is also "ACAT1" (and SOAT2's alias is "ACAT2"). These are entirely different proteins.

This collision has produced wrong-gene GO annotations on P24752 (see PMID:32944968 below).

Core enzymatic function (thiolase, EC 2.3.1.9)

T2 is a CoA-dependent thiolase catalyzing a reversible Claisen condensation / thiolytic cleavage.
- Reversible reaction: 2 acetyl-CoA <=> acetoacetyl-CoA + CoA (RHEA:21036; EC 2.3.1.9)
[UniProt P24752 CATALYTIC ACTIVITY].
- Also degrades the isoleucine-pathway intermediate 2-methylacetoacetyl-CoA:
propanoyl-CoA + acetyl-CoA <=> 2-methyl-3-oxobutanoyl-CoA + CoA (RHEA:30719) [UniProt P24752].
- UniProt FUNCTION: "The activity of the enzyme is reversible and it can also catalyze the
condensation of two acetyl-CoA molecules into acetoacetyl-CoA (PubMed:17371050). Thereby, it
plays a major role in ketone body metabolism" [UniProt P24752 FUNCTION].

PMID:17371050 (Haapalainen et al., Biochemistry 2007; crystal structures of human T2; ABSTRACT
ONLY in cache, full_text_available: false): "Mitochondrial acetoacetyl-coenzyme A (CoA) thiolase
(T2) is important in the pathways for the synthesis and degradation of ketone bodies as well as
for the degradation of 2-methylacetoacetyl-CoA." "A unique property of T2 is its activation by
potassium ions." "The potassium ion is bound near the CoA binding site and the catalytic site."
"A unique property of T2 is its ability to use 2-methyl-branched acetoacetyl-CoA as a substrate,
whereas the other structurally characterized thiolases cannot utilize the 2-methylated compounds."
"The kinetic measurements show that T2 can degrade acetoacetyl-CoA and 2-methylacetoacetyl-CoA
with similar catalytic efficiencies." [PMID:17371050 abstract].
-> Supports: acetyl-CoA C-acetyltransferase activity (GO:0003985), C-acetyltransferase activity
(GO:0016453), potassium ion binding (GO:0030955), homotetramer, ketone body + isoleucine roles.

Activation by potassium ions, not sodium [UniProt ACTIVITY REGULATION, ECO:0000269|PubMed:17371050].
Active sites: Cys126 (acyl-enzyme intermediate), His413 (proton donor/acceptor) [UniProt FT].

Subcellular location

Mitochondrion / mitochondrial matrix.
- PMID:1979337 (Fukao et al. 1990): "Pulse labeling followed by subcellular fractionation revealed
that the T2 proteins in the fibroblasts from these patients are present in the mitochondria."
[PMID:1979337 abstract]. -> Supports mitochondrion (GO:0005739).
- Mitochondrial matrix (GO:0005759) is the standard CC for a soluble matrix thiolase; supported by
Reactome TAS annotations and UniProt-IEA. Consistent with the homotetramer crystal structures
(soluble protein).

Ketone body metabolism (ketogenesis AND ketolysis)

T2 sits at the acetoacetyl-CoA <-> 2 acetyl-CoA node, central to both ketone body synthesis and
utilization. PMID:17371050: "important in the pathways for the synthesis and degradation of
ketone bodies." [PMID:17371050 abstract].
GOA captures: ketone body catabolic process (GO:0046952, IMP PMID:1979337) and ketone body
metabolic process (GO:1902224, IC) [GOA / UniProt GO xrefs].

Isoleucine catabolism + 3-ketothiolase / beta-ketothiolase deficiency (3KTD)

T2 catalyzes the final thiolytic step of isoleucine catabolism (2-methylacetoacetyl-CoA ->
propionyl-CoA + acetyl-CoA). Loss causes "beta-ketothiolase deficiency" / 3KTD (MIM:203750).
- PMID:9744475 (Fukao et al. 1998): "Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency is an
inborn error of ketone body and isoleucine catabolisms." [PMID:9744475 abstract].
- UniProt DISEASE: "3-ketothiolase deficiency (3KTD)... An autosomal recessive inborn error of
isoleucine catabolism characterized by intermittent ketoacidotic attacks... Urinary excretion of
2-methyl-3-hydroxybutyric acid, 2-methylacetoacetic acid, triglylglycine, butanone is increased."
[UniProt P24752 DISEASE].
- Disease-variant papers (all ABSTRACT ONLY in cache), each characterizing missense alleles with
reduced/abolished thiolase activity:
- PMID:1715688 (347Ala->Thr / A380T): unstable T2 protein.
- PMID:7728148 (N158D, T297M, A301P): "only the mutant T2 polypeptide with T297M appeared to
have a detectable residual activity"; expression confirmed reduced activity.
- PMID:9744475 (N93S, I312T, A333P): destabilizing missense, reduced residual thiolase activity.
- PMID:8103405 (two original 2-methylacetoacetic-aciduria families; "K(+)-activated enzyme,
mitochondrial acetoacetyl-coenzyme A thiolase (T2)"): unstable/translation-impairing alleles.
- PMID:1979337: original cloning + four 3KTD fibroblast lines.
These collectively support GO:0003985 (MF), GO:0006550 L-isoleucine catabolic process (BP), and
GO:0046952 ketone body catabolic process. The IMP/EXP annotations rest on patient-fibroblast and
expression assays (loss/reduction of thiolase activity in disease alleles).

WRONG-GENE annotations from PMID:32944968 (cholesterol acyltransferase / ER) -> REMOVE

PMID:32944968 (Wang et al., EMBO J 2020; "Cholesterol 25-Hydroxylase inhibits SARS-CoV-2...";
full text available). The paper studies CH25H/25HC activating the ER acyl-CoA:cholesterol
acyltransferase (ACAT) to deplete plasma-membrane cholesterol:
- "25HC inhibits viral membrane fusion by activating the ER-localized acyl-CoA:cholesterol
acyltransferase (ACAT) which leads to the depletion of accessible cholesterol from the plasma
membrane." [PMID:32944968 abstract].
- "the enzyme catalyzing the esterification of cholesterol, ACAT" ... knockdown "by lentiviral
vectors carrying ... shRNAs targeting ACAT1 and ACAT2." [PMID:32944968 results].
The "ACAT" here is the cholesterol-esterifying SOAT1/SOAT2 (EC 2.3.1.26), an ER enzyme — NOT
the mitochondrial thiolase P24752 (EC 2.3.1.9). The mitochondrial T2 has no cholesterol
O-acyltransferase activity and is not an ER protein. The two GOA annotations derived from this
paper on P24752 are therefore wrong-gene mis-attributions caused by the ACAT1/SOAT1 name collision:
- GO:0034736 cholesterol O-acyltransferase activity (IDA, PMID:32944968) -> REMOVE.
- GO:0005783 endoplasmic reticulum (IDA "is_active_in", PMID:32944968) -> REMOVE.

Moonlighting: ACAT1 tetramer as a protein acetyltransferase (acetylates IDH2/PDH)

A genuine, separate moonlighting activity of the mitochondrial T2 tetramer (Fan et al., Mol Cell
2016, PMID:27867011): tetrameric ACAT1 acetylates pyruvate dehydrogenase complex components (PDHA1,
PDP1) and IDH2 (K413), inhibiting them; relevant in cancer metabolism. Captured in GOA only as the
Reactome reaction "ACAT1 tetramer acetylates IDH2 dimer" (Reactome:R-HSA-9854415), used here as a
mitochondrial-matrix location TAS annotation. This protein-lysine-acetyltransferase activity is
NOT in the GOA MF set being reviewed; noted for suggested questions / proposed terms.
(Not citing as supporting_text since PMID:27867011 is not in the publications cache.)

Proteomics / exosome / mitochondrial-proteome localizations (non-core)

GO:0070062 extracellular exosome (HDA, PMID:23533145 prostatic-secretion exosomes; PMID:19056867
urinary exosomes): high-throughput vesicle proteomics; ubiquitous-contaminant-type CC, not the
functional location. KEEP_AS_NON_CORE.
GO:0005739 mitochondrion (HTP, PMID:34800366 quantitative mito proteome): supports mitochondrial
localization; consistent but less specific than matrix. KEEP_AS_NON_CORE / ACCEPT.

Electronic / ortholog-transfer BP terms (Ensembl GO_REF:0000107) — likely over-annotation

liver development (GO:0001889), response to hormone (GO:0009725), response to starvation
(GO:0042594), adipose tissue development (GO:0060612), metanephric proximal convoluted tubule
development (GO:0072229): all IEA transferred from rodent orthologs. These reflect tissue
expression/physiology contexts, not direct molecular roles of the thiolase. MARK_AS_OVER_ANNOTATED
or KEEP_AS_NON_CORE. (T2 is expressed kidney/liver-enhanced per HPA; "response to starvation" is
plausible given ketogenesis but is an indirect ortholog inference.)

Other MF terms

GO:0016453 C-acetyltransferase activity (IDA PMID:17371050): correct, slightly more general
parent of GO:0003985; KEEP_AS_NON_CORE.
GO:0016746 / GO:0016747 acyltransferase activity (InterPro IEA): correct but very general
parents; KEEP_AS_NON_CORE.
GO:0030955 potassium ion binding (IDA PMID:17371050): correct — T2 is K+-activated and the
crystal structure resolves bound K+; ACCEPT (cofactor/activator binding, supporting but not the
catalytic core MF).
GO:0120225 coenzyme A binding (IEA): correct — CoA is substrate/cosubstrate; KEEP_AS_NON_CORE.
GO:0019899 enzyme binding (IEA ortholog): generic; over-annotation/non-core.
GO:0042802 identical protein binding (IEA ortholog): T2 is a homotetramer, so self-association is
real, but "identical protein binding" is uninformative; KEEP_AS_NON_CORE.

CoA / acetyl-CoA process terms (PMID:17371050, BHF-UCL IDA)

acetyl-CoA biosynthetic process (GO:0006085), acetyl-CoA catabolic process (GO:0046356), coenzyme A
metabolic process (GO:0015936), coenzyme A biosynthetic process (GO:0015937), propionyl-CoA
biosynthetic process (GO:1902860): these are BHF-UCL curator inferences from the in-vitro
reversible thiolase reactions. They are chemically defensible (the reaction produces/consumes
acetyl-CoA and CoA, and the isoleucine branch yields propionyl-CoA precursor) but are reaction-level
restatements rather than the gene's physiological process. KEEP_AS_NON_CORE; the physiological BP
core is ketone body metabolism + isoleucine catabolism.

fatty acid beta-oxidation (GO:0006635)

UniProt PATHWAY: "Lipid metabolism; fatty acid beta-oxidation." and FUNCTION describes T2 as "one
of the enzymes that catalyzes the last step of the mitochondrial beta-oxidation pathway." However,
T2/ACAT1's physiologically dominant role is the acetoacetyl-CoA node (ketone bodies) and the
2-methyl-branched isoleucine intermediate; the unbranched long-chain 3-oxoacyl-CoA thiolysis of
classic beta-oxidation is mainly ACAA2 (medium/short-chain) and the MTP/HADHB (long-chain). The
beta-oxidation annotation (IEA via UniPathway) is defensible at the parent level but non-core for
this gene. KEEP_AS_NON_CORE.

Summary of decisions

Core MF: acetyl-CoA C-acetyltransferase activity (GO:0003985).
Core BP: ketone body metabolic process and L-isoleucine catabolic process.
Core CC: mitochondrial matrix (GO:0005759).
REMOVE (wrong gene, SOAT1/SOAT2 collision): GO:0034736 cholesterol O-acyltransferase activity
and GO:0005783 endoplasmic reticulum (both PMID:32944968).
Over-annotation (ortholog IEA tissue/physiology BP): liver dev, adipose dev, metanephric tubule
dev, response to hormone, (response to starvation borderline).
Non-core but correct: matrix/mito CC variants, K+/CoA binding, general transferase parents,
exosome CC, CoA/acetyl-CoA reaction-level BP terms, fatty acid beta-oxidation.

📄 View Raw YAML

id: P24752
gene_symbol: ACAT1
product_type: PROTEIN
status: COMPLETE
taxon:
  id: NCBITaxon:9606
  label: Homo sapiens
description: >-
  ACAT1 encodes the mitochondrial enzyme acetyl-CoA acetyltransferase / acetoacetyl-CoA
  thiolase (also called "T2"; EC 2.3.1.9), a soluble homotetramer of the mitochondrial
  matrix. It is a CoA-dependent, potassium-activated thiolase that catalyzes the reversible
  Claisen condensation/thiolysis at the acetoacetyl-CoA node (2 acetyl-CoA <-> acetoacetyl-CoA
  + CoA), giving it a central role in ketone body metabolism (both ketogenesis and
  ketolysis). It also degrades the 2-methyl-branched intermediate 2-methylacetoacetyl-CoA,
  catalyzing the final thiolytic step of L-isoleucine catabolism. Loss-of-function variants
  cause beta-ketothiolase deficiency (3-ketothiolase deficiency, 3KTD), an autosomal recessive
  inborn error of isoleucine and ketone body catabolism marked by ketoacidotic episodes and
  urinary excretion of 2-methyl-3-hydroxybutyrate, 2-methylacetoacetate and tiglylglycine.
  Despite sharing the historical nickname "ACAT1", this mitochondrial thiolase is distinct from
  the endoplasmic-reticulum cholesterol-esterifying enzyme sterol O-acyltransferase 1 (SOAT1,
  EC 2.3.1.26, also formerly nicknamed ACAT1); ACAT1/T2 has no cholesterol O-acyltransferase
  activity.
alternative_products:
- name: '1'
  id: P24752-1
- name: '2'
  id: P24752-2
  sequence_note: VSP_056844, VSP_056845
existing_annotations:
# ============================================================================
# MOLECULAR FUNCTION - THIOLASE / ACETYL-CoA C-ACETYLTRANSFERASE (CORE)
# ============================================================================
- term:
    id: GO:0003985
    label: acetyl-CoA C-acetyltransferase activity
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  qualifier: enables
  review:
    summary: >-
      Phylogenetic (IBA) assignment of the acetyl-CoA C-acetyltransferase (acetoacetyl-CoA
      thiolase, EC 2.3.1.9) activity. This is the well-established core catalytic function of
      ACAT1/T2 and is concordant with direct biochemical and structural evidence.
    action: ACCEPT
    reason: >-
      Core molecular function, supported across IBA, EXP, IDA, and IMP evidence and by crystal
      structures of human T2.
    supported_by:
    - reference_id: PMID:17371050
      supporting_text: >-
        Mitochondrial acetoacetyl-coenzyme A (CoA) thiolase (T2) is important in the pathways
        for the synthesis and degradation of ketone bodies as well as for the degradation of
        2-methylacetoacetyl-CoA.
      full_text_unavailable: true
- term:
    id: GO:0003985
    label: acetyl-CoA C-acetyltransferase activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  qualifier: enables
  review:
    summary: >-
      Electronic assignment (RHEA:21036 / EC 2.3.1.9 mapping) of the core acetyl-CoA
      C-acetyltransferase activity. Consistent with the IBA and experimental annotations.
    action: ACCEPT
    reason: >-
      Redundant with experimental evidence but correctly captures the core catalytic function.
- term:
    id: GO:0003985
    label: acetyl-CoA C-acetyltransferase activity
  evidence_type: EXP
  original_reference_id: PMID:1715688
  qualifier: enables
  review:
    summary: >-
      Experimental annotation from characterization of a 3KTD disease allele (A380T / "347Ala
      to Thr"), where expression analysis showed the mutation destabilizes T2 and impairs the
      thiolase enzyme. This links the EC 2.3.1.9 thiolase activity directly to ACAT1.
    action: ACCEPT
    reason: >-
      Core molecular function with experimental (disease-variant expression) support. The
      curator read the full text; the cached record is abstract-only.
    supported_by:
    - reference_id: PMID:1715688
      supporting_text: >-
        mitochondrial acetoacetyl-CoA thiolase (T2) biosynthesized in the patient's fibroblasts
        (GK06) was unstable
      full_text_unavailable: true
- term:
    id: GO:0003985
    label: acetyl-CoA C-acetyltransferase activity
  evidence_type: EXP
  original_reference_id: PMID:7728148
  qualifier: enables
  review:
    summary: >-
      Experimental annotation from characterization of 3KTD alleles (N158D, T297M, A301P); only
      T297M retained detectable residual thiolase activity, confirming that the assayed activity
      is the acetoacetyl-CoA thiolase function of ACAT1.
    action: ACCEPT
    reason: >-
      Core molecular function supported by mutant-enzyme expression assays.
    supported_by:
    - reference_id: PMID:7728148
      supporting_text: >-
        only the mutant T2 polypeptide with T297M appeared to have a detectable residual
        activity, in spite of its instability
      full_text_unavailable: true
- term:
    id: GO:0003985
    label: acetyl-CoA C-acetyltransferase activity
  evidence_type: EXP
  original_reference_id: PMID:9744475
  qualifier: enables
  review:
    summary: >-
      Experimental annotation from characterization of 3KTD missense alleles (N93S, I312T, A333P)
      with reduced residual thiolase activity, again establishing the acetoacetyl-CoA thiolase
      activity of ACAT1.
    action: ACCEPT
    reason: >-
      Core molecular function; the paper explicitly frames T2 deficiency as an error of "ketone
      body and isoleucine catabolisms".
    supported_by:
    - reference_id: PMID:9744475
      supporting_text: >-
        Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency is an inborn error of ketone body
        and isoleucine catabolisms.
      full_text_unavailable: true
- term:
    id: GO:0003985
    label: acetyl-CoA C-acetyltransferase activity
  evidence_type: IDA
  original_reference_id: PMID:17371050
  qualifier: enables
  review:
    summary: >-
      Direct assay annotation from the crystallographic/kinetic study of human T2, which measured
      degradation of acetoacetyl-CoA (and 2-methylacetoacetyl-CoA) and the reverse condensation
      of two acetyl-CoA molecules.
    action: ACCEPT
    reason: >-
      Core molecular function with direct biochemical and structural evidence (homotetramer,
      active-site residues, K+ activation).
    supported_by:
    - reference_id: PMID:17371050
      supporting_text: >-
        The kinetic measurements show that T2 can degrade acetoacetyl-CoA and
        2-methylacetoacetyl-CoA with similar catalytic efficiencies.
      full_text_unavailable: true
- term:
    id: GO:0003985
    label: acetyl-CoA C-acetyltransferase activity
  evidence_type: IMP
  original_reference_id: PMID:1979337
  qualifier: enables
  review:
    summary: >-
      IMP annotation from the original cDNA cloning paper, which analyzed T2 in fibroblasts from
      four 3-ketothiolase-deficiency patients (loss/reduction of the thiolase). Supports the core
      enzymatic function.
    action: ACCEPT
    reason: >-
      Core molecular function inferred from patient phenotype/enzyme deficiency.
    supported_by:
    - reference_id: PMID:1979337
      supporting_text: >-
        Complementary DNAs encoding the precursor of human hepatic mitochondrial acetoacetyl-CoA
        thiolase (T2) (EC 2.3.1.9) were cloned and sequenced.
- term:
    id: GO:0003985
    label: acetyl-CoA C-acetyltransferase activity
  evidence_type: IMP
  original_reference_id: PMID:8103405
  qualifier: enables
  review:
    summary: >-
      IMP annotation from molecular study of the two original 3KTD probands ("the K(+)-activated
      enzyme, mitochondrial acetoacetyl-coenzyme A thiolase (T2)"), whose mutant alleles impair
      the thiolase. Supports the core enzymatic function.
    action: ACCEPT
    reason: >-
      Core molecular function; redundant with other EXP/IMP annotations but correctly assigned.
    supported_by:
    - reference_id: PMID:8103405
      supporting_text: >-
        shown later to have a deficiency of the K(+)-activated enzyme, mitochondrial
        acetoacetyl-coenzyme A thiolase (T2)
      full_text_unavailable: true
- term:
    id: GO:0003985
    label: acetyl-CoA C-acetyltransferase activity
  evidence_type: TAS
  original_reference_id: PMID:1979337
  qualifier: enables
  review:
    summary: >-
      TAS restatement of the core acetyl-CoA C-acetyltransferase activity from the cloning paper.
    action: ACCEPT
    reason: >-
      Core molecular function; consistent with all other lines of evidence.
- term:
    id: GO:0016453
    label: C-acetyltransferase activity
  evidence_type: IDA
  original_reference_id: PMID:17371050
  qualifier: enables
  review:
    summary: >-
      Direct-assay annotation to the more general parent term C-acetyltransferase activity. Correct
      but less informative than the specific child GO:0003985.
    action: KEEP_AS_NON_CORE
    reason: >-
      Correct parent term; the specific acetyl-CoA C-acetyltransferase activity (GO:0003985) is the
      preferred core annotation.
- term:
    id: GO:0016746
    label: acyltransferase activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  qualifier: enables
  review:
    summary: >-
      InterPro-based assignment of the broad acyltransferase activity parent term (thiolase domain).
    action: KEEP_AS_NON_CORE
    reason: >-
      Technically correct but uninformative; superseded by the specific thiolase term.
- term:
    id: GO:0016747
    label: acyltransferase activity, transferring groups other than amino-acyl groups
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  qualifier: enables
  review:
    summary: >-
      InterPro-based assignment of an intermediate acyltransferase parent term based on the thiolase
      InterPro signatures.
    action: KEEP_AS_NON_CORE
    reason: >-
      Correct parent term; the specific acetyl-CoA C-acetyltransferase activity better captures the
      molecular function.
# ============================================================================
# MOLECULAR FUNCTION - COFACTOR / SUBSTRATE / GENERIC BINDING
# ============================================================================
- term:
    id: GO:0030955
    label: potassium ion binding
  evidence_type: IDA
  original_reference_id: PMID:17371050
  qualifier: enables
  review:
    summary: >-
      Direct annotation supported by the crystal structures, which resolve a bound potassium ion
      near the CoA-binding and catalytic sites; T2 is uniquely activated by K+ (not Na+).
    action: ACCEPT
    reason: >-
      Genuine, well-evidenced activator/cofactor binding that is a distinctive functional feature of
      T2, though it is supporting rather than the catalytic core function itself.
    supported_by:
    - reference_id: PMID:17371050
      supporting_text: >-
        The potassium ion is bound near the CoA binding site and the catalytic site.
      full_text_unavailable: true
- term:
    id: GO:0120225
    label: coenzyme A binding
  evidence_type: IEA
  original_reference_id: GO_REF:0000107
  qualifier: enables
  review:
    summary: >-
      Electronic (ortholog-transfer) annotation for coenzyme A binding. CoA is a substrate/cosubstrate
      of the thiolase reaction and the crystal structures define a CoA-binding site, so this is correct.
    action: KEEP_AS_NON_CORE
    reason: >-
      Correct substrate-binding annotation, but it is an intrinsic part of the catalytic mechanism
      already captured by the thiolase MF.
- term:
    id: GO:0019899
    label: enzyme binding
  evidence_type: IEA
  original_reference_id: GO_REF:0000107
  qualifier: enables
  review:
    summary: >-
      Generic "enzyme binding" transferred electronically from a rodent ortholog. Uninformative and
      not tied to any specific, verified interaction for human ACAT1.
    action: MARK_AS_OVER_ANNOTATED
    reason: >-
      Vague binding term with only ortholog-based electronic support; conveys no specific functional
      information.
- term:
    id: GO:0042802
    label: identical protein binding
  evidence_type: IEA
  original_reference_id: GO_REF:0000107
  qualifier: enables
  review:
    summary: >-
      Electronic annotation for self-association. ACAT1/T2 is a homotetramer, so self-interaction is
      real, but the generic term is uninformative and the catalytically relevant fact (homotetramer)
      is better captured by subunit/complex description.
    action: KEEP_AS_NON_CORE
    reason: >-
      Consistent with the homotetrameric quaternary structure but uninformative as a function term.
# ============================================================================
# MOLECULAR FUNCTION - WRONG GENE (SOAT1/SOAT2 NAME COLLISION) -> REMOVE
# ============================================================================
- term:
    id: GO:0034736
    label: cholesterol O-acyltransferase activity
  evidence_type: IDA
  original_reference_id: PMID:32944968
  qualifier: enables
  review:
    summary: >-
      This annotation mis-attributes a cholesterol-esterifying activity to the mitochondrial thiolase
      P24752. PMID:32944968 studies the ER acyl-CoA:cholesterol acyltransferase ("ACAT", EC 2.3.1.26),
      which is sterol O-acyltransferase SOAT1/SOAT2 - distinct genes that merely share the historical
      nickname "ACAT1/ACAT2". The paper's functional perturbation used "shRNAs targeting ACAT1 and
      ACAT2" (i.e., SOAT1/SOAT2), and describes the enzyme as ER-localized. The mitochondrial T2
      (EC 2.3.1.9) has no cholesterol O-acyltransferase activity.
    action: REMOVE
    reason: >-
      Wrong-gene mis-annotation arising from the ACAT1/SOAT1 name collision. Cholesterol esterification
      is an EC 2.3.1.26 activity of the ER enzyme SOAT1/SOAT2, not of the mitochondrial acetoacetyl-CoA
      thiolase P24752. This contradicts the established mitochondrial-matrix thiolase function and
      should not be propagated.
    supported_by:
    - reference_id: PMID:32944968
      supporting_text: >-
        25HC inhibits viral membrane fusion by activating the ER-localized acyl-CoA:cholesterol
        acyltransferase (ACAT) which leads to the depletion of accessible cholesterol from the plasma
        membrane.
    proposed_replacement_terms:
    - id: GO:0003985
      label: acetyl-CoA C-acetyltransferase activity
# ============================================================================
# CELLULAR COMPONENT - WRONG GENE (ER, SAME PMID) -> REMOVE
# ============================================================================
- term:
    id: GO:0005783
    label: endoplasmic reticulum
  evidence_type: IDA
  original_reference_id: PMID:32944968
  qualifier: is_active_in
  review:
    summary: >-
      ER localization derived from the same SOAT/cholesterol-acyltransferase study (PMID:32944968).
      The ER-localized enzyme in that paper is the cholesterol-esterifying SOAT1/SOAT2, not the
      mitochondrial matrix thiolase P24752. ACAT1/T2 is a soluble mitochondrial matrix protein.
    action: REMOVE
    reason: >-
      Wrong-gene mis-annotation (ACAT1/SOAT1 collision). The "is_active_in ER" claim belongs to the
      cholesterol acyltransferase SOAT1/SOAT2; the mitochondrial T2 acts in the mitochondrial matrix.
    supported_by:
    - reference_id: PMID:32944968
      supporting_text: >-
        25HC inhibits viral membrane fusion by activating the ER-localized acyl-CoA:cholesterol
        acyltransferase (ACAT) which leads to the depletion of accessible cholesterol from the plasma
        membrane.
    proposed_replacement_terms:
    - id: GO:0005759
      label: mitochondrial matrix
# ============================================================================
# CELLULAR COMPONENT - MITOCHONDRION / MITOCHONDRIAL MATRIX
# ============================================================================
- term:
    id: GO:0005739
    label: mitochondrion
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  qualifier: is_active_in
  review:
    summary: >-
      Phylogenetic localization to the mitochondrion. Correct; the more specific matrix term is
      preferred for the core localization.
    action: KEEP_AS_NON_CORE
    reason: >-
      Correct but less specific than mitochondrial matrix (GO:0005759).
- term:
    id: GO:0005739
    label: mitochondrion
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  qualifier: located_in
  review:
    summary: >-
      Electronic mitochondrial localization consistent with experimental evidence.
    action: KEEP_AS_NON_CORE
    reason: >-
      Correct but less specific than the matrix annotation.
- term:
    id: GO:0005739
    label: mitochondrion
  evidence_type: IDA
  original_reference_id: GO_REF:0000052
  qualifier: located_in
  review:
    summary: >-
      Immunofluorescence (HPA) mitochondrial localization. Correct localization.
    action: KEEP_AS_NON_CORE
    reason: >-
      Correct mitochondrial localization; matrix term is more specific.
- term:
    id: GO:0005739
    label: mitochondrion
  evidence_type: IDA
  original_reference_id: PMID:1979337
  qualifier: located_in
  review:
    summary: >-
      Direct localization from subcellular fractionation/pulse labeling in the original cloning paper,
      showing T2 protein resides in mitochondria. The more specific matrix term is preferred for the
      core localization, so this is kept as non-core for consistency with the other mitochondrion
      annotations.
    action: KEEP_AS_NON_CORE
    reason: >-
      Correct, experimentally supported mitochondrial localization, but less specific than the
      mitochondrial-matrix core annotation.
    supported_by:
    - reference_id: PMID:1979337
      supporting_text: >-
        Pulse labeling followed by subcellular fractionation revealed that the T2 proteins in the
        fibroblasts from these patients are present in the mitochondria.
- term:
    id: GO:0005739
    label: mitochondrion
  evidence_type: HTP
  original_reference_id: PMID:34800366
  qualifier: located_in
  review:
    summary: >-
      High-throughput quantitative mitochondrial-proteome localization. Consistent with the
      established mitochondrial localization of ACAT1.
    action: KEEP_AS_NON_CORE
    reason: >-
      Correct mitochondrial localization from proteomics; less specific than matrix.
- term:
    id: GO:0005759
    label: mitochondrial matrix
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  qualifier: located_in
  review:
    summary: >-
      Electronic assignment to the mitochondrial matrix, the correct compartment for this soluble
      homotetrameric thiolase.
    action: ACCEPT
    reason: >-
      Core cellular localization; concordant with Reactome curation and the soluble crystal structures.
- term:
    id: GO:0005759
    label: mitochondrial matrix
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-70844
  qualifier: located_in
  review:
    summary: >-
      Reactome localization of ACAT1 to the mitochondrial matrix in the isoleucine-catabolism reaction
      (2-methylacetoacetyl-CoA thiolysis).
    action: ACCEPT
    reason: >-
      Correct core localization from pathway curation.
- term:
    id: GO:0005759
    label: mitochondrial matrix
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-73916
  qualifier: located_in
  review:
    summary: >-
      Reactome localization to the matrix in the ketone-body condensation reaction
      (2 acetyl-CoA <=> acetoacetyl-CoA + CoA).
    action: ACCEPT
    reason: >-
      Correct core localization.
- term:
    id: GO:0005759
    label: mitochondrial matrix
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-74181
  qualifier: located_in
  review:
    summary: >-
      Reactome localization to the matrix in the ketolysis reaction (acetoacetyl-CoA + CoA <=> 2
      acetyl-CoA).
    action: ACCEPT
    reason: >-
      Correct core localization.
- term:
    id: GO:0005759
    label: mitochondrial matrix
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-9838035
  qualifier: located_in
  review:
    summary: >-
      Reactome matrix localization in the context of mitochondrial protein quality control (CLPXP
      substrate binding).
    action: ACCEPT
    reason: >-
      Correct localization; the matrix compartment is right even though the cited reaction concerns
      protease handling of matrix proteins.
- term:
    id: GO:0005759
    label: mitochondrial matrix
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-9838081
  qualifier: located_in
  review:
    summary: >-
      Reactome matrix localization in a LONP1-mediated matrix-protein degradation reaction.
    action: ACCEPT
    reason: >-
      Correct matrix localization.
- term:
    id: GO:0005759
    label: mitochondrial matrix
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-9838093
  qualifier: located_in
  review:
    summary: >-
      Reactome matrix localization in a LONP1 matrix-protein-binding reaction.
    action: ACCEPT
    reason: >-
      Correct matrix localization.
- term:
    id: GO:0005759
    label: mitochondrial matrix
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-9838289
  qualifier: located_in
  review:
    summary: >-
      Reactome matrix localization in a CLPXP matrix-protein-degradation reaction.
    action: ACCEPT
    reason: >-
      Correct matrix localization.
- term:
    id: GO:0005759
    label: mitochondrial matrix
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-9854415
  qualifier: located_in
  review:
    summary: >-
      Reactome matrix localization in the reaction "ACAT1 tetramer acetylates IDH2 dimer". This
      reflects a genuine moonlighting protein-lysine-acetyltransferase activity of the ACAT1
      homotetramer reported in cancer metabolism (acetylation of IDH2 at K413 / PDH components);
      here it is used only as a matrix-localization annotation.
    action: ACCEPT
    reason: >-
      Correct matrix localization. The associated acetyltransferase moonlighting activity is noted in
      proposed_new_terms/suggested_questions but is not part of the curated GOA MF set under review.
# ============================================================================
# CELLULAR COMPONENT - EXTRACELLULAR EXOSOME (NON-CORE)
# ============================================================================
- term:
    id: GO:0070062
    label: extracellular exosome
  evidence_type: HDA
  original_reference_id: PMID:23533145
  qualifier: located_in
  review:
    summary: >-
      High-throughput detection of ACAT1 in prostatic-secretion urinary exosome proteomics. This is a
      common finding for abundant mitochondrial/metabolic proteins in vesicle proteomes and does not
      reflect the functional site of the enzyme.
    action: KEEP_AS_NON_CORE
    reason: >-
      Proteomic vesicle-detection localization, not the functional mitochondrial-matrix site.
- term:
    id: GO:0070062
    label: extracellular exosome
  evidence_type: HDA
  original_reference_id: PMID:19056867
  qualifier: located_in
  review:
    summary: >-
      High-throughput detection of ACAT1 in urinary exosome proteomics. As above, not a functional
      localization.
    action: KEEP_AS_NON_CORE
    reason: >-
      Proteomic detection in secreted vesicles; non-core.
# ============================================================================
# BIOLOGICAL PROCESS - KETONE BODY METABOLISM (CORE)
# ============================================================================
- term:
    id: GO:0046952
    label: ketone body catabolic process
  evidence_type: IMP
  original_reference_id: PMID:1979337
  qualifier: involved_in
  review:
    summary: >-
      IMP annotation linking ACAT1 to ketone body catabolism (ketolysis), via patient fibroblast
      analysis in 3-ketothiolase deficiency. T2 catalyzes acetoacetyl-CoA + CoA <=> 2 acetyl-CoA, the
      key ketolytic thiolase step.
    action: ACCEPT
    reason: >-
      Core biological process; ketone body metabolism is a principal physiological role of T2.
    supported_by:
    - reference_id: PMID:17371050
      supporting_text: >-
        important in the pathways for the synthesis and degradation of ketone bodies
      full_text_unavailable: true
- term:
    id: GO:0006085
    label: acetyl-CoA biosynthetic process
  evidence_type: IDA
  original_reference_id: PMID:17371050
  qualifier: involved_in
  review:
    summary: >-
      Curator (BHF-UCL) IDA annotation reflecting that thiolytic cleavage of acetoacetyl-CoA yields
      acetyl-CoA. This is a reaction-level restatement of the thiolase activity rather than the
      gene's physiological process.
    action: KEEP_AS_NON_CORE
    reason: >-
      Chemically defensible product of the reaction, but the physiological process core is ketone body
      metabolism / isoleucine catabolism, not generic acetyl-CoA biosynthesis.
- term:
    id: GO:0046356
    label: acetyl-CoA catabolic process
  evidence_type: IDA
  original_reference_id: PMID:17371050
  qualifier: involved_in
  review:
    summary: >-
      BHF-UCL IDA annotation reflecting the condensation direction (consumption of acetyl-CoA to form
      acetoacetyl-CoA). Reaction-level restatement of the reversible thiolase chemistry.
    action: KEEP_AS_NON_CORE
    reason: >-
      Defensible at the reaction level; the physiological BP core is ketone body metabolism.
# ============================================================================
# BIOLOGICAL PROCESS - L-ISOLEUCINE CATABOLISM (CORE)
# ============================================================================
- term:
    id: GO:0006550
    label: L-isoleucine catabolic process
  evidence_type: TAS
  original_reference_id: PMID:17371050
  qualifier: involved_in
  review:
    summary: >-
      TAS annotation placing ACAT1 in isoleucine catabolism, via its unique ability to cleave the
      2-methyl-branched intermediate 2-methylacetoacetyl-CoA - a defining property of T2.
    action: ACCEPT
    reason: >-
      Core biological process. The 2-methyl-branched substrate specificity is a distinctive feature of
      T2 and the basis of beta-ketothiolase deficiency as an isoleucine-catabolism disorder.
    supported_by:
    - reference_id: PMID:17371050
      supporting_text: >-
        A unique property of T2 is its ability to use 2-methyl-branched acetoacetyl-CoA as a substrate
      full_text_unavailable: true
- term:
    id: GO:0006550
    label: L-isoleucine catabolic process
  evidence_type: IMP
  original_reference_id: PMID:1979337
  qualifier: involved_in
  review:
    summary: >-
      IMP annotation from patient-fibroblast analysis in 3-ketothiolase deficiency, an inborn error of
      isoleucine catabolism. Supports the isoleucine-catabolism role.
    action: ACCEPT
    reason: >-
      Core biological process supported by the disease phenotype.
- term:
    id: GO:0006550
    label: L-isoleucine catabolic process
  evidence_type: IMP
  original_reference_id: PMID:8103405
  qualifier: involved_in
  review:
    summary: >-
      IMP annotation from molecular study of the two original 2-methylacetoacetic-aciduria families
      with T2 deficiency, consistent with the isoleucine-catabolism role.
    action: ACCEPT
    reason: >-
      Core biological process; redundant but correctly assigned.
    supported_by:
    - reference_id: PMID:8103405
      supporting_text: >-
        first reported with 2-methylacetoacetic aciduria, and shown later to have a deficiency of the
        K(+)-activated enzyme, mitochondrial acetoacetyl-coenzyme A thiolase (T2)
      full_text_unavailable: true
# ============================================================================
# BIOLOGICAL PROCESS - CoA METABOLISM / PROPIONYL-CoA (NON-CORE, REACTION-LEVEL)
# ============================================================================
- term:
    id: GO:0015936
    label: coenzyme A metabolic process
  evidence_type: IDA
  original_reference_id: PMID:17371050
  qualifier: involved_in
  review:
    summary: >-
      BHF-UCL IDA annotation reflecting that CoA is consumed/released in the thiolase reaction.
      Reaction-level rather than a distinct physiological process.
    action: KEEP_AS_NON_CORE
    reason: >-
      CoA is intrinsic to the catalytic mechanism; not an independent core process.
- term:
    id: GO:0015937
    label: coenzyme A biosynthetic process
  evidence_type: IDA
  original_reference_id: PMID:17371050
  qualifier: involved_in
  review:
    summary: >-
      BHF-UCL IDA annotation reflecting CoA release during thiolytic cleavage. This is not genuine CoA
      biosynthesis (the de novo CoA biosynthetic pathway is unrelated); it is a reaction-level artifact.
    action: MARK_AS_OVER_ANNOTATED
    reason: >-
      ACAT1 does not participate in coenzyme A biosynthesis; releasing free CoA as a thiolase product is
      not the biosynthetic pathway. Over-annotation.
- term:
    id: GO:1902860
    label: propionyl-CoA biosynthetic process
  evidence_type: IDA
  original_reference_id: PMID:17371050
  qualifier: involved_in
  review:
    summary: >-
      BHF-UCL IDA annotation reflecting that thiolysis of 2-methylacetoacetyl-CoA in the isoleucine
      pathway yields propionyl-CoA (plus acetyl-CoA). This is a product of the isoleucine-catabolism
      step rather than a separate core process.
    action: KEEP_AS_NON_CORE
    reason: >-
      Defensible as a product of the isoleucine-catabolism reaction; the physiological BP is better
      captured by L-isoleucine catabolic process.
# ============================================================================
# BIOLOGICAL PROCESS - FATTY ACID BETA-OXIDATION (NON-CORE)
# ============================================================================
- term:
    id: GO:0006635
    label: fatty acid beta-oxidation
  evidence_type: IEA
  original_reference_id: GO_REF:0000041
  qualifier: involved_in
  review:
    summary: >-
      UniPathway-derived electronic annotation to fatty acid beta-oxidation. UniProt lists T2 under
      this pathway as a thiolase, but the unbranched long-/medium-chain 3-oxoacyl-CoA thiolysis of
      classic beta-oxidation is principally performed by ACAA2 and the MTP (HADHA/HADHB); ACAT1/T2's
      dominant roles are the acetoacetyl-CoA (ketone body) node and the 2-methyl-branched isoleucine
      intermediate.
    action: KEEP_AS_NON_CORE
    reason: >-
      Defensible at the pathway-parent level but not the core physiological role of this thiolase.
# ============================================================================
# BIOLOGICAL PROCESS - ORTHOLOG-TRANSFER TISSUE/PHYSIOLOGY TERMS (OVER-ANNOTATION)
# ============================================================================
- term:
    id: GO:0001889
    label: liver development
  evidence_type: IEA
  original_reference_id: GO_REF:0000107
  qualifier: involved_in
  review:
    summary: >-
      Electronic ortholog-transfer (Ensembl Compara) annotation. ACAT1 is liver-enhanced in expression,
      but there is no evidence it has a developmental role in the liver; this reflects expression context
      rather than function.
    action: MARK_AS_OVER_ANNOTATED
    reason: >-
      Ortholog-based IEA with no mechanistic link to liver development for a metabolic thiolase.
- term:
    id: GO:0009725
    label: response to hormone
  evidence_type: IEA
  original_reference_id: GO_REF:0000107
  qualifier: involved_in
  review:
    summary: >-
      Electronic ortholog-transfer annotation. Generic stimulus-response term without specific evidence
      for human ACAT1.
    action: MARK_AS_OVER_ANNOTATED
    reason: >-
      Vague, ortholog-based; not a core function.
- term:
    id: GO:0042594
    label: response to starvation
  evidence_type: IEA
  original_reference_id: GO_REF:0000107
  qualifier: involved_in
  review:
    summary: >-
      Electronic ortholog-transfer annotation. Plausible in spirit (ketogenesis is induced by fasting and
      T2 contributes to ketone body metabolism), but the specific "response to starvation" process is an
      indirect ortholog inference rather than a demonstrated role of ACAT1.
    action: KEEP_AS_NON_CORE
    reason: >-
      Biologically plausible via ketogenesis but only electronically inferred from orthologs; non-core.
- term:
    id: GO:0060612
    label: adipose tissue development
  evidence_type: IEA
  original_reference_id: GO_REF:0000107
  qualifier: involved_in
  review:
    summary: >-
      Electronic ortholog-transfer annotation. No evidence for a developmental role of ACAT1 in adipose
      tissue; reflects expression/physiology context.
    action: MARK_AS_OVER_ANNOTATED
    reason: >-
      Ortholog-based IEA with no mechanistic support for a developmental function.
- term:
    id: GO:0072229
    label: metanephric proximal convoluted tubule development
  evidence_type: IEA
  original_reference_id: GO_REF:0000107
  qualifier: involved_in
  review:
    summary: >-
      Highly specific developmental term transferred electronically from a rodent ortholog. ACAT1 is
      kidney-enhanced in expression, but there is no evidence for a direct role in nephron development.
    action: MARK_AS_OVER_ANNOTATED
    reason: >-
      Over-specific ortholog-based IEA with no mechanistic basis for this metabolic enzyme.
references:
- id: GO_REF:0000002
  title: Gene Ontology annotation through association of InterPro records with GO terms
  findings: []
- id: GO_REF:0000033
  title: Annotation inferences using phylogenetic trees
  findings: []
- id: GO_REF:0000041
  title: Gene Ontology annotation based on UniPathway vocabulary mapping
  findings: []
- id: GO_REF:0000052
  title: Gene Ontology annotation based on curation of immunofluorescence data
  findings: []
- id: GO_REF:0000107
  title: Automatic transfer of experimentally verified manual GO annotation data to orthologs using
    Ensembl Compara
  findings: []
- id: GO_REF:0000120
  title: Combined Automated Annotation using Multiple IEA Methods
  findings: []
- id: PMID:1715688
  title: Evidence for a structural mutation (347Ala to Thr) in a German family with 3-ketothiolase
    deficiency.
  findings:
  - statement: >-
      Characterized a 3KTD missense allele (A380T / "347Ala to Thr" in the mature subunit) shown by
      expression analysis to destabilize the T2 protein, linking EC 2.3.1.9 thiolase activity to ACAT1.
    reference_section_type: ABSTRACT
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      PubMed-verified disease-variant study of mitochondrial acetoacetyl-CoA thiolase (T2). Abstract-only
      in cache; supports the thiolase MF and disease association.
- id: PMID:17371050
  title: 'Crystallographic and kinetic studies of human mitochondrial acetoacetyl-CoA thiolase: the
    importance of potassium and chloride ions for its structure and function.'
  findings:
  - statement: >-
      High-resolution crystal structures of human T2 (apo and CoA complex, with/without K+). Defines the
      homotetramer, the CoA- and K+-binding sites near the catalytic site, and K+ activation.
    reference_section_type: ABSTRACT
  - statement: >-
      T2 is important for synthesis and degradation of ketone bodies and for degradation of
      2-methylacetoacetyl-CoA; it uniquely accepts 2-methyl-branched acetoacetyl-CoA and degrades
      acetoacetyl-CoA and 2-methylacetoacetyl-CoA with similar catalytic efficiencies.
    reference_section_type: ABSTRACT
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      PubMed-verified primary structural/kinetic study of human ACAT1/T2. Abstract-only in cache
      (full_text_available: false); the abstract directly supports MF, K+ binding, ketone body and
      isoleucine roles.
- id: PMID:19056867
  title: Large-scale proteomics and phosphoproteomics of urinary exosomes.
  findings:
  - statement: >-
      Proteomic profiling of human urinary exosomes; basis for the extracellular-exosome localization
      annotation. Not a functional localization for the mitochondrial thiolase.
    reference_section_type: ABSTRACT
  reference_review:
    relevance: LOW
    correctness: VERIFIED
    review_notes: >-
      High-throughput vesicle proteomics; supports only an incidental exosome detection.
- id: PMID:1979337
  title: Molecular cloning and sequence of the complementary DNA encoding human mitochondrial
    acetoacetyl-coenzyme A thiolase and study of the variant enzymes in cultured fibroblasts from
    patients with 3-ketothiolase deficiency.
  findings:
  - statement: >-
      Cloned/sequenced the 427-aa human T2 precursor (33-residue leader + mature subunit) and showed by
      subcellular fractionation that T2 protein localizes to mitochondria in patient fibroblasts.
    reference_section_type: ABSTRACT
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      PubMed-verified original cloning + localization study; supports mitochondrial localization and the
      thiolase function via 3KTD patient analysis.
- id: PMID:23533145
  title: In-depth proteomic analyses of exosomes isolated from expressed prostatic secretions in urine.
  findings:
  - statement: >-
      Proteomic analysis of prostatic-secretion urinary exosomes; basis for an extracellular-exosome
      localization annotation. Incidental, not functional.
    reference_section_type: ABSTRACT
  reference_review:
    relevance: LOW
    correctness: VERIFIED
    review_notes: >-
      High-throughput vesicle proteomics; supports only incidental exosome detection.
- id: PMID:32944968
  title: Cholesterol 25-Hydroxylase inhibits SARS-CoV-2 and other coronaviruses by depleting membrane
    cholesterol.
  findings:
  - statement: >-
      Studies CH25H/25HC activating the ER acyl-CoA:cholesterol acyltransferase ("ACAT", EC 2.3.1.26) -
      i.e. sterol O-acyltransferase SOAT1/SOAT2 - to deplete plasma-membrane cholesterol and block
      coronavirus fusion. Functional perturbation used shRNAs targeting "ACAT1 and ACAT2" (SOAT1/SOAT2).
    reference_section_type: ABSTRACT
  reference_review:
    relevance: NONE
    correctness: MISCITED
    review_notes: >-
      The "ACAT" in this paper is the ER cholesterol-esterifying enzyme SOAT1/SOAT2 (EC 2.3.1.26), not the
      mitochondrial acetoacetyl-CoA thiolase P24752 (EC 2.3.1.9). The cholesterol O-acyltransferase
      (GO:0034736) and endoplasmic reticulum (GO:0005783) annotations placed on P24752 from this paper are
      wrong-gene mis-attributions caused by the ACAT1/SOAT1 name collision; both are recommended for REMOVE.
- id: PMID:34800366
  title: Quantitative high-confidence human mitochondrial proteome and its dynamics in cellular context.
  findings:
  - statement: >-
      High-throughput quantitative mitochondrial proteome; supports mitochondrial localization of ACAT1.
    reference_section_type: ABSTRACT
  reference_review:
    relevance: MEDIUM
    correctness: VERIFIED
    review_notes: >-
      Confirms mitochondrial localization by high-throughput proteomics.
- id: PMID:7728148
  title: Molecular, biochemical, and clinical characterization of mitochondrial acetoacetyl-coenzyme A
    thiolase deficiency in two further patients.
  findings:
  - statement: >-
      Characterized 3KTD alleles (N158D, T297M, A301P); expression analyses showed reduced/abolished T2
      activity, with only T297M retaining detectable residual activity.
    reference_section_type: ABSTRACT
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      PubMed-verified disease-variant characterization; supports thiolase MF and isoleucine/ketone-body
      catabolism roles. Abstract-only in cache.
- id: PMID:8103405
  title: Molecular studies of mitochondrial acetoacetyl-coenzyme A thiolase deficiency in the two original
    families.
  findings:
  - statement: >-
      Molecular characterization of the two original 2-methylacetoacetic-aciduria probands, confirmed as
      deficient in the K+-activated mitochondrial acetoacetyl-CoA thiolase (T2); identified inactivating
      alleles.
    reference_section_type: ABSTRACT
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      PubMed-verified; supports MF and the isoleucine/ketone-body catabolism role via disease. Abstract-only.
- id: PMID:9744475
  title: Characterization of N93S, I312T, and A333P missense mutations in two Japanese families with
    mitochondrial acetoacetyl-CoA thiolase deficiency.
  findings:
  - statement: >-
      Characterized 3KTD missense alleles (N93S, I312T, A333P) with reduced residual thiolase activity;
      states explicitly that T2 deficiency is an inborn error of ketone body and isoleucine catabolisms.
    reference_section_type: ABSTRACT
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      PubMed-verified disease-variant study; directly supports the ketone body + isoleucine catabolism
      framing. Abstract-only in cache.
- id: PMID:27867011
  title: Tetrameric Acetyl-CoA Acetyltransferase 1 Is Important for Tumor Growth.
  findings:
  - statement: >-
      Reports that the tetrameric form of mitochondrial ACAT1 acts as a protein-lysine acetyltransferase,
      acetylating and inhibiting pyruvate dehydrogenase (PDHA1) and PDH phosphatase (PDP1), and that
      tetrameric ACAT1 promotes tumor growth. Basis for the moonlighting acetyltransferase activity noted
      in proposed_new_terms.
    supporting_text: >-
      We identified mitochondrial acetyl-CoA acetyltransferase 1 (ACAT1) and SIRT3 as upstream
      acetyltransferase and deacetylase, respectively, of PDHA1 and PDP1
  reference_review:
    relevance: MEDIUM
    correctness: VERIFIED
    review_notes: >-
      Now fetched into the publications cache and verified against full text: the paper identifies ACAT1
      as the upstream acetyltransferase of PDHA1 and PDP1, confirming the moonlighting protein-lysine
      acetyltransferase activity (Mol Cell 2016, Fan et al.).
- id: Reactome:R-HSA-70844
  title: alpha-methylacetoacetyl-CoA + CoA => propionyl-CoA + acetyl-CoA
  findings:
  - statement: >-
      Reactome reaction for the final thiolytic step of isoleucine catabolism catalyzed by ACAT1 in the
      mitochondrial matrix; supports the matrix localization and isoleucine role.
- id: Reactome:R-HSA-73916
  title: 2 acetyl-CoA <=> acetoacetyl-CoA + CoA
  findings:
  - statement: >-
      Reactome reaction for the ketone-body condensation direction catalyzed by ACAT1; supports matrix
      localization and ketone body metabolism.
- id: Reactome:R-HSA-74181
  title: acetoacetyl-CoA + CoA <=> 2 acetyl-CoA
  findings:
  - statement: >-
      Reactome reaction for the ketolytic thiolysis direction catalyzed by ACAT1; supports matrix
      localization and ketone body catabolism.
- id: Reactome:R-HSA-9838035
  title: CLPXP binds mitochondrial matrix proteins
  findings:
  - statement: >-
      Reactome quality-control reaction in which ACAT1, as a matrix protein, is a CLPXP substrate;
      supports mitochondrial-matrix localization.
- id: Reactome:R-HSA-9838081
  title: LONP1 degrades mitochondrial matrix proteins
  findings:
  - statement: >-
      Reactome quality-control reaction; ACAT1 is annotated as a matrix protein substrate, supporting
      matrix localization.
- id: Reactome:R-HSA-9838093
  title: LONP1 binds mitochondrial matrix proteins
  findings:
  - statement: >-
      Reactome quality-control reaction; supports ACAT1 matrix localization.
- id: Reactome:R-HSA-9838289
  title: CLPXP degrades mitochondrial matrix proteins
  findings:
  - statement: >-
      Reactome quality-control reaction; supports ACAT1 matrix localization.
- id: Reactome:R-HSA-9854415
  title: ACAT1 tetramer acetylates IDH2 dimer
  findings:
  - statement: >-
      Reactome reaction capturing the moonlighting protein-lysine-acetyltransferase activity of the ACAT1
      homotetramer (acetylation of IDH2 at K413), localized to the mitochondrial matrix. Used here as a
      matrix-localization annotation; the acetyltransferase activity itself is not in the curated GOA MF
      set under review.
core_functions:
- description: >-
    Potassium-activated mitochondrial-matrix acetoacetyl-CoA thiolase (EC 2.3.1.9) that catalyzes the
    reversible thiolytic cleavage/condensation at the acetoacetyl-CoA node (acetoacetyl-CoA + CoA <-> 2
    acetyl-CoA), central to ketone body metabolism (ketogenesis and ketolysis).
  supported_by:
  - reference_id: PMID:17371050
    supporting_text: >-
      important in the pathways for the synthesis and degradation of ketone bodies
    full_text_unavailable: true
  - reference_id: PMID:9744475
    supporting_text: >-
      Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency is an inborn error of ketone body and
      isoleucine catabolisms.
    full_text_unavailable: true
  molecular_function:
    id: GO:0003985
    label: acetyl-CoA C-acetyltransferase activity
  directly_involved_in:
  - id: GO:0046952
    label: ketone body catabolic process
  locations:
  - id: GO:0005759
    label: mitochondrial matrix
- description: >-
    Catalyzes the final thiolytic step of L-isoleucine catabolism, cleaving the 2-methyl-branched
    intermediate 2-methylacetoacetyl-CoA into propionyl-CoA and acetyl-CoA - a substrate specificity
    unique among the structurally characterized thiolases. Loss of this activity causes beta-ketothiolase
    (3-ketothiolase) deficiency.
  supported_by:
  - reference_id: PMID:17371050
    supporting_text: >-
      A unique property of T2 is its ability to use 2-methyl-branched acetoacetyl-CoA as a substrate
    full_text_unavailable: true
  - reference_id: PMID:9744475
    supporting_text: >-
      Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency is an inborn error of ketone body and
      isoleucine catabolisms.
    full_text_unavailable: true
  molecular_function:
    id: GO:0003985
    label: acetyl-CoA C-acetyltransferase activity
  directly_involved_in:
  - id: GO:0006550
    label: L-isoleucine catabolic process
  locations:
  - id: GO:0005759
    label: mitochondrial matrix
proposed_new_terms:
- proposed_name: ACAT1 tetramer protein-lysine-N-acetyltransferase activity
  proposed_definition: >-
    A protein-lysine N6-acetyltransferase activity exerted by the tetrameric form of mitochondrial
    acetyl-CoA acetyltransferase 1 (ACAT1/T2), distinct from its acetoacetyl-CoA thiolase activity,
    that transfers an acetyl group from acetyl-CoA to specific lysine residues of mitochondrial enzymes
    (e.g. IDH2 K413 and pyruvate dehydrogenase components), thereby regulating their activity.
  justification: >-
    Tetrameric ACAT1 reportedly moonlights as a protein-lysine acetyltransferase that acetylates and
    inhibits IDH2 and PDH components (Fan et al., PMID:27867011; Reactome:R-HSA-9854415). This activity is
    distinct from the canonical thiolase MF and is not captured by any existing GOA molecular-function
    annotation for P24752. An existing term such as GO:0061733 (peptide-lysine-N-acetyltransferase
    activity) may already suffice; a dedicated term is proposed only if expert review of the full-text
    evidence concludes a more specific concept is warranted.
suggested_questions:
- question: >-
    Should the moonlighting protein-acetyltransferase activity of the ACAT1 homotetramer (acetylation of
    IDH2/PDH; PMID:27867011) be added as a separate molecular function annotation, distinct from the core
    thiolase activity?
- question: >-
    The cholesterol O-acyltransferase (GO:0034736) and endoplasmic reticulum (GO:0005783) annotations on
    P24752 derive from a study of the ER enzyme SOAT1/SOAT2. Can UniProt/GOA add a guard against the
    ACAT1/SOAT1 name-collision so such wrong-gene annotations are not propagated to the mitochondrial
    thiolase?
- question: >-
    Is the "fatty acid beta-oxidation" pathway annotation appropriate for ACAT1, given that unbranched
    long-chain 3-oxoacyl-CoA thiolysis is principally performed by ACAA2 and the MTP (HADHA/HADHB)?
suggested_experiments:
- description: >-
    Express recombinant human ACAT1/T2 and directly assay for cholesterol O-acyltransferase (sterol
    esterification, EC 2.3.1.26) activity alongside SOAT1 as a positive control.
  hypothesis: >-
    Recombinant mitochondrial ACAT1/T2 will show no cholesterol-esterifying activity, confirming that
    GO:0034736 is a wrong-gene mis-annotation belonging to SOAT1/SOAT2.
- description: >-
    Quantify ketone body and isoleucine-pathway flux (acetoacetyl-CoA turnover, 2-methylacetoacetyl-CoA
    cleavage, urinary 2-methyl-3-hydroxybutyrate/tiglylglycine) in ACAT1-knockout versus wild-type cells
    or patient fibroblasts.
  hypothesis: >-
    ACAT1 loss will impair both ketolysis and the final thiolytic step of isoleucine catabolism, producing
    the characteristic 2-methyl-branched metabolite accumulation of beta-ketothiolase deficiency.
- description: >-
    Reconstitute the ACAT1-tetramer-mediated acetylation of IDH2 in vitro and in cells, and test whether
    K+ activation and tetramer formation are required for the acetyltransferase (versus thiolase) activity.
  hypothesis: >-
    The protein-acetyltransferase moonlighting activity depends on the tetrameric assembly of ACAT1 and is
    mechanistically separable from its acetoacetyl-CoA thiolase activity.

ACAT1

Gene Description

Proposed New Ontology Terms

ACAT1 tetramer protein-lysine-N-acetyltransferase activity