HADHA encodes the alpha subunit of the mitochondrial trifunctional protein (MTP/TFP), a heterotetrameric complex (alpha2-beta2) with HADHB that is bound to the matrix face of the mitochondrial inner membrane and catalyzes the last three steps of long-chain fatty acid beta-oxidation. The alpha subunit carries TWO of these activities: long-chain enoyl-CoA hydratase (LCEH, EC 4.2.1.17), which hydrates 2-trans-enoyl-CoA to (3S)-3-hydroxyacyl-CoA, and long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD, EC 1.1.1.211), which oxidizes (3S)-3-hydroxyacyl-CoA to 3-oxoacyl-CoA using NAD+. The third step (thiolytic cleavage to acetyl-CoA) is carried by the beta subunit (HADHB). The enzyme is specific for long-chain (C10-C16) acyl-CoA substrates, including saturated and unsaturated species. Independently of the beta subunit, the alpha subunit also exhibits a monolysocardiolipin acyltransferase activity that participates in cardiolipin remodeling. Deficiency of HADHA causes isolated LCHAD deficiency and generalized mitochondrial trifunctional protein deficiency, presenting with hypoketotic hypoglycemia, cardiomyopathy, peripheral neuropathy and pigmentary retinopathy, and associated with maternal HELLP syndrome / acute fatty liver of pregnancy.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004300 enoyl-CoA hydratase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic (IBA) annotation assigning enoyl-CoA hydratase activity (EC 4.2.1.17) to HADHA. This is one of the two correct core catalytic activities of the alpha subunit: it hydrates 2-trans-enoyl-CoA to (3S)-3-hydroxyacyl-CoA in step 2 of each beta-oxidation cycle. Directly demonstrated for the human alpha subunit by expression studies (PMID:8135828) and confirmed structurally (PMID:30850536). Reason: Core molecular function of HADHA, well supported by direct expression and structural evidence. Supporting Evidence: PMID:8135828 Expression of this cDNA in mammalian cells yielded a polypeptide with the long-chain enoyl-CoA hydratase and long-chain 3-hydroxyacyl-CoA dehydrogenase activities. |
| GO:0004300 enoyl-CoA hydratase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic (IEA) annotation of enoyl-CoA hydratase activity based on InterPro/EC mapping. Consistent with the IBA and direct experimental evidence. Reason: Redundant with the experimental annotation but correctly captures the core hydratase function of the alpha subunit. |
| GO:0004300 enoyl-CoA hydratase activity | EXP PMID:1550553 Human liver long-chain 3-hydroxyacyl-coenzyme A dehydrogenas... | ACCEPT | Summary: Experimental annotation of enoyl-CoA hydratase activity. PMID:1550553 purified the human liver multifunctional enzyme and showed it possesses 2-enoyl-CoA hydratase activity in addition to dehydrogenase and thiolase. Although this early paper did not separate subunit-specific activities, PMID:8135828 subsequently localized the hydratase to the alpha subunit (HADHA). Reason: Core molecular function with direct experimental support; subunit assignment confirmed by PMID:8135828. Supporting Evidence: PMID:1550553 the enzyme possesses 2-enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase activities which cannot be separated from the dehydrogenase. |
| GO:0004300 enoyl-CoA hydratase activity | TAS PMID:8135828 Structural analysis of cDNAs for subunits of human mitochond... | ACCEPT | Summary: TAS annotation of enoyl-CoA hydratase activity citing PMID:8135828, the paper that definitively showed expression of the alpha-subunit cDNA yields hydratase (and dehydrogenase) activity. Reason: Core molecular function with strong direct evidence assigning the hydratase to the alpha subunit specifically. Supporting Evidence: PMID:8135828 Expression of this cDNA in mammalian cells yielded a polypeptide with the long-chain enoyl-CoA hydratase and long-chain 3-hydroxyacyl-CoA dehydrogenase activities. |
| GO:0016509 long-chain (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic (IBA) annotation assigning long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD, EC 1.1.1.211) activity to HADHA. This is the second core catalytic activity of the alpha subunit (step 3 of beta-oxidation, oxidation of (3S)-3-hydroxyacyl-CoA to 3-oxoacyl-CoA using NAD+). The long-chain (rather than generic) term correctly reflects the C10-C16 substrate specificity of the enzyme. Reason: Core molecular function of HADHA, supported by direct expression studies and long-chain substrate specificity data. Supporting Evidence: PMID:8135828 Expression of this cDNA in mammalian cells yielded a polypeptide with the long-chain enoyl-CoA hydratase and long-chain 3-hydroxyacyl-CoA dehydrogenase activities. |
| GO:0016509 long-chain (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic (IEA) annotation of long-chain 3-hydroxyacyl-CoA dehydrogenase activity based on RHEA/EC mapping. Consistent with the IBA and direct experimental evidence. Reason: Redundant with experimental annotations but correctly captures the core dehydrogenase function with the appropriate long-chain specificity. |
| GO:0016509 long-chain (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity | EXP PMID:1550553 Human liver long-chain 3-hydroxyacyl-coenzyme A dehydrogenas... | ACCEPT | Summary: Experimental annotation. PMID:1550553 purified the long-chain-specific 3-hydroxyacyl-CoA dehydrogenase from human liver mitochondrial membranes and showed maximum activity with C10-C16 substrates and inactivity with acetoacetyl-CoA, defining the long-chain specificity that distinguishes HADHA from the short-chain HADH. Reason: Core molecular function with direct experimental support and explicit long-chain specificity. Supporting Evidence: PMID:1550553 gives maximum activity with 3-ketoacyl-CoA substrates of C10 to C16 acyl-chain length but is inactive with acetoacetyl-CoA. |
| GO:0016509 long-chain (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity | EXP PMID:8135828 Structural analysis of cDNAs for subunits of human mitochond... | ACCEPT | Summary: Experimental annotation citing the definitive subunit-assignment paper. Expression of the alpha-subunit cDNA yielded long-chain 3-hydroxyacyl-CoA dehydrogenase activity. Reason: Core molecular function with strong direct evidence assigning the dehydrogenase to the alpha subunit. Supporting Evidence: PMID:8135828 Expression of this cDNA in mammalian cells yielded a polypeptide with the long-chain enoyl-CoA hydratase and long-chain 3-hydroxyacyl-CoA dehydrogenase activities. |
| GO:0016509 long-chain (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity | EXP PMID:8163672 Mitochondrial trifunctional protein deficiency. Catalytic he... | ACCEPT | Summary: Experimental annotation from the patient catalytic-heterogeneity study, which assayed the trifunctional protein's 3-hydroxyacyl-CoA dehydrogenase activity (with medium- to long-chain substrates) in fibroblasts from two LCHAD-deficiency patients. Reason: Supports the dehydrogenase activity of the alpha subunit; the long-chain specific assignment is consistent with the gene's role in LCHAD deficiency. Supporting Evidence: PMID:8163672 We examined the enzyme protein and biosynthesis of human trifunctional protein harboring enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase activity in cultured skin fibroblasts from two patients with long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency. |
| GO:0003857 (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity | IEA GO_REF:0000120 | MODIFY | Summary: Electronic annotation of the generic (chain-length-unspecified) 3-hydroxyacyl-CoA dehydrogenase activity. Correct in essence, but HADHA is long-chain specific, so the more specific term GO:0016509 better captures the molecular function. Reason: Generalize/replace with the long-chain-specific term GO:0016509, which is already present and is the appropriate level of specificity for HADHA (C10-C16 substrates). Proposed replacements: long-chain (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity |
| GO:0003857 (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity | TAS PMID:8135828 Structural analysis of cDNAs for subunits of human mitochond... | MODIFY | Summary: TAS annotation of the generic 3-hydroxyacyl-CoA dehydrogenase activity citing PMID:8135828. The activity is correct for HADHA, but the long-chain specific term GO:0016509 is preferred given the demonstrated C10-C16 specificity. Reason: Generalize/replace with the long-chain-specific term GO:0016509. Proposed replacements: long-chain (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity Supporting Evidence: PMID:8135828 Expression of this cDNA in mammalian cells yielded a polypeptide with the long-chain enoyl-CoA hydratase and long-chain 3-hydroxyacyl-CoA dehydrogenase activities. |
| GO:0018812 3-hydroxyacyl-CoA dehydratase activity | IEA GO_REF:0000116 | MODIFY | Summary: RHEA-based electronic annotation. GO:0018812 describes the same chemical reaction as enoyl-CoA hydratase (RHEA:16105, 3-hydroxyacyl-CoA <=> 2-enoyl-CoA + H2O) written in the dehydration (reverse) direction. This is the alpha subunit's hydratase activity captured under a reverse-direction label. Reason: The conventional molecular-function term for this reaction in HADHA is enoyl-CoA hydratase activity (GO:0004300, EC 4.2.1.17, physiological hydration direction); the dehydratase label is the reverse framing of the same reaction. Proposed replacements: enoyl-CoA hydratase activity |
| GO:0018812 3-hydroxyacyl-CoA dehydratase activity | EXP PMID:1550553 Human liver long-chain 3-hydroxyacyl-coenzyme A dehydrogenas... | MODIFY | Summary: Experimental annotation derived from the hydratase activity demonstrated in PMID:1550553. As above, this is the same reaction as enoyl-CoA hydratase written in the reverse direction. Reason: Replace with the conventional hydratase term GO:0004300 (EC 4.2.1.17). Proposed replacements: enoyl-CoA hydratase activity Supporting Evidence: PMID:1550553 the enzyme possesses 2-enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase activities which cannot be separated from the dehydrogenase. |
| GO:0018812 3-hydroxyacyl-CoA dehydratase activity | EXP PMID:8135828 Structural analysis of cDNAs for subunits of human mitochond... | MODIFY | Summary: Experimental annotation from the subunit-assignment paper. The alpha subunit's hydratase activity is captured here under the reverse-direction dehydratase label. Reason: Replace with the conventional hydratase term GO:0004300 (EC 4.2.1.17). Proposed replacements: enoyl-CoA hydratase activity Supporting Evidence: PMID:8135828 Expression of this cDNA in mammalian cells yielded a polypeptide with the long-chain enoyl-CoA hydratase and long-chain 3-hydroxyacyl-CoA dehydrogenase activities. |
| GO:0003985 acetyl-CoA C-acetyltransferase activity | TAS PMID:8135828 Structural analysis of cDNAs for subunits of human mitochond... | REMOVE | Summary: This annotation assigns thiolase/acetyltransferase activity to HADHA, but PMID:8135828 - the very paper cited - showed that the 3-ketoacyl-CoA thiolase activity is carried by the BETA subunit (HADHB), not the alpha subunit. Expression of the alpha-subunit cDNA yielded only hydratase and dehydrogenase activities; only the beta-subunit cDNA yielded thiolase activity. The crystal structure (PMID:30850536) likewise places the 3-ketothiolase active site in the beta subunit. Reason: Thiolase (acetyl-CoA C-acetyltransferase) activity belongs to HADHB, not HADHA, and is contradicted by the cited paper itself and by the structural data. This is an old PINC annotation that mis-attributed a beta-subunit activity to the alpha subunit. Supporting Evidence: PMID:8135828 Expression of this cDNA in mammalian cells yielded a polypeptide with the long-chain enoyl-CoA hydratase and long-chain 3-hydroxyacyl-CoA dehydrogenase activities. PMID:30850536 employing 2-enoyl-CoA hydratase (ECH), 3-hydroxyl-CoA dehydrogenase (HAD), and 3-ketothiolase (KT) activities consecutively. |
| GO:0052816 long-chain fatty acyl-CoA hydrolase activity | IDA PMID:22586271 Acyl coenzyme A thioesterase Them5/Acot15 is involved in car... | REMOVE | Summary: This IDA annotates HADHA with long-chain fatty acyl-CoA hydrolase (thioesterase) activity citing PMID:22586271. That paper (full text available) is a structural and functional study of the Hotdog-fold mitochondrial thioesterase Them5/Acot15 and its paralog Them4; it contains no mention of HADHA, the trifunctional protein, or P40939 anywhere in the abstract or full text. The thioesterase activity it describes belongs to Them5, not HADHA. HADHA is a hydratase/dehydrogenase, not an acyl-CoA thioesterase. Reason: Wrong-paper attribution. The cited reference does not assay HADHA and does not support a thioesterase/hydrolase activity for this protein; the activity is that of Them5/Acot15. Supporting Evidence: PMID:22586271 Here, we present a structural and functional analysis of a new mammalian mitochondrial thioesterase, Them5. |
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: Root-level catalytic activity term from InterPro mapping. Correct but entirely uninformative. Reason: Too general; the specific hydratase and dehydrogenase terms capture the molecular function. |
| GO:0016491 oxidoreductase activity | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: Broad oxidoreductase parent term from InterPro mapping, reflecting the dehydrogenase domain. Reason: Correct but too general; GO:0016509 is the appropriate specific term. |
| GO:0016616 oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: Intermediate-specificity parent of the 3-hydroxyacyl-CoA dehydrogenase activity, from InterPro mapping. Reason: Correct parent term; the specific child GO:0016509 should be used for core function. |
| GO:0070403 NAD+ binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: NAD+ binding from InterPro mapping, reflecting the NAD-binding Rossmann domain of the dehydrogenase. A cofactor-binding capability that supports but does not by itself describe the catalytic function. Reason: Correct molecular characteristic (the dehydrogenase uses NAD+), but subsidiary to the catalytic dehydrogenase activity. |
| GO:0005515 protein binding | IPI PMID:20562859 Network organization of the human autophagy system. | KEEP AS NON CORE | Summary: IPI from an autophagy-system network study (HADHA detected interacting with autophagy machinery, including GABARAP/ATG8 family members). Reason: Generic protein binding is uninformative about molecular function; per curation guidelines it is not endorsed as core. |
| GO:0005515 protein binding | IPI PMID:28514442 Architecture of the human interactome defines protein commun... | KEEP AS NON CORE | Summary: IPI based on interaction with HADHB (P55084), the obligate beta-subunit partner in the MTP complex. Reason: Reflects obligate complex formation, which is better captured by the complex-membership annotation GO:0016507; generic protein binding is uninformative. |
| GO:0005515 protein binding | IPI PMID:29915090 Cryo-EM structure of human mitochondrial trifunctional prote... | KEEP AS NON CORE | Summary: IPI from the cryo-EM structure study, reflecting HADHA-HADHB (P55084) interaction within the TFP tetramer. Reason: Obligate complex interaction better represented by GO:0016507. |
| GO:0005515 protein binding | IPI PMID:30850536 Crystal structure of human mitochondrial trifunctional prote... | KEEP AS NON CORE | Summary: IPI from the crystal structure study, reflecting HADHA-HADHB (P55084) interaction in the alpha2-beta2 complex. Reason: Obligate complex interaction better represented by GO:0016507. |
| GO:0005515 protein binding | IPI PMID:32814053 Interactome Mapping Provides a Network of Neurodegenerative ... | KEEP AS NON CORE | Summary: IPI from a neurodegenerative-disease interactome mapping study. Reason: Generic protein binding term; uninformative about molecular function. |
| GO:0005515 protein binding | IPI PMID:33961781 Dual proteome-scale networks reveal cell-specific remodeling... | KEEP AS NON CORE | Summary: IPI from a dual proteome-scale interactome study, again reflecting interaction with HADHB (P55084). Reason: Obligate complex interaction better represented by GO:0016507. |
| GO:0005515 protein binding | IPI PMID:40205054 Multimodal cell maps as a foundation for structural and func... | KEEP AS NON CORE | Summary: IPI from a multimodal cell-maps study, reflecting interaction with HADHB (P55084). Reason: Obligate complex interaction better represented by GO:0016507. |
| GO:0006635 fatty acid beta-oxidation | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic annotation placing HADHA in the fatty acid beta-oxidation pathway. The MTP complex, of which HADHA is the catalytic alpha subunit, performs the last three steps of long-chain fatty acid beta-oxidation. Reason: Core biological process, supported by direct functional, genetic and structural data. Supporting Evidence: PMID:29915090 The mitochondrial trifunctional protein (TFP) catalyzes three reactions in the fatty acid beta-oxidation process. |
| GO:0006635 fatty acid beta-oxidation | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic annotation of fatty acid beta-oxidation, consistent with IBA and direct evidence. Reason: Redundant but correctly captures the core biological process. |
| GO:0006635 fatty acid beta-oxidation | IDA PMID:29915090 Cryo-EM structure of human mitochondrial trifunctional prote... | ACCEPT | Summary: IDA (ComplexPortal/UniProt) from the cryo-EM structural characterization of the TFP complex in beta-oxidation. Reason: Core biological process with direct structural evidence for the functional beta-oxidation complex. Supporting Evidence: PMID:29915090 The mitochondrial trifunctional protein (TFP) catalyzes three reactions in the fatty acid beta-oxidation process. |
| GO:0006635 fatty acid beta-oxidation | IMP PMID:31604922 TFPa/HADHA is required for fatty acid beta-oxidation and car... | ACCEPT | Summary: IMP from HADHA-deficient human iPSC-derived cardiomyocytes, which demonstrated that HADHA is required for fatty acid beta-oxidation (loss of HADHA produced reduced FAO and a metabolic/electrophysiological disease phenotype). Reason: Core biological process supported by loss-of-function (mutant phenotype) evidence in a human cell model. Supporting Evidence: PMID:31604922 HADHA (tri-functional protein alpha), a monolysocardiolipin acyltransferase-like enzyme, is required for fatty acid beta-oxidation and cardiolipin remodeling, essential for functional mitochondria in human |
| GO:0035965 cardiolipin acyl-chain remodeling | IDA PMID:23152787 Human trifunctional protein alpha links cardiolipin remodeli... | ACCEPT | Summary: IDA showing that the alpha subunit of TFP has monolysocardiolipin acyltransferase activity that remodels cardiolipin. Purified recombinant alphaTFP acylated MLCL to CL with linoleoyl-, oleoyl- and palmitoyl-CoA, and its expression increased linoleate/oleate incorporation into CL in cells, linking beta-oxidation to cardiolipin remodeling. This is an activity of the alpha subunit independent of the beta subunit. Reason: Well-supported, alpha-subunit-specific moonlighting function in cardiolipin remodeling. Retained as a genuine (secondary) biological process for HADHA. Supporting Evidence: PMID:23152787 Purified human recombinant Ξ±TFP exhibited acyl-CoA acyltransferase activity in the acylation of MLCL to CL with linoleoyl-CoA, oleoyl-CoA and palmitoyl-CoA as substrates. |
| GO:0035965 cardiolipin acyl-chain remodeling | IMP PMID:31604922 TFPa/HADHA is required for fatty acid beta-oxidation and car... | ACCEPT | Summary: IMP from HADHA-deficient iPSC cardiomyocytes showing defective cardiolipin remodeling on loss of HADHA, corroborating the acyltransferase activity reported in PMID:23152787. Reason: Mutant-phenotype evidence supporting HADHA's role in cardiolipin remodeling. Supporting Evidence: PMID:31604922 HADHA (tri-functional protein alpha), a monolysocardiolipin acyltransferase-like enzyme, is required for fatty acid beta-oxidation and cardiolipin remodeling, essential for functional mitochondria in human |
| GO:0006631 fatty acid metabolic process | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: Broad parent term of fatty acid beta-oxidation from InterPro mapping. Reason: Correct but too general; GO:0006635 (fatty acid beta-oxidation) is the appropriate specific term. |
| GO:0005743 mitochondrial inner membrane | IEA GO_REF:0000044 | ACCEPT | Summary: Electronic annotation of mitochondrial inner membrane localization based on UniProt subcellular location. The MTP complex is membrane-bound on the matrix face of the inner membrane. Reason: Core cellular localization, supported by structural and direct evidence. |
| GO:0005743 mitochondrial inner membrane | IDA PMID:29915090 Cryo-EM structure of human mitochondrial trifunctional prote... | ACCEPT | Summary: IDA (ComplexPortal) from the cryo-EM study, which showed that a concave surface of the TFP tetramer associates with the membrane. Reason: Core cellular localization with direct structural evidence. Supporting Evidence: PMID:29915090 A concave surface of the TFP tetramer interacts with the detergent molecules in the structure, suggesting that this region is involved in associating with the membrane. |
| GO:0005743 mitochondrial inner membrane | TAS Reactome:R-HSA-1482775 | ACCEPT | Summary: TAS from Reactome (MLCL acylation to CL by HADH at the inner membrane). Correct localization. Reason: Correct inner membrane localization from pathway database curation. |
| GO:0005743 mitochondrial inner membrane | TAS Reactome:R-HSA-77271 | ACCEPT | Summary: TAS from Reactome for a beta-oxidation reaction at the mitochondrial inner membrane. Reason: Correct inner membrane localization for the beta-oxidation machinery. |
| GO:0005743 mitochondrial inner membrane | TAS Reactome:R-HSA-77277 | ACCEPT | Summary: TAS from Reactome for the enoyl-CoA hydratase reaction (HADHA's own activity) at the inner membrane. Reason: Correct inner membrane localization for the hydratase reaction carried by HADHA. |
| GO:0005743 mitochondrial inner membrane | TAS Reactome:R-HSA-77283 | ACCEPT | Summary: TAS from Reactome for the 3-hydroxyacyl-CoA dehydrogenase reaction (HADHA's own activity) at the inner membrane. Reason: Correct inner membrane localization for the dehydrogenase reaction carried by HADHA. |
| GO:0005743 mitochondrial inner membrane | TAS Reactome:R-HSA-77301 | ACCEPT | Summary: TAS from Reactome for a C16 hydratase reaction at the inner membrane. Reason: Correct inner membrane localization. |
| GO:0005743 mitochondrial inner membrane | TAS Reactome:R-HSA-77303 | ACCEPT | Summary: TAS from Reactome for a C16 dehydrogenase reaction at the inner membrane. Reason: Correct inner membrane localization. |
| GO:0005743 mitochondrial inner membrane | TAS Reactome:R-HSA-77304 | ACCEPT | Summary: TAS from Reactome for a thiolase reaction at the inner membrane. Localization is correct for HADHA via complex membership even though the thiolase step is catalyzed by HADHB. Reason: Correct inner membrane localization for the TFP complex. |
| GO:0005743 mitochondrial inner membrane | TAS Reactome:R-HSA-77309 | ACCEPT | Summary: TAS from Reactome for a beta-oxidation reaction at the inner membrane. Reason: Correct inner membrane localization. |
| GO:0005743 mitochondrial inner membrane | TAS Reactome:R-HSA-77321 | ACCEPT | Summary: TAS from Reactome for a beta-oxidation reaction at the inner membrane. Reason: Correct inner membrane localization. |
| GO:0005743 mitochondrial inner membrane | TAS Reactome:R-HSA-77329 | ACCEPT | Summary: TAS from Reactome for a beta-oxidation reaction at the inner membrane. Reason: Correct inner membrane localization. |
| GO:0005743 mitochondrial inner membrane | TAS Reactome:R-HSA-77340 | ACCEPT | Summary: TAS from Reactome for a beta-oxidation reaction at the inner membrane. Reason: Correct inner membrane localization. |
| GO:0016507 mitochondrial fatty acid beta-oxidation multienzyme complex | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic annotation placing HADHA as part of the mitochondrial TFP complex. HADHA is the alpha subunit of the alpha2-beta2 heterotetramer. Reason: Core cellular component; HADHA is an obligate subunit of the TFP complex whose structure has been determined by cryo-EM and X-ray crystallography. Supporting Evidence: PMID:30850536 The biological unit of the protein is Ξ±2Ξ²2 |
| GO:0016507 mitochondrial fatty acid beta-oxidation multienzyme complex | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro-based electronic annotation of TFP complex membership, consistent with IBA and structural evidence. Reason: Redundant but correctly captures core complex membership. |
| GO:0016507 mitochondrial fatty acid beta-oxidation multienzyme complex | IPI PMID:29915090 Cryo-EM structure of human mitochondrial trifunctional prote... | ACCEPT | Summary: IPI (ComplexPortal) from the cryo-EM structure directly demonstrating the alpha2-beta2 tetrameric complex. Reason: Core cellular component with direct structural evidence. Supporting Evidence: PMID:29915090 Here we report a 4.2-Γ
cryo-electron microscopy Ξ±2Ξ²2 tetrameric structure of the human TFP. |
| GO:0005739 mitochondrion | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: General mitochondrial localization from electronic mapping. Correct but less specific than the inner membrane annotation. Reason: Correct but subsumed by the more specific GO:0005743 / GO:0016507. |
| GO:0005739 mitochondrion | IDA GO_REF:0000052 | KEEP AS NON CORE | Summary: IDA from HPA immunofluorescence confirming mitochondrial localization. Reason: Correct but less specific than inner membrane localization. |
| GO:0005739 mitochondrion | EXP PMID:29915090 Cryo-EM structure of human mitochondrial trifunctional prote... | KEEP AS NON CORE | Summary: Experimental mitochondrial localization associated with the structural study. Reason: Correct but the more specific inner membrane / complex terms are preferred for core annotation. |
| GO:0005739 mitochondrion | IDA PMID:23152787 Human trifunctional protein alpha links cardiolipin remodeli... | KEEP AS NON CORE | Summary: IDA mitochondrial localization from the cardiolipin-remodeling study. Reason: Correct but less specific than inner membrane localization. |
| GO:0005739 mitochondrion | HTP PMID:34800366 Quantitative high-confidence human mitochondrial proteome an... | KEEP AS NON CORE | Summary: HTP mitochondrial localization from a quantitative mitochondrial proteome study. Reason: Correct mitochondrial localization from proteomics; less specific than inner membrane. |
| GO:0005739 mitochondrion | HDA PMID:20833797 Phosphoproteome analysis of functional mitochondria isolated... | KEEP AS NON CORE | Summary: HDA mitochondrial localization from a muscle mitochondrial phosphoproteome study. Reason: Correct mitochondrial localization; less specific than inner membrane. |
| GO:0042645 mitochondrial nucleoid | IDA PMID:18063578 The layered structure of human mitochondrial DNA nucleoids. | KEEP AS NON CORE | Summary: IDA from a mitochondrial nucleoid proteomics study. HADHA was identified in native nucleoid preparations, but such metabolic proteins were not observed to cross-link to mtDNA, indicating peripheral association rather than a core nucleoid role. Reason: Peripheral/co-purifying association, not a functional nucleoid component; the core localization is the inner membrane. Supporting Evidence: PMID:18063578 Several other metabolic proteins and chaperones identified in native nucleoids, including ATAD3, were not observed to cross-link to mtDNA. |
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Download this section (compressed HTML)Q: Is the monolysocardiolipin acyltransferase / cardiolipin remodeling activity of the alpha subunit a genuine independent physiological function in vivo, or a secondary moonlighting activity? Should it be modeled with a specific acyltransferase molecular-function term (EC 2.3.1.-) in addition to GO:0035965?
Q: Should the generic dehydrogenase (GO:0003857) and dehydratase (GO:0018812) annotations on HADHA be superseded by the long-chain-specific terms (GO:0016509, GO:0004300) to better reflect the demonstrated C10-C16 substrate specificity?
Experiment: Reconstitute recombinant HADHA alone and as the alpha2-beta2 complex with HADHB and assay enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities across a chain-length series (C4-C20) to quantitatively define substrate specificity.
Hypothesis: HADHA exhibits hydratase and dehydrogenase activity restricted to long-chain (>=C10) acyl-CoA substrates, distinguishing it from the short-chain enzymes ECHS1 and HADH.
Experiment: Test whether the MLCL acyltransferase / cardiolipin remodeling activity of HADHA can be genetically separated from its hydratase/dehydrogenase activities using active-site point mutants in HADHA-deficient cardiomyocytes.
Hypothesis: The cardiolipin acyltransferase activity is mechanistically distinct from the beta-oxidation catalytic sites and can be uncoupled, indicating a bona fide moonlighting function.
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