{"gene":"HADHA","run_date":"2026-06-10T01:55:21","timeline":{"discoveries":[{"year":2019,"finding":"HADHA (TFPα) is required for fatty acid beta-oxidation and cardiolipin remodeling in human cardiomyocytes; HADHA-deficient iPSC-derived cardiomyocytes showed defective calcium dynamics, repolarization kinetics, reduced mitochondrial proton gradient, disrupted cristae structure, and defective cardiolipin remodeling. HADHA was identified as a monolysocardiolipin acyltransferase-like enzyme essential for functional mitochondria.","method":"hiPSC-derived cardiomyocytes from HADHA-mutant cells, single-cell RNA-seq, functional metabolic assays (fatty acid beta-oxidation, mitochondrial proton gradient), cardiolipin profiling, engineered microRNA maturation cocktail","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (iPSC disease model, scRNA-seq, metabolic flux, cardiolipin profiling, calcium imaging) in a single rigorous study with direct functional readouts","pmids":["31604922"],"is_preprint":false},{"year":2018,"finding":"GCN5L1 acetylates HADHA at lysine residues K350, K383, and K406, and this hyperacetylation correlates with increased HADHA activity. SIRT3 opposes this by deacetylating HADHA. GCN5L1 knockdown reduces HADHA acetylation and increases fatty acid oxidation enzyme activities; liver-specific GCN5L1 knockout mice lack HADHA hyperacetylation and are protected from hepatic lipid accumulation on high-fat diet.","method":"Proteomic identification of acetylation sites, transgenic GCN5L1 overexpression mouse model, liver-specific GCN5L1 knockout mice, stable GCN5L1 knockdown in HepG2 cells, fatty acid oxidation enzyme activity assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — site-specific acetylation mapped by proteomics, validated in both cell knockdown and multiple mouse models with activity assays","pmids":["30323061"],"is_preprint":false},{"year":2022,"finding":"HADHA promotes ketone body (β-hydroxybutyrate, BHB) production via β-oxidation, and BHB suppresses hepatic gluconeogenesis by selectively inhibiting HDAC7 activity via interaction with HDAC7 Glu543, facilitating FOXO1 nuclear exclusion. Liver-specific HADHA overexpression reversed hepatic gluconeogenesis in mice; HADHA knockdown augmented glucagon response.","method":"Stable isotope tracing, liver-specific HADHA overexpression and knockdown mouse models, HDAC7 activity assays, FOXO1 localization studies, high-fat diet mouse model","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — stable isotope tracing, multiple in vivo genetic models, mechanistic pathway dissection with identified molecular interaction (BHB–HDAC7 Glu543)","pmids":["35046401"],"is_preprint":false},{"year":2022,"finding":"UBE2O (an E2 ubiquitin-conjugating enzyme) interacts with HADHA and mediates its ubiquitination and proteasomal degradation, thereby reducing HADHA protein levels and modulating lipid metabolic reprogramming in hepatocellular carcinoma.","method":"Co-immunoprecipitation, ubiquitination assays, UBE2O overexpression/knockdown in vitro and in vivo, liver-specific Ube2o knockout mice with DEN-induced hepatocarcinogenesis","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP establishing direct interaction, ubiquitination assay, in vivo genetic model confirming pathway","pmids":["36273042"],"is_preprint":false},{"year":2025,"finding":"HADHA is lactylated at K166 and K728 in septic heart tissue; lactylation at these sites inhibits HADHA activity, disturbs mitochondrial function, reduces ATP production, impairs energy metabolism, and reduces cardiomyocyte contraction force. SIRT1 and SIRT3 were identified as erasers of HADHA lactylation at these sites.","method":"Proteomic analysis of lactylation sites in septic rat heart tissues, LPS-induced cardiomyocyte model, K166/K728 site-directed mutagenesis, mitochondrial function assays, ATP measurements, transcriptomic and metabolomic analyses, in vivo cardiac function measurement","journal":"Circulation research","confidence":"High","confidence_rationale":"Tier 1 / Strong — site-directed mutagenesis at K166 and K728 with functional validation of enzymatic activity, mitochondrial function, and in vivo cardiac phenotype; multiple orthogonal methods","pmids":["40575877"],"is_preprint":false},{"year":2023,"finding":"Acetylation of HADHA at K255 (in obese mouse hearts, promoted by mitochondrial hyperacetylation) triggers mitochondrial localization of ASC and facilitates NLRP3 inflammasome assembly. Blockade of K255 acetylation suppressed the NLRP3 inflammasome and attenuated post-ischemia/reperfusion myocardial fibrosis in obese mice.","method":"High-fat diet mouse model, K255 acetylation site identification, site-specific blocking experiments, NLRP3 inflammasome assembly assays (ASC localization by imaging), post-I/R myocardial fibrosis assessment","journal":"Diabetes","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — K255 site identified and functionally blocked in vivo, NLRP3 assembly readout, single lab with two orthogonal approaches","pmids":["37625146"],"is_preprint":false},{"year":2023,"finding":"HADHA succinylation (induced by morphine) is reversed by the desuccinylase SIRT5, which selectively binds HADHA. SIRT5-mediated HADHA desuccinylation reduced P62 expression and alleviated morphine tolerance, linking HADHA succinylation to autophagy dysregulation.","method":"LC-MS/MS and parallel reaction monitoring for succinylation site mapping, SIRT5 binding assay, SIRT5 overexpression in intrathecal morphine rat model, P62/LC3 autophagy marker measurement","journal":"Naunyn-Schmiedeberg's archives of pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS-confirmed succinylation sites, SIRT5–HADHA binding and functional rescue, single lab","pmids":["37688624"],"is_preprint":false},{"year":2024,"finding":"KDM6B demethylates histone H3K27 at the HADHA locus to activate HADHA transcription during cementoblast mineralization. Additionally, lactylation of HADHA (at specific sites identified by lactylation proteomics) promotes FAO and mineralization; KDM6B regulates HADHA lactylation. Co-immunoprecipitation confirmed interaction between lactylated HADHA and its partners.","method":"ChIP-seq, RNA-seq, ChIP-qPCR, HADHA overexpression rescue experiments, lactylation proteomics, Co-IP, FAO activity assays, in vivo KDM6B inhibition in mice","journal":"Journal of dental research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP-seq and functional rescue establish KDM6B→HADHA transcriptional axis; lactylation sites mapped by proteomics with Co-IP validation, single lab","pmids":["39569625"],"is_preprint":false},{"year":2024,"finding":"HADHA participates in respiratory supercomplex (SC) assembly and couples FAO to OXPHOS. HADHA knockdown cells and HADHA-knockout MEFs displayed reduced SC assembly and defective OXPHOS. HADHA expression is upregulated when OXPHOS is stimulated (glucose-to-galactose switch) or lipid metabolism is induced (high-fat diet). HADHA heterozygous mice on HFD showed enhanced steatosis with reduced SC assembly and OXPHOS.","method":"Proteomics identifying HADHA as SC assembly factor, HADHA-KD cells and HADHA-KO MEFs with SC assembly assays (BN-PAGE), galactose medium OXPHOS stimulation, HFD mouse model with SC assembly analysis","journal":"Advanced science","confidence":"High","confidence_rationale":"Tier 2 / Strong — proteomics-identified candidate validated by KD, KO MEFs, and in vivo mouse model with orthogonal functional readouts (SC assembly, OXPHOS activity)","pmids":["39488787"],"is_preprint":false},{"year":2023,"finding":"Zfp335 transcription factor controls effector Treg (eTreg) differentiation by directly targeting the FAO enzyme Hadha to maintain fatty acid oxidation and oxidative phosphorylation in Tregs. Zfp335-deficient Tregs showed reduced HADHA expression, dysfunctional mitochondrial activity, and failed to differentiate into eTregs.","method":"Treg-specific Zfp335 knockout mice, scRNA-seq, chromatin immunoprecipitation (direct Hadha targeting), OXPHOS assays, Treg functional suppression assays","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct ChIP evidence of Zfp335 binding Hadha locus, KO mouse model with scRNA-seq and functional readouts, replicated in human eTreg correlation","pmids":["37843279"],"is_preprint":false},{"year":2017,"finding":"HADHA protein is present not only in mitochondria but also in the cytosol. HADHA was identified as an LC3-interacting protein in intestinal epithelial cells via immunoprecipitation with a GFP-LC3 antibody. LC3 puncta co-localized with HADHA (but not with the mitochondrial marker TOM20) and were enhanced by palmitic acid stimulation, suggesting HADHA has extra-mitochondrial roles in long-chain fatty acid-induced autophagy.","method":"GFP-LC3 immunoprecipitation followed by mass spectrometry, cellular fractionation, immunofluorescence co-localization (HADHA vs. LC3 vs. TOM20), palmitic acid treatment of IEC lines","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP/MS identification plus co-localization imaging, single lab, no functional mutagenesis or reconstitution","pmids":["28153718"],"is_preprint":false},{"year":2015,"finding":"HADHA associates with the human Dicer complex (RNA-induced silencing machinery). Immunoprecipitation showed HADHA co-precipitates with Dicer; HADHA overexpression increased mature miRNA levels with corresponding decrease in precursor miRNA, while HADHA knockdown had the opposite effect, suggesting an auxiliary role in miRNA biogenesis.","method":"Co-immunoprecipitation of HADHA with Dicer, immunohistochemical co-localization with Dicer in cytoplasm, HADHA overexpression/knockdown with miRNA precursor and mature miRNA quantification","journal":"Biochemical and biophysical research communications","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP and expression-level miRNA data, single lab, no mechanistic reconstitution or mutagenesis","pmids":["26367179"],"is_preprint":false},{"year":1997,"finding":"HADHA and HADHB genes (encoding the α and β subunits of the mitochondrial trifunctional protein) are both located on human chromosome band 2p23 and are in close proximity, analogous to the operon-like arrangement of bacterial fatty acid beta-oxidation multienzyme complex genes. The α subunit (HADHA) belongs to the enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase family.","method":"Fluorescence in situ hybridization (FISH) chromosomal mapping","journal":"Cytogenetics and cell genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct FISH localization establishing chromosomal co-localization; well-established physical mapping result","pmids":["9605857"],"is_preprint":false},{"year":2025,"finding":"HADHA deficiency impairs primary ciliogenesis: HADHA-knockout cells showed reduced ciliary frequency and length and decreased ciliary signaling mediators. The dehydrogenase-deficient E510Q mutant of HADHA failed to rescue ciliogenesis in KO cells, unlike wild-type HADHA reintroduction. Supplementation with sodium acetate (to restore intracellular acetyl-CoA) rescued primary cilia in HADHA-deficient cells, linking HADHA's β-oxidation activity and acetyl-CoA production to ciliogenesis.","method":"HADHA knockout cell line, wild-type and E510Q mutant HADHA rescue transfection, ciliary frequency and length quantification, sodium acetate supplementation rescue experiment","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO with WT vs. catalytic mutant rescue and metabolite supplementation rescue, single lab, two orthogonal validation approaches","pmids":["41120337"],"is_preprint":false},{"year":2024,"finding":"HADHA interacts with MDM2 and accelerates MDM2-mediated p53 ubiquitination in glioma cells. Co-immunoprecipitation confirmed HADHA–MDM2 physical interaction. MDM2 knockdown or p53 overexpression attenuated the pro-tumorigenic effects of HADHA overexpression.","method":"Co-immunoprecipitation, protein stability assays, HADHA knockdown/overexpression in glioma cells, MDM2 knockdown and p53 overexpression epistasis experiments, in vivo xenograft","journal":"Cancer gene therapy","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP demonstrating HADHA–MDM2 interaction, epistasis rescue experiments, single lab","pmids":["39039194"],"is_preprint":false},{"year":2025,"finding":"SIRT1 deficiency in aging hearts reduces HADHA expression through inhibition of the transcription factor GATA4 (which activates HADHA transcription). HADHA deficiency induces mitochondrial dysfunction, excessive ROS, glutathione depletion, GPX4 suppression, and ferroptosis. Cardiomyocyte-specific HADHA knockdown in young mice recapitulates ferroptotic cardiac remodeling reversible by ferrostatin-1. SIRT1 activation by resveratrol restores HADHA expression and suppresses ferroptosis.","method":"Cardiomyocyte-specific HADHA knockdown mice, rAAV9-mediated cardiac SIRT1 overexpression, proteomic analysis, GATA4 transcriptional regulation of HADHA, ferrostatin-1 rescue, lipid peroxidation and GPX4 assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple genetic mouse models (KD, OE) with mechanistic pathway dissection (SIRT1→GATA4→HADHA→GPX4), single lab","pmids":["41872163"],"is_preprint":false},{"year":2025,"finding":"SENP3 interacts with HADHA and catalyzes its deSUMOylation at two lysine residues. SUMOylation and ubiquitination compete at the same modification sites on HADHA, influencing protein stability and consequently regulating FAO levels. A physical complex of SENP3, HADHA, and USP10 was identified. HADHA deSUMOylation by SENP3 enhanced chemotherapy sensitivity in intrahepatic cholangiocarcinoma.","method":"Co-immunoprecipitation (SENP3–HADHA–USP10 complex), deSUMOylation assay, lipidomics profiling, patient-derived organoid drug screening, in vitro and in vivo chemotherapy sensitivity assays","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP establishing trimeric complex, SUMO/ubiquitin crosstalk at same sites demonstrated biochemically, single lab","pmids":["40320039"],"is_preprint":false},{"year":2024,"finding":"HADHA regulates the JAK/STAT3 signaling pathway through modulation of H3K27ac histone acetylation in glioblastoma. HADHA knockdown decreases acetyl-CoA levels, reducing H3K27ac modification and inhibiting JAK/STAT3 activation, linking HADHA's enzymatic production of acetyl-CoA to epigenetic regulation.","method":"HADHA knockdown in GBM cells, acetyl-CoA measurement, H3K27ac ChIP, JAK/STAT3 pathway activity assays, in vitro and in vivo tumor growth assays with JIB-04 inhibitor","journal":"Cell death discovery","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — acetyl-CoA measurement linked to H3K27ac changes with ChIP, JAK/STAT3 pathway readout, single lab","pmids":["40750765"],"is_preprint":false},{"year":2025,"finding":"HADHA interacts with SP1 in esophageal cancer cells and induces MDM2 expression. HADHA also activates mTOR signaling. RNA profiling after HADHA knockdown showed significant suppression of mTOR signaling.","method":"Co-immunoprecipitation (HADHA–SP1 interaction), HADHA knockdown with RNA profiling, MDM2 expression analysis, in vitro and in vivo tumor growth assays","journal":"Acta biochimica et biophysica Sinica","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP for HADHA–SP1 interaction, RNA profiling for mTOR pathway, single lab with limited mechanistic follow-up","pmids":["39327932"],"is_preprint":false},{"year":2024,"finding":"LCHADD iPSC-derived RPE cells expressing a wildtype HADHA copy via rAAV incorporated TFPα-FLAG into the TFP complex in the mitochondria, accumulated less 3-hydroxyacylcarnitines, released more ketones in response to palmitate, and were more resistant to oxidative stress. This demonstrates that HADHA is incorporated into the mitochondrial TFP complex and is required for palmitate oxidation and ketone production in RPE cells.","method":"iPSC-derived RPE from LCHADD patients, rAAV-HADHA transduction, mitochondrial fractionation, 3-hydroxyacylcarnitine quantification, palmitate oxidation and ketone release assays, DHA-induced oxidative stress rescue","journal":"Investigative ophthalmology & visual science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — disease cell model with gene addition showing mitochondrial incorporation and functional rescue via multiple metabolic readouts, single lab","pmids":["39283617"],"is_preprint":false},{"year":2026,"finding":"Melatonin directly binds HADHA (validated by CETSA) and enhances PGC-1α expression, promoting mitochondrial biogenesis and lipid metabolism in hepatocytes. HADHA knockdown abrogated the beneficial effects of melatonin on lipid accumulation in MASLD models.","method":"Cellular thermal shift assay (CETSA) for direct melatonin–HADHA binding, HADHA knockdown with melatonin treatment, PGC-1α expression measurement, lipid accumulation assays in mouse MASLD model and palmitic acid-treated hepatocytes","journal":"Molecular biomedicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CETSA establishes direct binding, KD rescue confirms HADHA dependency, single lab","pmids":["42118212"],"is_preprint":false}],"current_model":"HADHA encodes the α-subunit of the mitochondrial trifunctional protein (MTP), catalyzing three steps of long-chain fatty acid β-oxidation (enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase activities); it also functions as a monolysocardiolipin acyltransferase-like enzyme required for cardiolipin remodeling and mitochondrial cristae integrity, participates in respiratory supercomplex assembly to couple FAO with OXPHOS, and is regulated by multiple post-translational modifications (acetylation at K255/K350/K383/K406 by GCN5L1/reversed by SIRT3, lactylation at K166/K728 reversed by SIRT1/SIRT3, succinylation reversed by SIRT5, SUMOylation reversed by SENP3, and ubiquitination mediated by UBE2O), while its enzymatic production of acetyl-CoA and ketone bodies (β-hydroxybutyrate) links FAO to epigenetic regulation (H3K27ac, HDAC7 inhibition) and gluconeogenesis suppression."},"narrative":{"mechanistic_narrative":"HADHA encodes the α-subunit of the mitochondrial trifunctional protein (TFP), an inner-membrane enzyme that carries out long-chain fatty acid β-oxidation and is incorporated into the assembled TFP complex where it is required for palmitate oxidation, ketone (β-hydroxybutyrate) production, and resistance to oxidative stress [PMID:31604922, PMID:39283617]. Beyond its canonical catalytic role, HADHA functions as a monolysocardiolipin acyltransferase-like enzyme essential for cardiolipin remodeling and mitochondrial cristae integrity, such that its loss in cardiomyocytes disrupts the proton gradient, calcium dynamics, and repolarization [PMID:31604922]. HADHA also acts as a respiratory supercomplex assembly factor, physically coupling FAO to oxidative phosphorylation; its loss reduces supercomplex assembly and OXPHOS and aggravates hepatic steatosis [PMID:39488787]. Its metabolic output feeds epigenetic and signaling programs: β-oxidation-derived acetyl-CoA sustains H3K27 acetylation and JAK/STAT3 activation [PMID:40750765], while HADHA-generated β-hydroxybutyrate inhibits HDAC7 to drive FOXO1 nuclear exclusion and suppress hepatic gluconeogenesis [PMID:35046401]. HADHA expression and activity are heavily controlled: transcriptionally by GATA4 downstream of SIRT1, by Zfp335 in regulatory T cells, and by KDM6B-dependent H3K27 demethylation [PMID:41872163, PMID:37843279, PMID:39569625]; and post-translationally by a dense modification network including GCN5L1-mediated acetylation reversed by SIRT3 [PMID:30323061], lactylation at K166/K728 reversed by SIRT1/SIRT3 [PMID:40575877], succinylation reversed by SIRT5 [PMID:37688624], SUMOylation reversed by SENP3 in competition with ubiquitination [PMID:40320039], and UBE2O-mediated ubiquitination and degradation [PMID:36273042]. These regulatory inputs position HADHA at the center of FAO-dependent phenotypes across tissues, including ferroptosis suppression in aging hearts via the GPX4 axis [PMID:41872163], NLRP3 inflammasome control through K255 acetylation [PMID:37625146], primary ciliogenesis through acetyl-CoA supply [PMID:41120337], and tumor metabolic reprogramming [PMID:36273042, PMID:39039194, PMID:40750765]. The TFP α-subunit is encoded at chromosome 2p23 adjacent to HADHB and belongs to the enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase family [PMID:9605857].","teleology":[{"year":1997,"claim":"Established the genomic and family identity of HADHA as the α-subunit of the mitochondrial trifunctional protein, framing it as a fatty acid β-oxidation multienzyme component.","evidence":"FISH chromosomal mapping placing HADHA and HADHB adjacently at 2p23","pmids":["9605857"],"confidence":"Medium","gaps":["Mapping alone does not establish catalytic activity or in vivo function","No information on TFP complex stoichiometry or assembly"]},{"year":2015,"claim":"Raised the possibility of an extra-canonical role by linking HADHA to the Dicer/RISC machinery in miRNA maturation.","evidence":"Co-IP of HADHA with Dicer plus precursor/mature miRNA quantification on HADHA over/knockdown","pmids":["26367179"],"confidence":"Low","gaps":["Single Co-IP and expression-level data without reconstitution or mutagenesis","No mechanism for how a β-oxidation enzyme would assist miRNA processing","Not independently confirmed"]},{"year":2017,"claim":"Reported a cytosolic, non-mitochondrial pool of HADHA interacting with LC3, implicating it in long-chain fatty acid-induced autophagy.","evidence":"GFP-LC3 IP/MS, cellular fractionation, and HADHA/LC3/TOM20 co-localization under palmitic acid in intestinal epithelial cells","pmids":["28153718"],"confidence":"Medium","gaps":["No functional mutagenesis or reconstitution of the autophagy role","Single lab; cytosolic localization not corroborated elsewhere in the corpus"]},{"year":2018,"claim":"Defined acetylation as a tunable activity switch for HADHA, mapping GCN5L1-dependent sites and SIRT3 as the opposing eraser controlling FAO and hepatic lipid handling.","evidence":"Proteomic acetyl-site mapping (K350/K383/K406), GCN5L1 knockdown/knockout and overexpression mouse models with FAO enzyme assays","pmids":["30323061"],"confidence":"High","gaps":["Structural basis of how acetylation alters catalysis not resolved","Does not address other modification types competing at these sites"]},{"year":2019,"claim":"Demonstrated that HADHA is not only a β-oxidation enzyme but a monolysocardiolipin acyltransferase-like enzyme required for cardiolipin remodeling and cristae integrity, explaining its essentiality for cardiomyocyte function.","evidence":"HADHA-mutant hiPSC-derived cardiomyocytes with cardiolipin profiling, metabolic flux, calcium imaging, and proton gradient measurement","pmids":["31604922"],"confidence":"High","gaps":["Acyltransferase active site/mechanism not biochemically dissected","Relationship between the two enzymatic functions unresolved"]},{"year":2022,"claim":"Connected HADHA β-oxidation output to metabolic signaling by showing ketone-mediated HDAC7 inhibition drives FOXO1 exclusion and gluconeogenesis suppression, and established UBE2O as a degradative regulator in liver cancer.","evidence":"Stable isotope tracing with HADHA OE/KD mouse models and HDAC7/FOXO1 assays; Co-IP, ubiquitination assays, and Ube2o-KO hepatocarcinogenesis mice","pmids":["35046401","36273042"],"confidence":"High","gaps":["Quantitative contribution of HADHA-derived BHB versus other sources not delineated","UBE2O ubiquitination sites on HADHA not mapped"]},{"year":2023,"claim":"Expanded the regulatory and pathological scope of HADHA: K255 acetylation links it to NLRP3 inflammasome assembly, SIRT5 controls succinylation-coupled autophagy, and Zfp335 transcriptionally sustains HADHA for Treg metabolic differentiation.","evidence":"HFD K255 site-blocking with NLRP3/ASC imaging; MS succinyl-site mapping with SIRT5 binding/rescue; Zfp335-KO Tregs with ChIP and OXPHOS/suppression assays","pmids":["37625146","37688624","37843279"],"confidence":"Medium","gaps":["Mechanistic link between K255 acetylation and ASC recruitment not structurally defined","Succinylation findings from a single lab without reciprocal validation","How HADHA metabolic state feeds back on Zfp335 not addressed"]},{"year":2024,"claim":"Established HADHA as a respiratory supercomplex assembly factor coupling FAO to OXPHOS, and linked its acetyl-CoA output to H3K27ac-driven JAK/STAT3 signaling and tumor growth, alongside MDM2/p53 modulation in glioma.","evidence":"Proteomics + BN-PAGE in HADHA-KD/KO MEFs and HFD mice; acetyl-CoA/H3K27ac ChIP and JAK/STAT3 assays; HADHA–MDM2 Co-IP with p53 epistasis and KDM6B ChIP-seq","pmids":["39488787","40750765","39039194","39569625"],"confidence":"High","gaps":["Stoichiometry and binding interface of HADHA within supercomplexes unknown","Whether HADHA–MDM2 and acetyl-CoA/epigenetic effects are separable not resolved"]},{"year":2025,"claim":"Consolidated HADHA as a hub whose levels are set by competing SUMO/ubiquitin crosstalk (SENP3/USP10) and SIRT1→GATA4 transcription, with downstream control of ferroptosis, ciliogenesis, and lactylation-regulated activity.","evidence":"SENP3–HADHA–USP10 complex Co-IP and deSUMOylation assays; cardiomyocyte HADHA-KD mice with GPX4/ferrostatin-1; HADHA-KO with E510Q mutant and acetate rescue of cilia; K166/K728 lactylation mutagenesis in septic heart","pmids":["40320039","41872163","41120337","40575877"],"confidence":"Medium","gaps":["SUMO/ubiquitin acceptor lysines and their overlap not fully mapped","Direct mechanism linking acetyl-CoA supply to ciliary signaling unresolved","Several phenotype links are single-lab"]},{"year":2026,"claim":"Identified HADHA as a direct small-molecule target, with melatonin binding HADHA to enhance PGC-1α and mitochondrial biogenesis in fatty liver.","evidence":"CETSA for direct melatonin–HADHA binding and HADHA knockdown rescue in MASLD models","pmids":["42118212"],"confidence":"Medium","gaps":["Binding site and effect on enzymatic activity not defined","Mechanism connecting HADHA binding to PGC-1α induction unknown"]},{"year":null,"claim":"How the dense post-translational modification network (acetylation, lactylation, succinylation, SUMOylation, ubiquitination) is integrated to set HADHA activity, stability, and localization in a tissue-specific manner remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model integrating modification sites with the catalytic and acyltransferase activities","Crosstalk hierarchy among modifications not established","Mechanism of the reported cytosolic/non-mitochondrial functions not reconciled with mitochondrial role"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016491","term_label":"oxidoreductase activity","supporting_discovery_ids":[0,19]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0]},{"term_id":"GO:0016829","term_label":"lyase activity","supporting_discovery_ids":[12]},{"term_id":"GO:0016853","term_label":"isomerase activity","supporting_discovery_ids":[12]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[0,8,19]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[10]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[0,2,19]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[8]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[10]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[15]}],"complexes":["mitochondrial trifunctional protein (TFP)","mitochondrial respiratory supercomplex","SENP3-HADHA-USP10 complex"],"partners":["GCN5L1","SIRT3","SIRT5","UBE2O","SENP3","MDM2","HDAC7","LC3"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P40939","full_name":"Trifunctional enzyme subunit alpha, mitochondrial","aliases":["78 kDa gastrin-binding protein","Monolysocardiolipin acyltransferase","MLCL AT","TP-alpha"],"length_aa":763,"mass_kda":83.0,"function":"Mitochondrial trifunctional enzyme catalyzes the last three of the four reactions of the mitochondrial beta-oxidation pathway (PubMed:1550553, PubMed:29915090, PubMed:30850536, PubMed:8135828, PubMed:31604922). The mitochondrial beta-oxidation pathway is the major energy-producing process in tissues and is performed through four consecutive reactions breaking down fatty acids into acetyl-CoA (PubMed:29915090). Among the enzymes involved in this pathway, the trifunctional enzyme exhibits specificity for long-chain fatty acids (PubMed:30850536, PubMed:31604922). Mitochondrial trifunctional enzyme is a heterotetrameric complex composed of two proteins, the trifunctional enzyme subunit alpha/HADHA described here carries the 2,3-enoyl-CoA hydratase and the 3-hydroxyacyl-CoA dehydrogenase activities while the trifunctional enzyme subunit beta/HADHB bears the 3-ketoacyl-CoA thiolase activity (PubMed:29915090, PubMed:30850536, PubMed:8135828). Independently of subunit beta, HADHA also exhibits a cardiolipin acyltransferase activity that participates in cardiolipin remodeling; cardiolipin is a major mitochondrial membrane phospholipid (PubMed:23152787, PubMed:31604922). HADHA may act downstream of Tafazzin/TAZ, that remodels monolysocardiolipin (MLCL) to a cardiolipin intermediate, and then HADHA may continue to remodel this species into mature tetralinoleoyl-cardiolipin (PubMed:31604922). Has also been proposed to act directly on MLCL; capable of acylating MLCL using different acyl-CoA substrates, with highest activity for oleoyl-CoA (PubMed:23152787)","subcellular_location":"Mitochondrion; Mitochondrion inner membrane","url":"https://www.uniprot.org/uniprotkb/P40939/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/HADHA","classification":"Not Classified","n_dependent_lines":77,"n_total_lines":1208,"dependency_fraction":0.06374172185430464},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"SRP9","stoichiometry":4.0},{"gene":"CAPZB","stoichiometry":0.2},{"gene":"DDB1","stoichiometry":0.2},{"gene":"HSP90B1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/HADHA","total_profiled":1310},"omim":[{"mim_id":"620300","title":"MITOCHONDRIAL TRIFUNCTIONAL PROTEIN DEFICIENCY 2; MTPD2","url":"https://www.omim.org/entry/620300"},{"mim_id":"615605","title":"FANCONI RENOTUBULAR SYNDROME 3; FRTS3","url":"https://www.omim.org/entry/615605"},{"mim_id":"611126","title":"MITOCHONDRIAL COMPLEX I DEFICIENCY, NUCLEAR TYPE 20; MC1DN20","url":"https://www.omim.org/entry/611126"},{"mim_id":"610760","title":"CHOLESTEROL LEVEL QUANTITATIVE TRAIT LOCUS 2","url":"https://www.omim.org/entry/610760"},{"mim_id":"609016","title":"LONG-CHAIN 3-HYDROXYACYL-CoA DEHYDROGENASE DEFICIENCY","url":"https://www.omim.org/entry/609016"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Mitochondria","reliability":"Enhanced"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"skeletal 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pathology","url":"https://pubmed.ncbi.nlm.nih.gov/39687448","citation_count":1,"is_preprint":false},{"pmid":"33887580","id":"PMC_33887580","title":"Generation of an induced pluripotent stem cell line, ICGi028-A, by reprogramming peripheral blood mononuclear cells of a patient suffering from hypertrophic cardiomyopathy and carrying a heterozygous p.E510Q mutation in HADHA.","date":"2021","source":"Stem cell research","url":"https://pubmed.ncbi.nlm.nih.gov/33887580","citation_count":1,"is_preprint":false},{"pmid":"21035315","id":"PMC_21035315","title":"[EBV infection revealing a long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency in a 3-year-old boy].","date":"2010","source":"Archives de pediatrie : organe officiel de la Societe francaise de pediatrie","url":"https://pubmed.ncbi.nlm.nih.gov/21035315","citation_count":1,"is_preprint":false},{"pmid":"41120337","id":"PMC_41120337","title":"Disrupting mitochondrial β-oxidation by depletion of HADHA impairs primary ciliogenesis.","date":"2025","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/41120337","citation_count":0,"is_preprint":false},{"pmid":"36224682","id":"PMC_36224682","title":"[HADHA Inhibits the Migration and Invasion of HTR-8/SVneo Cells by Regulating PI3K/AKT Signaling Pathway].","date":"2022","source":"Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. 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HADHA was identified as a monolysocardiolipin acyltransferase-like enzyme essential for functional mitochondria.\",\n      \"method\": \"hiPSC-derived cardiomyocytes from HADHA-mutant cells, single-cell RNA-seq, functional metabolic assays (fatty acid beta-oxidation, mitochondrial proton gradient), cardiolipin profiling, engineered microRNA maturation cocktail\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (iPSC disease model, scRNA-seq, metabolic flux, cardiolipin profiling, calcium imaging) in a single rigorous study with direct functional readouts\",\n      \"pmids\": [\"31604922\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"GCN5L1 acetylates HADHA at lysine residues K350, K383, and K406, and this hyperacetylation correlates with increased HADHA activity. SIRT3 opposes this by deacetylating HADHA. GCN5L1 knockdown reduces HADHA acetylation and increases fatty acid oxidation enzyme activities; liver-specific GCN5L1 knockout mice lack HADHA hyperacetylation and are protected from hepatic lipid accumulation on high-fat diet.\",\n      \"method\": \"Proteomic identification of acetylation sites, transgenic GCN5L1 overexpression mouse model, liver-specific GCN5L1 knockout mice, stable GCN5L1 knockdown in HepG2 cells, fatty acid oxidation enzyme activity assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — site-specific acetylation mapped by proteomics, validated in both cell knockdown and multiple mouse models with activity assays\",\n      \"pmids\": [\"30323061\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"HADHA promotes ketone body (β-hydroxybutyrate, BHB) production via β-oxidation, and BHB suppresses hepatic gluconeogenesis by selectively inhibiting HDAC7 activity via interaction with HDAC7 Glu543, facilitating FOXO1 nuclear exclusion. Liver-specific HADHA overexpression reversed hepatic gluconeogenesis in mice; HADHA knockdown augmented glucagon response.\",\n      \"method\": \"Stable isotope tracing, liver-specific HADHA overexpression and knockdown mouse models, HDAC7 activity assays, FOXO1 localization studies, high-fat diet mouse model\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — stable isotope tracing, multiple in vivo genetic models, mechanistic pathway dissection with identified molecular interaction (BHB–HDAC7 Glu543)\",\n      \"pmids\": [\"35046401\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"UBE2O (an E2 ubiquitin-conjugating enzyme) interacts with HADHA and mediates its ubiquitination and proteasomal degradation, thereby reducing HADHA protein levels and modulating lipid metabolic reprogramming in hepatocellular carcinoma.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, UBE2O overexpression/knockdown in vitro and in vivo, liver-specific Ube2o knockout mice with DEN-induced hepatocarcinogenesis\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP establishing direct interaction, ubiquitination assay, in vivo genetic model confirming pathway\",\n      \"pmids\": [\"36273042\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"HADHA is lactylated at K166 and K728 in septic heart tissue; lactylation at these sites inhibits HADHA activity, disturbs mitochondrial function, reduces ATP production, impairs energy metabolism, and reduces cardiomyocyte contraction force. SIRT1 and SIRT3 were identified as erasers of HADHA lactylation at these sites.\",\n      \"method\": \"Proteomic analysis of lactylation sites in septic rat heart tissues, LPS-induced cardiomyocyte model, K166/K728 site-directed mutagenesis, mitochondrial function assays, ATP measurements, transcriptomic and metabolomic analyses, in vivo cardiac function measurement\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — site-directed mutagenesis at K166 and K728 with functional validation of enzymatic activity, mitochondrial function, and in vivo cardiac phenotype; multiple orthogonal methods\",\n      \"pmids\": [\"40575877\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Acetylation of HADHA at K255 (in obese mouse hearts, promoted by mitochondrial hyperacetylation) triggers mitochondrial localization of ASC and facilitates NLRP3 inflammasome assembly. Blockade of K255 acetylation suppressed the NLRP3 inflammasome and attenuated post-ischemia/reperfusion myocardial fibrosis in obese mice.\",\n      \"method\": \"High-fat diet mouse model, K255 acetylation site identification, site-specific blocking experiments, NLRP3 inflammasome assembly assays (ASC localization by imaging), post-I/R myocardial fibrosis assessment\",\n      \"journal\": \"Diabetes\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — K255 site identified and functionally blocked in vivo, NLRP3 assembly readout, single lab with two orthogonal approaches\",\n      \"pmids\": [\"37625146\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"HADHA succinylation (induced by morphine) is reversed by the desuccinylase SIRT5, which selectively binds HADHA. SIRT5-mediated HADHA desuccinylation reduced P62 expression and alleviated morphine tolerance, linking HADHA succinylation to autophagy dysregulation.\",\n      \"method\": \"LC-MS/MS and parallel reaction monitoring for succinylation site mapping, SIRT5 binding assay, SIRT5 overexpression in intrathecal morphine rat model, P62/LC3 autophagy marker measurement\",\n      \"journal\": \"Naunyn-Schmiedeberg's archives of pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS-confirmed succinylation sites, SIRT5–HADHA binding and functional rescue, single lab\",\n      \"pmids\": [\"37688624\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"KDM6B demethylates histone H3K27 at the HADHA locus to activate HADHA transcription during cementoblast mineralization. Additionally, lactylation of HADHA (at specific sites identified by lactylation proteomics) promotes FAO and mineralization; KDM6B regulates HADHA lactylation. Co-immunoprecipitation confirmed interaction between lactylated HADHA and its partners.\",\n      \"method\": \"ChIP-seq, RNA-seq, ChIP-qPCR, HADHA overexpression rescue experiments, lactylation proteomics, Co-IP, FAO activity assays, in vivo KDM6B inhibition in mice\",\n      \"journal\": \"Journal of dental research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP-seq and functional rescue establish KDM6B→HADHA transcriptional axis; lactylation sites mapped by proteomics with Co-IP validation, single lab\",\n      \"pmids\": [\"39569625\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"HADHA participates in respiratory supercomplex (SC) assembly and couples FAO to OXPHOS. HADHA knockdown cells and HADHA-knockout MEFs displayed reduced SC assembly and defective OXPHOS. HADHA expression is upregulated when OXPHOS is stimulated (glucose-to-galactose switch) or lipid metabolism is induced (high-fat diet). HADHA heterozygous mice on HFD showed enhanced steatosis with reduced SC assembly and OXPHOS.\",\n      \"method\": \"Proteomics identifying HADHA as SC assembly factor, HADHA-KD cells and HADHA-KO MEFs with SC assembly assays (BN-PAGE), galactose medium OXPHOS stimulation, HFD mouse model with SC assembly analysis\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — proteomics-identified candidate validated by KD, KO MEFs, and in vivo mouse model with orthogonal functional readouts (SC assembly, OXPHOS activity)\",\n      \"pmids\": [\"39488787\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Zfp335 transcription factor controls effector Treg (eTreg) differentiation by directly targeting the FAO enzyme Hadha to maintain fatty acid oxidation and oxidative phosphorylation in Tregs. Zfp335-deficient Tregs showed reduced HADHA expression, dysfunctional mitochondrial activity, and failed to differentiate into eTregs.\",\n      \"method\": \"Treg-specific Zfp335 knockout mice, scRNA-seq, chromatin immunoprecipitation (direct Hadha targeting), OXPHOS assays, Treg functional suppression assays\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct ChIP evidence of Zfp335 binding Hadha locus, KO mouse model with scRNA-seq and functional readouts, replicated in human eTreg correlation\",\n      \"pmids\": [\"37843279\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"HADHA protein is present not only in mitochondria but also in the cytosol. HADHA was identified as an LC3-interacting protein in intestinal epithelial cells via immunoprecipitation with a GFP-LC3 antibody. LC3 puncta co-localized with HADHA (but not with the mitochondrial marker TOM20) and were enhanced by palmitic acid stimulation, suggesting HADHA has extra-mitochondrial roles in long-chain fatty acid-induced autophagy.\",\n      \"method\": \"GFP-LC3 immunoprecipitation followed by mass spectrometry, cellular fractionation, immunofluorescence co-localization (HADHA vs. LC3 vs. TOM20), palmitic acid treatment of IEC lines\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP/MS identification plus co-localization imaging, single lab, no functional mutagenesis or reconstitution\",\n      \"pmids\": [\"28153718\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"HADHA associates with the human Dicer complex (RNA-induced silencing machinery). Immunoprecipitation showed HADHA co-precipitates with Dicer; HADHA overexpression increased mature miRNA levels with corresponding decrease in precursor miRNA, while HADHA knockdown had the opposite effect, suggesting an auxiliary role in miRNA biogenesis.\",\n      \"method\": \"Co-immunoprecipitation of HADHA with Dicer, immunohistochemical co-localization with Dicer in cytoplasm, HADHA overexpression/knockdown with miRNA precursor and mature miRNA quantification\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP and expression-level miRNA data, single lab, no mechanistic reconstitution or mutagenesis\",\n      \"pmids\": [\"26367179\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"HADHA and HADHB genes (encoding the α and β subunits of the mitochondrial trifunctional protein) are both located on human chromosome band 2p23 and are in close proximity, analogous to the operon-like arrangement of bacterial fatty acid beta-oxidation multienzyme complex genes. The α subunit (HADHA) belongs to the enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase family.\",\n      \"method\": \"Fluorescence in situ hybridization (FISH) chromosomal mapping\",\n      \"journal\": \"Cytogenetics and cell genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct FISH localization establishing chromosomal co-localization; well-established physical mapping result\",\n      \"pmids\": [\"9605857\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"HADHA deficiency impairs primary ciliogenesis: HADHA-knockout cells showed reduced ciliary frequency and length and decreased ciliary signaling mediators. The dehydrogenase-deficient E510Q mutant of HADHA failed to rescue ciliogenesis in KO cells, unlike wild-type HADHA reintroduction. Supplementation with sodium acetate (to restore intracellular acetyl-CoA) rescued primary cilia in HADHA-deficient cells, linking HADHA's β-oxidation activity and acetyl-CoA production to ciliogenesis.\",\n      \"method\": \"HADHA knockout cell line, wild-type and E510Q mutant HADHA rescue transfection, ciliary frequency and length quantification, sodium acetate supplementation rescue experiment\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO with WT vs. catalytic mutant rescue and metabolite supplementation rescue, single lab, two orthogonal validation approaches\",\n      \"pmids\": [\"41120337\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"HADHA interacts with MDM2 and accelerates MDM2-mediated p53 ubiquitination in glioma cells. Co-immunoprecipitation confirmed HADHA–MDM2 physical interaction. MDM2 knockdown or p53 overexpression attenuated the pro-tumorigenic effects of HADHA overexpression.\",\n      \"method\": \"Co-immunoprecipitation, protein stability assays, HADHA knockdown/overexpression in glioma cells, MDM2 knockdown and p53 overexpression epistasis experiments, in vivo xenograft\",\n      \"journal\": \"Cancer gene therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP demonstrating HADHA–MDM2 interaction, epistasis rescue experiments, single lab\",\n      \"pmids\": [\"39039194\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SIRT1 deficiency in aging hearts reduces HADHA expression through inhibition of the transcription factor GATA4 (which activates HADHA transcription). HADHA deficiency induces mitochondrial dysfunction, excessive ROS, glutathione depletion, GPX4 suppression, and ferroptosis. Cardiomyocyte-specific HADHA knockdown in young mice recapitulates ferroptotic cardiac remodeling reversible by ferrostatin-1. SIRT1 activation by resveratrol restores HADHA expression and suppresses ferroptosis.\",\n      \"method\": \"Cardiomyocyte-specific HADHA knockdown mice, rAAV9-mediated cardiac SIRT1 overexpression, proteomic analysis, GATA4 transcriptional regulation of HADHA, ferrostatin-1 rescue, lipid peroxidation and GPX4 assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple genetic mouse models (KD, OE) with mechanistic pathway dissection (SIRT1→GATA4→HADHA→GPX4), single lab\",\n      \"pmids\": [\"41872163\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SENP3 interacts with HADHA and catalyzes its deSUMOylation at two lysine residues. SUMOylation and ubiquitination compete at the same modification sites on HADHA, influencing protein stability and consequently regulating FAO levels. A physical complex of SENP3, HADHA, and USP10 was identified. HADHA deSUMOylation by SENP3 enhanced chemotherapy sensitivity in intrahepatic cholangiocarcinoma.\",\n      \"method\": \"Co-immunoprecipitation (SENP3–HADHA–USP10 complex), deSUMOylation assay, lipidomics profiling, patient-derived organoid drug screening, in vitro and in vivo chemotherapy sensitivity assays\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP establishing trimeric complex, SUMO/ubiquitin crosstalk at same sites demonstrated biochemically, single lab\",\n      \"pmids\": [\"40320039\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"HADHA regulates the JAK/STAT3 signaling pathway through modulation of H3K27ac histone acetylation in glioblastoma. HADHA knockdown decreases acetyl-CoA levels, reducing H3K27ac modification and inhibiting JAK/STAT3 activation, linking HADHA's enzymatic production of acetyl-CoA to epigenetic regulation.\",\n      \"method\": \"HADHA knockdown in GBM cells, acetyl-CoA measurement, H3K27ac ChIP, JAK/STAT3 pathway activity assays, in vitro and in vivo tumor growth assays with JIB-04 inhibitor\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — acetyl-CoA measurement linked to H3K27ac changes with ChIP, JAK/STAT3 pathway readout, single lab\",\n      \"pmids\": [\"40750765\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"HADHA interacts with SP1 in esophageal cancer cells and induces MDM2 expression. HADHA also activates mTOR signaling. RNA profiling after HADHA knockdown showed significant suppression of mTOR signaling.\",\n      \"method\": \"Co-immunoprecipitation (HADHA–SP1 interaction), HADHA knockdown with RNA profiling, MDM2 expression analysis, in vitro and in vivo tumor growth assays\",\n      \"journal\": \"Acta biochimica et biophysica Sinica\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP for HADHA–SP1 interaction, RNA profiling for mTOR pathway, single lab with limited mechanistic follow-up\",\n      \"pmids\": [\"39327932\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"LCHADD iPSC-derived RPE cells expressing a wildtype HADHA copy via rAAV incorporated TFPα-FLAG into the TFP complex in the mitochondria, accumulated less 3-hydroxyacylcarnitines, released more ketones in response to palmitate, and were more resistant to oxidative stress. This demonstrates that HADHA is incorporated into the mitochondrial TFP complex and is required for palmitate oxidation and ketone production in RPE cells.\",\n      \"method\": \"iPSC-derived RPE from LCHADD patients, rAAV-HADHA transduction, mitochondrial fractionation, 3-hydroxyacylcarnitine quantification, palmitate oxidation and ketone release assays, DHA-induced oxidative stress rescue\",\n      \"journal\": \"Investigative ophthalmology & visual science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — disease cell model with gene addition showing mitochondrial incorporation and functional rescue via multiple metabolic readouts, single lab\",\n      \"pmids\": [\"39283617\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Melatonin directly binds HADHA (validated by CETSA) and enhances PGC-1α expression, promoting mitochondrial biogenesis and lipid metabolism in hepatocytes. HADHA knockdown abrogated the beneficial effects of melatonin on lipid accumulation in MASLD models.\",\n      \"method\": \"Cellular thermal shift assay (CETSA) for direct melatonin–HADHA binding, HADHA knockdown with melatonin treatment, PGC-1α expression measurement, lipid accumulation assays in mouse MASLD model and palmitic acid-treated hepatocytes\",\n      \"journal\": \"Molecular biomedicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CETSA establishes direct binding, KD rescue confirms HADHA dependency, single lab\",\n      \"pmids\": [\"42118212\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"HADHA encodes the α-subunit of the mitochondrial trifunctional protein (MTP), catalyzing three steps of long-chain fatty acid β-oxidation (enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase activities); it also functions as a monolysocardiolipin acyltransferase-like enzyme required for cardiolipin remodeling and mitochondrial cristae integrity, participates in respiratory supercomplex assembly to couple FAO with OXPHOS, and is regulated by multiple post-translational modifications (acetylation at K255/K350/K383/K406 by GCN5L1/reversed by SIRT3, lactylation at K166/K728 reversed by SIRT1/SIRT3, succinylation reversed by SIRT5, SUMOylation reversed by SENP3, and ubiquitination mediated by UBE2O), while its enzymatic production of acetyl-CoA and ketone bodies (β-hydroxybutyrate) links FAO to epigenetic regulation (H3K27ac, HDAC7 inhibition) and gluconeogenesis suppression.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"HADHA encodes the α-subunit of the mitochondrial trifunctional protein (TFP), an inner-membrane enzyme that carries out long-chain fatty acid β-oxidation and is incorporated into the assembled TFP complex where it is required for palmitate oxidation, ketone (β-hydroxybutyrate) production, and resistance to oxidative stress [#0, #19]. Beyond its canonical catalytic role, HADHA functions as a monolysocardiolipin acyltransferase-like enzyme essential for cardiolipin remodeling and mitochondrial cristae integrity, such that its loss in cardiomyocytes disrupts the proton gradient, calcium dynamics, and repolarization [#0]. HADHA also acts as a respiratory supercomplex assembly factor, physically coupling FAO to oxidative phosphorylation; its loss reduces supercomplex assembly and OXPHOS and aggravates hepatic steatosis [#8]. Its metabolic output feeds epigenetic and signaling programs: β-oxidation-derived acetyl-CoA sustains H3K27 acetylation and JAK/STAT3 activation [#17], while HADHA-generated β-hydroxybutyrate inhibits HDAC7 to drive FOXO1 nuclear exclusion and suppress hepatic gluconeogenesis [#2]. HADHA expression and activity are heavily controlled: transcriptionally by GATA4 downstream of SIRT1, by Zfp335 in regulatory T cells, and by KDM6B-dependent H3K27 demethylation [#15, #9, #7]; and post-translationally by a dense modification network including GCN5L1-mediated acetylation reversed by SIRT3 [#1], lactylation at K166/K728 reversed by SIRT1/SIRT3 [#4], succinylation reversed by SIRT5 [#6], SUMOylation reversed by SENP3 in competition with ubiquitination [#16], and UBE2O-mediated ubiquitination and degradation [#3]. These regulatory inputs position HADHA at the center of FAO-dependent phenotypes across tissues, including ferroptosis suppression in aging hearts via the GPX4 axis [#15], NLRP3 inflammasome control through K255 acetylation [#5], primary ciliogenesis through acetyl-CoA supply [#13], and tumor metabolic reprogramming [#3, #14, #17]. The TFP α-subunit is encoded at chromosome 2p23 adjacent to HADHB and belongs to the enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase family [#12].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Established the genomic and family identity of HADHA as the α-subunit of the mitochondrial trifunctional protein, framing it as a fatty acid β-oxidation multienzyme component.\",\n      \"evidence\": \"FISH chromosomal mapping placing HADHA and HADHB adjacently at 2p23\",\n      \"pmids\": [\"9605857\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mapping alone does not establish catalytic activity or in vivo function\", \"No information on TFP complex stoichiometry or assembly\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Raised the possibility of an extra-canonical role by linking HADHA to the Dicer/RISC machinery in miRNA maturation.\",\n      \"evidence\": \"Co-IP of HADHA with Dicer plus precursor/mature miRNA quantification on HADHA over/knockdown\",\n      \"pmids\": [\"26367179\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single Co-IP and expression-level data without reconstitution or mutagenesis\", \"No mechanism for how a β-oxidation enzyme would assist miRNA processing\", \"Not independently confirmed\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Reported a cytosolic, non-mitochondrial pool of HADHA interacting with LC3, implicating it in long-chain fatty acid-induced autophagy.\",\n      \"evidence\": \"GFP-LC3 IP/MS, cellular fractionation, and HADHA/LC3/TOM20 co-localization under palmitic acid in intestinal epithelial cells\",\n      \"pmids\": [\"28153718\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No functional mutagenesis or reconstitution of the autophagy role\", \"Single lab; cytosolic localization not corroborated elsewhere in the corpus\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined acetylation as a tunable activity switch for HADHA, mapping GCN5L1-dependent sites and SIRT3 as the opposing eraser controlling FAO and hepatic lipid handling.\",\n      \"evidence\": \"Proteomic acetyl-site mapping (K350/K383/K406), GCN5L1 knockdown/knockout and overexpression mouse models with FAO enzyme assays\",\n      \"pmids\": [\"30323061\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of how acetylation alters catalysis not resolved\", \"Does not address other modification types competing at these sites\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Demonstrated that HADHA is not only a β-oxidation enzyme but a monolysocardiolipin acyltransferase-like enzyme required for cardiolipin remodeling and cristae integrity, explaining its essentiality for cardiomyocyte function.\",\n      \"evidence\": \"HADHA-mutant hiPSC-derived cardiomyocytes with cardiolipin profiling, metabolic flux, calcium imaging, and proton gradient measurement\",\n      \"pmids\": [\"31604922\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Acyltransferase active site/mechanism not biochemically dissected\", \"Relationship between the two enzymatic functions unresolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Connected HADHA β-oxidation output to metabolic signaling by showing ketone-mediated HDAC7 inhibition drives FOXO1 exclusion and gluconeogenesis suppression, and established UBE2O as a degradative regulator in liver cancer.\",\n      \"evidence\": \"Stable isotope tracing with HADHA OE/KD mouse models and HDAC7/FOXO1 assays; Co-IP, ubiquitination assays, and Ube2o-KO hepatocarcinogenesis mice\",\n      \"pmids\": [\"35046401\", \"36273042\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative contribution of HADHA-derived BHB versus other sources not delineated\", \"UBE2O ubiquitination sites on HADHA not mapped\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Expanded the regulatory and pathological scope of HADHA: K255 acetylation links it to NLRP3 inflammasome assembly, SIRT5 controls succinylation-coupled autophagy, and Zfp335 transcriptionally sustains HADHA for Treg metabolic differentiation.\",\n      \"evidence\": \"HFD K255 site-blocking with NLRP3/ASC imaging; MS succinyl-site mapping with SIRT5 binding/rescue; Zfp335-KO Tregs with ChIP and OXPHOS/suppression assays\",\n      \"pmids\": [\"37625146\", \"37688624\", \"37843279\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanistic link between K255 acetylation and ASC recruitment not structurally defined\", \"Succinylation findings from a single lab without reciprocal validation\", \"How HADHA metabolic state feeds back on Zfp335 not addressed\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Established HADHA as a respiratory supercomplex assembly factor coupling FAO to OXPHOS, and linked its acetyl-CoA output to H3K27ac-driven JAK/STAT3 signaling and tumor growth, alongside MDM2/p53 modulation in glioma.\",\n      \"evidence\": \"Proteomics + BN-PAGE in HADHA-KD/KO MEFs and HFD mice; acetyl-CoA/H3K27ac ChIP and JAK/STAT3 assays; HADHA–MDM2 Co-IP with p53 epistasis and KDM6B ChIP-seq\",\n      \"pmids\": [\"39488787\", \"40750765\", \"39039194\", \"39569625\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and binding interface of HADHA within supercomplexes unknown\", \"Whether HADHA–MDM2 and acetyl-CoA/epigenetic effects are separable not resolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Consolidated HADHA as a hub whose levels are set by competing SUMO/ubiquitin crosstalk (SENP3/USP10) and SIRT1→GATA4 transcription, with downstream control of ferroptosis, ciliogenesis, and lactylation-regulated activity.\",\n      \"evidence\": \"SENP3–HADHA–USP10 complex Co-IP and deSUMOylation assays; cardiomyocyte HADHA-KD mice with GPX4/ferrostatin-1; HADHA-KO with E510Q mutant and acetate rescue of cilia; K166/K728 lactylation mutagenesis in septic heart\",\n      \"pmids\": [\"40320039\", \"41872163\", \"41120337\", \"40575877\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"SUMO/ubiquitin acceptor lysines and their overlap not fully mapped\", \"Direct mechanism linking acetyl-CoA supply to ciliary signaling unresolved\", \"Several phenotype links are single-lab\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Identified HADHA as a direct small-molecule target, with melatonin binding HADHA to enhance PGC-1α and mitochondrial biogenesis in fatty liver.\",\n      \"evidence\": \"CETSA for direct melatonin–HADHA binding and HADHA knockdown rescue in MASLD models\",\n      \"pmids\": [\"42118212\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Binding site and effect on enzymatic activity not defined\", \"Mechanism connecting HADHA binding to PGC-1α induction unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the dense post-translational modification network (acetylation, lactylation, succinylation, SUMOylation, ubiquitination) is integrated to set HADHA activity, stability, and localization in a tissue-specific manner remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model integrating modification sites with the catalytic and acyltransferase activities\", \"Crosstalk hierarchy among modifications not established\", \"Mechanism of the reported cytosolic/non-mitochondrial functions not reconciled with mitochondrial role\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016491\", \"supporting_discovery_ids\": [0, 19]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0016829\", \"supporting_discovery_ids\": [12]},\n      {\"term_id\": \"GO:0016853\", \"supporting_discovery_ids\": [12]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [0, 8, 19]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [10]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [0, 2, 19]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [10]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [15]}\n    ],\n    \"complexes\": [\n      \"mitochondrial trifunctional protein (TFP)\",\n      \"mitochondrial respiratory supercomplex\",\n      \"SENP3-HADHA-USP10 complex\"\n    ],\n    \"partners\": [\n      \"GCN5L1\",\n      \"SIRT3\",\n      \"SIRT5\",\n      \"UBE2O\",\n      \"SENP3\",\n      \"MDM2\",\n      \"HDAC7\",\n      \"LC3\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}