Silencing lncRNA-DARS-AS1 suppresses nonsmall cell lung cancer progression by stimulating miR-302a-3p to inhibit ACAT1 expression.

Long noncoding RNAs (LncRNAs) have been gaining attention as potential therapeutic targets for lung cancer. In this study, we investigated the expression and biological behavior of lncRNA DARS-AS1, its predicted interacting partner miR-302a-3p, and ACAT1 in nonsmall cell lung cancer (NSCLC). The transcript level of DARS-AS1, miR-302a-3p, and ACAT1 was analyzed using qRT-PCR. Endogenous expression of ACAT1 and the expression of-and changes in-AKT/ERK pathway-related proteins were determined using western blotting. MTS, Transwell, and apoptosis experiments were used to investigate the behavior of cells. The subcellular localization of DARS-AS1 was verified using FISH, and its binding site was verified using dual-luciferase reporter experiments. The binding of DARS-AS1 to miR-302a-3p was verified using RNA co-immunoprecipitation. In vivo experiments were performed using a xenograft model to determine the effect of DARS-AS1 knockout on ACAT1 and NSCLC. lncRNA DARS-AS1 was upregulated in NSCLC cell lines and tissues and the expression of lncRNA DARS-AS1 was negatively correlated with survival of patients with NSCLC. Knockdown of DARS-AS1 inhibited the malignant behaviors of NSCLC via upregulating miR-302a-3p. miR-302a-3p induced suppression of malignancy through regulating oncogene ACAT1. This study demonstrates that the DARS-AS1-miR-302a-3p-ACAT1 pathway plays a key role in NSCLC.

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

Mitochondrial acetyl-CoA acetyltransferase 1 (ACAT1) regulates pyruvate dehydrogenase complex (PDC) by acetylating pyruvate dehydrogenase (PDH) and PDH phosphatase. How ACAT1 is "hijacked" to contribute to the Warburg effect in human cancer remains unclear. We found that active, tetrameric ACAT1 is commonly upregulated in cells stimulated by EGF and in diverse human cancer cells, where ACAT1 tetramers, but not monomers, are phosphorylated and stabilized by enhanced Y407 phosphorylation. Moreover, we identified arecoline hydrobromide (AH) as a covalent ACAT1 inhibitor that binds to and disrupts only ACAT1 tetramers. The resultant AH-bound ACAT1 monomers cannot reform tetramers. Inhibition of tetrameric ACAT1 by abolishing Y407 phosphorylation or AH treatment results in decreased ACAT1 activity, leading to increased PDC flux and oxidative phosphorylation with attenuated cancer cell proliferation and tumor growth. These findings provide a mechanistic understanding of how oncogenic events signal through distinct acetyltransferases to regulate cancer metabolism and suggest ACAT1 as an anti-cancer target.

[Clinical analysis and genetic diagnosis of three children with Isoleucine metabolic disorders due to variants of HSD17B10 and ACAT1 genes].

OBJECTIVE: To explore the clinical, biochemical and genetic characteristics of three children with Isoleucine metabolic disorders due to variants of HSD17B10 and ACAT1 genes. METHODS: Two children with 17ß hydroxysteroid dehydrogenase 10 (HSD17B10) deficiency and a child with ß-ketothiolase deficiency (BKD) diagnosed at Shanghai Children's Hospital between 2014 and 2021 were selected as the study subjects. Clinical data of the children were collected. The children were subjected to blood acylcarnitine, urinary organic acid and genetic testing, and candidate variants were analyzed with bioinformatic tools. RESULTS: The main symptoms of the three children had included epilepsy, developmental delay, hypotonia and acidosis. Their blood acylcarnitine methylcrotonyl carnitine (C5:1), 3-hydroxyisovalerylcarnitine (C5-OH) and 3-hydroxybutylcarnitine (C4OH) were increased to various extents, and urine organic acids including methyl crotonylglycine and 2-methyl-3-hydroxybutyric acid were significantly increased. Child 1 and child 2 were respectively found to harbor a c.347G>A (p.R116Q) variant and a c.274G>A (p.A92T) variant of the HSD17B10 gene, and child 3 was found to harbor compound heterozygous variants of the ACAT1 gene, namely c.547G>A (p.G183R) and a c.331G>C (p.A111P). Among these, the c.274G>A (p.A92T) and c.331G>C (p.A111P) variants were unreported previously. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), they were respectively classified as variant of unknown significance (PP3_Strong+PM2_supporting) and likely pathogenic (PM3+PM2_Supporting+PP3_Moderate+PP4). CONCLUSION: Both the HSD17B10 deficiency and BKD can lead to Isoleucine metabolism disorders, which may be difficult to distinguish clinically. Genetic testing can further confirm the diagnosis. Discoveries of the HSD17B10: c.274G>A (p.A92T) variant and the ACAT1: c.331G>C (p.A111P) variant have enriched the mutational spectrum of the two diseases.

ACAT1-mediated METTL3 acetylation inhibits cell migration and invasion in triple negative breast cancer.

Triple-negative breast cancer (TNBC) is a heterogeneous and aggressive disease with poor prognosis. Acetylation modifications affect a great number of biological processes of malignant tumors. The current study aims at revealing the role of acetylation-related mechanism in TNBC progression. Methyltransferase like-3 (METTL3) was found to be downregulated in TNBC cells via quantitative polymerase chain reaction (qPCR) and western blot analyses. Co-Immunoprecipitation (Co-IP) and GST pulldown assays revealed the interaction between acetyl-CoA acetyltransferase 1 (ACAT1) and METTL3. Through further immunoprecipitation (IP) assay, we determined that ACAT1 stabilizes METTL3 protein via inhibiting the degradation of ubiquitin-proteasome. Functionally, ACAT1 inhibits TNBC cell migration and invasion. Moreover, nuclear receptor subfamily 2 group F member 6 (NR2F6) regulates ACAT1 expression at transcriptional level. Finally, we demonstrated that NR2F6/ACAT/METTL3 axis suppresses the migration and invasion of TNBC cells via METTL3. In conclusion, NR2F6 transcriptionally activates ACAT1 and promotes the suppressive effects of ACAT1-mediated METTL3 acetylation on TNBC cell migration and invasion.

Effects of UBE3A on Cell and Liver Metabolism through the Ubiquitination of PDHA1 and ACAT1.

Nonalcoholic fatty liver disease (NAFLD) is substantiated by the reprogramming of liver metabolic pathways that disrupts the homeostasis of lipid and glucose metabolism and thus promotes the progression of the disease. The metabolic pathways associated with NAFLD are regulated at different levels from gene transcription to various post-translational modifications including ubiquitination. Here, we used a novel orthogonal ubiquitin transfer platform to identify pyruvate dehydrogenase A1 (PDHA1) and acetyl-CoA acetyltransferase 1 (ACAT1), two important enzymes that regulate glycolysis and ketogenesis, as substrates of E3 ubiquitin ligase UBE3A/E6AP. We found that overexpression of UBE3A accelerated the degradation of PDHA1 and promoted glycolytic activities in HEK293 cells. Furthermore, a high-fat diet suppressed the expression of UBE3A in the mouse liver, which was associated with increased ACAT1 protein levels, while forced expression of UBE3A in the mouse liver resulted in decreased ACAT1 protein contents. As a result, the mice with forced expression of UBE3A in the liver exhibited enhanced accumulation of triglycerides, cholesterol, and ketone bodies. These results reveal the role of UBE3A in NAFLD development by inducing the degradation of ACAT1 in the liver and promoting lipid storage. Overall, our work uncovers an important mechanism underlying the regulation of glycolysis and lipid metabolism through UBE3A-mediated ubiquitination of PDHA1 and ACAT1 to regulate their stabilities and enzymatic activities in the cell.

Identification of Six Thiolases and Their Effects on Fatty Acid and Ergosterol Biosynthesis in Aspergillus oryzae.

Thiolase plays important roles in lipid metabolism. It can be divided into degradative thiolases (thioase I) and biosynthetic thiolases (thiolases II), which are involved in fatty acid ß-oxidation and acetoacetyl-CoA biosynthesis, respectively. The Saccharomyces cerevisiae genome harbors only one gene each for thioase I and thiolase II, namely, Pot1 and Erg10, respectively. In this study, six thiolases (named AoErg10A to AoErg10F) were identified in Aspergillus oryzae genome using bioinformatics analysis. Quantitative reverse transcription-PCR (qRT-PCR) indicated that the expression of these six thiolases varied at different growth times and under different forms of abiotic stress. Subcellular localization analysis showed that AoErg10A was located in the cytoplasm, AoErg10B and AoErg10C were in the mitochondria, and AoErg10D, AoErg10E, and AoErg10F were in the peroxisome. Yeast heterologous complementation assays revealed that AoErg10A, AoErg10D, AoErg10E, AoErg10F, and cytoplasmic AoErg10B (AoErg10BΔMTS) recovered the phenotypes of S. cerevisiae erg10 weak and lethal mutants and that only AoErg10D, AoErg10E, and AoErg10F recovered the phenotype of the pot1 mutant that cannot use oleic acid as the carbon source. Overexpression of AoErg10s affected either the growth speed or the sporulation of the transgenic strains. In addition, the fatty acid and ergosterol content changed in all the AoErg10-overexpressing strains. This study revealed the function of six thiolases in A. oryzae and their effect on growth and fatty acid and ergosterol biosynthesis, which may lay the foundation for genetic engineering for lipid metabolism in A. oryzae or other fungi. IMPORTANCE Thiolases, including thioase I and thiolase II, play important roles in lipid metabolism. Aspergillus oryzae, one of the most industrially important filamentous fungi, has been widely used for manufacturing oriental fermented food such as sauce, miso, and sake for a long time. In addition, A. oryzae has a high capability in production of high lipid content and has been used for lipid production. Thus, it is very important to investigate the function of thiolases in A. oryzae. In this study, six thiolase (named AoErg10A to AoErg10F) were identified by bioinformatics analysis. Unlike other reported thiolases in fungi, three of the six thiolases showed dual functions of thioase I and thiolase II in S. cerevisiae, indicating that the lipid metabolism is more complex in A. oryzae. The reveal of function of these thiolases in A. oryzae can lay the foundation for genetic engineering for lipid metabolism in A. oryzae or other fungi.

Autophagy inhibition and reactive oxygen species elimination by acetyl-CoA acetyltransferase 1 through fused in sarcoma protein to promote prostate cancer.

BACKGROUND: Prostate cancer is a major health issue affecting the male population worldwide, and its etiology remains relatively unknown. As presented on the Gene Expression Profiling Interactive Analysis database, acetyl-CoA acetyltransferase 1 (ACAT1) acts as a prostate cancer-promoting factor. ACAT1 expression in prostate cancer tissues is considerably higher than that in normal tissues, leading to a poor prognosis in patients with prostate cancer. Here, we aimed to study the role of the ACAT1-fused in sarcoma (FUS) complex in prostate cancer and identify new targets for the diagnosis and treatment of the disease. METHODS: We conducted immunohistochemical analysis of 57 clinical samples and in vitro and in vivo experiments using a mouse model and plasmid constructs to determine the expression of ACAT1 in prostate cancer. RESULTS: The relationship between the expression of ACAT1 and the Gleason score was significant. The expression of ACAT1 was higher in tissues with a Gleason score of > 7 than in tissues with a Gleason score of ≤7 (P = 0.0011). In addition, we revealed that ACAT1 can interact with the FUS protein. CONCLUSIONS: In prostate cancer, ACAT1 promotes the expression of P62 and Nrf2 through FUS and affects reactive oxygen species scavenging. These effects are due to the inhibition of autophagy by ACAT1. That is, ACAT1 promotes prostate cancer by inhibiting autophagy and eliminating active oxygen species. The expression of ACAT1 is related to prostate cancer. Studying the underlying mechanism may provide a new perspective on the treatment of prostate cancer.

Assessment of the diagnostic and prognostic relevance of ACAT1 and CE levels in plasma, peritoneal fluid and tumor tissue of epithelial ovarian cancer patients - a pilot study.

BACKGROUND: Abnormal accumulation of acyl-CoA cholesterol acyltransferase-1 (ACAT1) and ACAT1-mediated cholesterol esterified with fatty acids (CE) contribute to cancer progression in various cancers. Our findings of increased CE and ACAT1 levels in epithelial ovarian cancer (EOC) cell lines prompted us to investigate whether such an increase occurs in primary clinical samples obtained from human subjects diagnosed with EOC. We evaluated the diagnostic/prognostic potential of ACAT1 and CE in EOC by: 1) assessing ACAT1 and CE levels in plasma, peritoneal fluid, and ovarian/tumor tissues; 2) assessing diagnostic performance by Receiver Operating Characteristic (ROC) analysis; and 3) comparing expression of ACAT1 and CE with that of tumor proliferation marker, Ki67. METHODS: ACAT1 protein levels in plasma, peritoneal fluid and tissue were measured via enzyme-linked immunosorbent assay. Tissue expression of ACAT1 and Ki67 proteins were confirmed by immunohistochemistry and mRNA transcript levels were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR). CE levels were assessed in plasma, peritoneal fluid (colorimetric assay) and in tissue (thin layer chromatography). RESULTS: Preoperative levels of ACAT1 and CE on the day of surgery were significantly higher in tissue and peritoneal fluid from EOC patients vs. the non-malignant group, which included subjects with benign tumors and normal ovaries; however, no significant differences were observed in plasma. In tissue and peritoneal fluid, positive correlations were observed between CE and ACAT1 levels, as well as between ACAT1/CE and Ki67. CONCLUSIONS: ACAT1 and CE accumulation may be linked to the aggressive potential of EOC; therefore, these mediators may be useful biomarkers for EOC prognosis and target-specific treatments.

Potentiating the antitumour response of CD8(+) T cells by modulating cholesterol metabolism.

CD8(+) T cells have a central role in antitumour immunity, but their activity is suppressed in the tumour microenvironment. Reactivating the cytotoxicity of CD8(+) T cells is of great clinical interest in cancer immunotherapy. Here we report a new mechanism by which the antitumour response of mouse CD8(+) T cells can be potentiated by modulating cholesterol metabolism. Inhibiting cholesterol esterification in T cells by genetic ablation or pharmacological inhibition of ACAT1, a key cholesterol esterification enzyme, led to potentiated effector function and enhanced proliferation of CD8(+) but not CD4(+) T cells. This is due to the increase in the plasma membrane cholesterol level of CD8(+) T cells, which causes enhanced T-cell receptor clustering and signalling as well as more efficient formation of the immunological synapse. ACAT1-deficient CD8(+) T cells were better than wild-type CD8(+) T cells at controlling melanoma growth and metastasis in mice. We used the ACAT inhibitor avasimibe, which was previously tested in clinical trials for treating atherosclerosis and showed a good human safety profile, to treat melanoma in mice and observed a good antitumour effect. A combined therapy of avasimibe plus an anti-PD-1 antibody showed better efficacy than monotherapies in controlling tumour progression. ACAT1, an established target for atherosclerosis, is therefore also a potential target for cancer immunotherapy.

Engineering potassium activation into biosynthetic thiolase.

Activation of enzymes by monovalent cations (M+) is a widespread phenomenon in biology. Despite this, there are few structure-based studies describing the underlying molecular details. Thiolases are a ubiquitous and highly conserved family of enzymes containing both K+-activated and K+-independent members. Guided by structures of naturally occurring K+-activated thiolases, we have used a structure-based approach to engineer K+-activation into a K+-independent thiolase. To our knowledge, this is the first demonstration of engineering K+-activation into an enzyme, showing the malleability of proteins to accommodate M+ ions as allosteric regulators. We show that a few protein structural features encode K+-activation in this class of enzyme. Specifically, two residues near the substrate-binding site are sufficient for K+-activation: A tyrosine residue is required to complete the K+ coordination sphere, and a glutamate residue provides a compensating charge for the bound K+ ion. Further to these, a distal residue is important for positioning a K+-coordinating water molecule that forms a direct hydrogen bond to the substrate. The stability of a cation-π interaction between a positively charged residue and the substrate is determined by the conformation of the loop surrounding the substrate-binding site. Our results suggest that this cation-π interaction effectively overrides K+-activation, and is, therefore, destabilised in K+-activated thiolases. Evolutionary conservation of these amino acids provides a promising signature sequence for predicting K+-activation in thiolases. Together, our structural, biochemical and bioinformatic work provide important mechanistic insights into how enzymes can be allosterically activated by M+ ions.

OCT1 - a yeast mitochondrial thiolase involved in the 3-oxoadipate pathway.

The 3-oxoacyl-CoA thiolases catalyze the last step of the fatty acid ß-oxidation pathway. In yeasts and plants, this pathway takes place exclusively in peroxisomes, whereas in animals it occurs in both peroxisomes and mitochondria. In contrast to baker's yeast Saccharomyces cerevisiae, yeast species from the Debaryomycetaceae family also encode a thiolase with predicted mitochondrial localization. These yeasts are able to utilize a range of hydroxyaromatic compounds via the 3-oxoadipate pathway the last step of which is catalyzed by 3-oxoadipyl-CoA thiolase and presumably occurs in mitochondria. In this work, we studied Oct1p, an ortholog of this enzyme from Candida parapsilosis. We found that the cells grown on a 3-oxoadipate pathway substrate exhibit increased levels of the OCT1 mRNA. Deletion of both OCT1 alleles impairs the growth of C. parapsilosis cells on 3-oxoadipate pathway substrates and this defect can be rescued by expression of the OCT1 gene from a plasmid vector. Subcellular localization experiments and LC-MS/MS analysis of enriched organellar fraction-proteins confirmed the presence of Oct1p in mitochondria. Phylogenetic profiling of Oct1p revealed an intricate evolutionary pattern indicating multiple horizontal gene transfers among different fungal groups.

Production of D-lactic acid containing polyhydroxyalkanoate polymers in yeast Saccharomyces cerevisiae.

Polyhydroxyalkanoates (PHAs) provide biodegradable and bio-based alternatives to conventional plastics. Incorporation of 2-hydroxy acid monomers into polymer, in addition to 3-hydroxy acids, offers possibility to tailor the polymer properties. In this study, poly(D-lactic acid) (PDLA) and copolymer P(LA-3HB) were produced and characterized for the first time in the yeast Saccharomyces cerevisiae. Expression of engineered PHA synthase PhaC1437Ps6-19, propionyl-CoA transferase Pct540Cp, acetyl-CoA acetyltransferase PhaA, and acetoacetyl-CoA reductase PhaB1 resulted in accumulation of 3.6% P(LA-3HB) and expression of engineered enzymes PhaC1Pre and PctMe resulted in accumulation of 0.73% PDLA of the cell dry weight (CDW). According to NMR, P(LA-3HB) contained D-lactic acid repeating sequences. For reference, expression of PhaA, PhaB1, and PHA synthase PhaC1 resulted in accumulation 11% poly(hydroxybutyrate) (PHB) of the CDW. Weight average molecular weights of these polymers were comparable to similar polymers produced by bacterial strains, 24.6, 6.3, and 1 130 kDa for P(LA-3HB), PDLA, and PHB, respectively. The results suggest that yeast, as a robust and acid tolerant industrial production organism, could be suitable for production of 2-hydroxy acid containing PHAs from sugars or from 2-hydroxy acid containing raw materials. Moreover, the wide substrate specificity of PHA synthase enzymes employed increases the possibilities for modifying copolymer properties in yeast in the future.

IpdAB, a virulence factor in Mycobacterium tuberculosis, is a cholesterol ring-cleaving hydrolase.

Mycobacterium tuberculosis (Mtb) grows on host-derived cholesterol during infection. IpdAB, found in all steroid-degrading bacteria and a determinant of pathogenicity, has been implicated in the hydrolysis of the last steroid ring. Phylogenetic analyses revealed that IpdAB orthologs form a clade of CoA transferases (CoTs). In a coupled assay with a thiolase, IpdAB transformed the cholesterol catabolite (R)-2-(2-carboxyethyl)-3-methyl-6-oxocyclohex-1-ene-1-carboxyl-CoA (COCHEA-CoA) and CoASH to 4-methyl-5-oxo-octanedioyl-CoA (MOODA-CoA) and acetyl-CoA with high specificity (kcat/Km = 5.8 ± 0.8 × 104 M-1⋅s-1). The structure of MOODA-CoA was consistent with IpdAB hydrolyzing COCHEA-CoA to a ß-keto-thioester, a thiolase substrate. Contrary to characterized CoTs, IpdAB exhibited no activity toward small CoA thioesters. Further, IpdAB lacks the catalytic glutamate residue that is conserved in the ß-subunit of characterized CoTs and a glutamyl-CoA intermediate was not trapped during turnover. By contrast, Glu105A, conserved in the α-subunit of IpdAB, was essential for catalysis. A crystal structure of the IpdAB·COCHEA-CoA complex, solved to 1.4 Å, revealed that Glu105A is positioned to act as a catalytic base. Upon titration with COCHEA-CoA, the E105AA variant accumulated a yellow-colored species (λmax = 310 nm; Kd = 0.4 ± 0.2 µM) typical of ß-keto enolates. In the presence of D2O, IpdAB catalyzed the deuteration of COCHEA-CoA adjacent to the hydroxylation site at rates consistent with kcat Based on these data and additional IpdAB variants, we propose a retro-Claisen condensation-like mechanism for the IpdAB-mediated hydrolysis of COCHEA-CoA. This study expands the range of known reactions catalyzed by the CoT superfamily and provides mechanistic insight into an important determinant of Mtb pathogenesis.

[Analysis of ACAT1 gene variants in a patient with ß-ketothiolase deficiency].

OBJECTIVE: To explore the genetic etiology of a child suspected for ß-ketothiolase deficiency by neonatal screening. METHODS: All coding exons and flanking sequences of the ACAT1 gene were subjected to targeted capture and high-throughput sequencing. Suspected variants were verified by Sanger sequencing and bioinformatic analysis. RESULTS: The child was found to harbor compound heterozygous variants of the ACAT1 gene, namely c.121-3C>G and c.275G>A (p. Gly92Asp). The c.121-3C>G variant was also detected in his father and two sisters, while the c.275G>A (p. Gly92Asp) was a de novo variant. A c.334+ 172C>G (rs12226047) polymorphism was also detected in his mother and two sisters. Sanger sequencing has verified that the c.275G>A (p. Gly92Asp) and c.334+172C>G (rs12226047) variants are located on the same chromosome. Bioinformatics analysis suggested both c.121-3C>G and c.275G>A (p.G92D) variants to be damaging. Based on the American College of Medical Genetics and Genomics standards and guidelines, the c.275G>A variant of the ACAT1 gene was predicted to be pathogenic (PS2+ PM2+ PM3+ PP3+PP4), the c.121-3C>G variant to be likely pathogenic (PM2+ PM3+ PP3+PP4). CONCLUSION: The c.121-3C>G and c.275G>A variants of the ACAT1 gene probably underlay the pathogenesis of the child. Above finding has enriched the variant spectrum of the ACAT1 gene.

Identification of a four-gene metabolic signature predicting overall survival for hepatocellular carcinoma.

While hundreds of consistently altered metabolic genes had been identified in hepatocellular carcinoma (HCC), the prognostic role of them remains to be further elucidated. Messenger RNA expression profiles and clinicopathological data were downloaded from The Cancer Genome Atlas-Liver Hepatocellular Carcinoma and GSE14520 data set from the Gene Expression Omnibus database. Univariate Cox regression analysis and lasso Cox regression model established a novel four-gene metabolic signature (including acetyl-CoA acetyltransferase 1, glutamic-oxaloacetic transaminase 2, phosphatidylserine synthase 2, and uridine-cytidine kinase 2) for HCC prognosis prediction. Patients in the high-risk group shown significantly poorer survival than patients in the low-risk group. The signature was significantly correlated with other negative prognostic factors such as higher α-fetoprotein. The signature was found to be an independent prognostic factor for HCC survival. Nomogram including the signature shown some clinical net benefit for overall survival prediction. Furthermore, gene set enrichment analyses revealed several significantly enriched pathways, which might help explain the underlying mechanisms. Our study identified a novel robust four-gene metabolic signature for HCC prognosis prediction. The signature might reflect the dysregulated metabolic microenvironment and provided potential biomarkers for metabolic therapy and treatment response prediction in HCC.

Aspergillus fumigatus Mitochondrial Acetyl Coenzyme A Acetyltransferase as an Antifungal Target.

Ergosterol plays an important role in maintaining cell membrane sterol homeostasis in fungi, and as such, it is considered an effective target in antifungal chemotherapy. In yeast, the enzyme acetyl-coenzyme A (CoA) acetyltransferase (ERG10) catalyzes the Claisen condensation of two acetyl-CoA molecules to acetoacetyl-CoA in the ergosterol biosynthesis pathway and is reported as being critical for cell viability. Using yeast ERG10 for alignment, two orthologues, AfERG10A (AFUB_000550) and AfERG10B (AFUB_083570), were discovered in the opportunistic fungal pathogen Aspergillus fumigatus Despite the essentiality of AfERG10B having been previously validated, the biological function of AfERG10A remains unclear. In this study, we have characterized recombinant AfERG10A as a functional acetyl-CoA acetyltransferase catalyzing both synthetic and degradative reactions. Unexpectedly, AfERG10A localizes to the mitochondria in A. fumigatus, as shown by C-terminal green fluorescent protein (GFP) tag fusion. Both knockout and inducible promoter strategies demonstrate that Aferg10A is essential for the survival of A. fumigatus The reduced expression of Aferg10A leads to severe morphological defects and increased susceptibility to oxidative and cell wall stresses. Although the catalytic mechanism of acetyl-CoA acetyltransferase family is highly conserved, the crystal structure of AfERG10A and its complex with CoA are solved, revealing four substitutions within the CoA binding site that are different from human orthologues. Taken together, our combination of genetic and structural studies demonstrates that mitochondrial AfERG10A is essential for A. fumigatus cell viability and could be a potential drug target to feed the antifungal drug development pipeline.IMPORTANCE A growing number of people worldwide are suffering from invasive aspergillosis caused by the human opportunistic fungal pathogen A. fumigatus Current therapeutic options rely on a limited repertoire of antifungals. Ergosterol is an essential component of the fungal cell membrane as well as a target of current antifungals. Approximately 20 enzymes are involved in ergosterol biosynthesis, of which acetyl-CoA acetyltransferase (ACAT) is the first enzyme. Two ACATs in A. fumigatus are AfErg10A and AfErg10B. However, the biological function of AfErg10A is yet to be investigated. In this study, we showed that AfErg10A is localized in the mitochondria and is essential for A. fumigatus survival and morphological development. In combination with structural studies, we validated AfErg10A as a potential drug target that will facilitate the development of novel antifungals and improve the efficiency of existing drugs.

Molecular cloning, characterization, and heterologous expression of an acetyl-CoA acetyl transferase gene from Sanghuangporus baumii.

Acetyl-CoA C-acetyltransferase synthase gene (AACT) cDNA, DNA and promoter were cloned from Sanghuangporus baumii. The gene ORF (1260 bp) encoded 419 amino acids. The AACT DNA includes five exons (1-84 bp, 140-513 bp, 570-1027 bp, 1090-1282 bp, 1344-1494 bp) and four introns (85-139 bp, 514-569 bp, 1028-1089 bp, 1283-1343 bp). The molecular weight of AACT protein is 43.40 kDa, it is hydrophilic with a theoretical isoelectric point of 8.96. Furthermore, The region of the transcription start site is 1997-2047 bp of AACT promoter, and it contained promoter elements (TATA Boxs, CAAT Boxs, CAAT-box, ABRE, G-Boxs, Sp1, MSA-like, LTR). AACT recombinant protein (43.40 KDa + Tag protein 22.68 KDa) was subjected in SDS-PAGE. AACT the transcription levels of in different development stages were investigated. The expression of AACT in primordia (2.4-fold) and 15 d mycelia (2.3- fold) were significantly higher than 9 d mycelia (contral). The expression level of the AACT downstream genes and triterpenoids content were determined at different developmental stages. Triterpenoid content reached its peak on day 15(7.21 mg/g).

The 3-ketoacyl-CoA thiolase: an engineered enzyme for carbon chain elongation of chemical compounds.

Because of their function of catalyzing the rearrangement of the carbon chains, thiolases have attracted increasing attentions over the past decades. The 3-ketoacyl-CoA thiolase (KAT) is a member of the thiolase, which is capable of catalyzing the Claisen condensation reaction between the two acyl-CoAs, thereby achieving carbon chain elongation. In this way, diverse value-added compounds might be synthesized starting from simple small CoA thioesters. However, most KATs are hampered by low stability and poor substrate specificity, which has hindered the development of large-scale biosynthesis. In this review, the common characteristics in the three-dimensional structure of KATs from different sources are summarized. Moreover, structure-guided rational engineering is discussed as a strategy for enhancing the performance of KATs. Finally, we reviewed the metabolic engineering applications of KATs for producing various energy-storage molecules, such as n-butanol, fatty acids, dicarboxylic acids, and polyhydroxyalkanoates. KEY POINTS: • Summarize the structural characteristics and catalyzation mechanisms of KATs. • Review on the rational engineering to enhance the performance of KATs. • Discuss the applications of KATs for producing energy-storage molecules.

Downregulation of Acat1 by miR-21 may participate in liver fibrosis upon chronic DDT exposure.

The main objective of the present study was to investigate the toxic effect of long-term exposure to DDT (2,2-dichlorodiphenyl-1,1,1-trichloroethane) on rat livers. Female Wistar rats were treated with once-weekly i.p. doses of DDT (10 and 50 mg/kg) for 12 weeks. Histological analysis revealed significant changes in the liver structure, especially at a dose of 50 mg/kg, which consistent with a fibrotic state. Long-term DDT exposure increased micro RNA-21 (miR-21) level and decreased Acetyl-CoA acetyltransferase 1 (Acat1) mRNA and protein levels in a dose-dependent manner. A dual-luciferase reporter assay confirmed the regulation of the rat Acat1 3'-UTR by miR-21. Previous studies have described the involvement of ACAT1 in fibrogenesis; thus, regulation of the Acat1 gene by miR-21 may play a role in DDT exposure-mediated liver fibrosis.

Mutation update on ACAT1 variants associated with mitochondrial acetoacetyl-CoA thiolase (T2) deficiency.

Mitochondrial acetoacetyl-CoA thiolase (T2, encoded by the ACAT1 gene) deficiency is an inherited disorder of ketone body and isoleucine metabolism. It typically manifests with episodic ketoacidosis. The presence of isoleucine-derived metabolites is the key marker for biochemical diagnosis. To date, 105 ACAT1 variants have been reported in 149 T2-deficient patients. The 56 disease-associated missense ACAT1 variants have been mapped onto the crystal structure of T2. Almost all these missense variants concern residues that are completely or partially buried in the T2 structure. Such variants are expected to cause T2 deficiency by having lower in vivo T2 activity because of lower folding efficiency and/or stability. Expression and activity data of 30 disease-associated missense ACAT1 variants have been measured by expressing them in human SV40-transformed fibroblasts. Only two variants (p.Cys126Ser and p.Tyr219His) appear to have equal stability as wild-type. For these variants, which are inactive, the side chains point into the active site. In patients with T2 deficiency, the genotype does not correlate with the clinical phenotype but exerts a considerable effect on the biochemical phenotype. This could be related to variable remaining residual T2 activity in vivo and has important clinical implications concerning disease management and newborn screening.