[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.

Dynamics of the Trypanosoma cruzi infection in adipose tissue: Assessing gene expression of PNPLA2, FASN, and ACAT1 under Benzonidazole treatment and indirect mononuclear immune cells interaction.

Trypanosoma cruzi is a parasite with a high capacity to adapt to the host. Animal models have already demonstrated that the tropism of this parasite occurs not only in cardiac/digestive tissues but also in adipose tissue (AT). That said, the consequences ofT. cruziinfection for AT and the implications of treatment with Benzonidazole in this tissue are under discussion. Here, we tested the hypothesis that T. cruzi infection in adipose tissue upon treatment with Benzonidazole (Bz) and the interaction of mononuclear immune cells (PBMC) influences the relative expression of ACAT1, FASN, and PNPLA2 genes. Thus, stem cells derived from adipose tissue (ADSC) after adipogenic differentiation were indirectly cultivated with PBMC after infection with the T. cruzi Y strain and treatment with Bz. We use the TcSAT-IAM system and RT-qPCR to evaluate the parasite load and the relative quantification (ΔCt) of the ACAT1, FASN, and PNPLA2 genes. Our results demonstrate that treatment with Bz did not reduce adipocyte infection in the presence (p-value: 0.5796) or absence (p-value: 0.1854) of cultivation with PBMC. In addition, even though there is no statistical difference when compared to the control group (AT), T. cruzi induces the FASN expression (Rq: 14.00). However, treatment with Bz in AT suggests the increases of PNPLA2 expression levels (Rq: 12.58), even in the absence of T. cruzi infection. During indirect cultivation with PBMC, T. cruzi smooths the expression of PNPLA2 (Rq: 0.824) and instigates the expression of ACAT1 (Rq: 1.632) and FASN (Rq: 1.394). Furthermore, the treatment with Bz during infection induces PNPLA2 expression (Rq: 1.871), maintaining FASN expression levels (Rq: 1.334). Given this, our results indicate that treatment with Benzonidazole did not decrease T. cruzi infection in adipose tissue. However, treating the adipocyte cells with Bz during the interaction with PBMC cells influences the lipid pathways scenario, inducing lipolytic metabolism through the expression of PNPLA2.

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.

Elevated serum ß-hydroxybutyrate, a circulating ketone metabolite, accelerates colorectal cancer proliferation and metastasis via ACAT1.

Colorectal cancer (CRC) ranks third in incidence and second in mortality worldwide. Metabolic disorders are known to be closely associated with CRC. Functional metabolomics aims to translate metabolomics-derived biomarkers to disease mechanisms. Previous work based on untargeted liquid chromatography identified 30 differential metabolites of CRC. Among them, only ß-hydroxybutyrate (BHB) was elevated in CRC. Here, we first confirm the increased level of ß-hydroxybutyrate by targeted metabolomic analysis using an independent cohort of 400 serum samples by UPLC-QQQ-MS/MS analysis. Using appropriate cell and animal models, we find that treatment with pathological levels of ß-hydroxybutyrate expedites CRC proliferation and metastasis. Out of four major rate-limiting enzymes of ketolysis, only acetyl-coenzyme A acetyltransferase1 (ACAT1) expression is increased in paired human CRC tissues. These findings suggest probable clinical relevance for the functional implications of ß-hydroxybutyrate in CRC. We demonstrate that ß-hydroxybutyrate may exert its tumorigenic effects via regulation of ACAT1, due to induction of downstream isocitrate dehydrogenase1 (IDH1) acetylation. Genetic silencing of ACAT1 significantly suppresses the progression of CRC and abrogates the effects of ß-hydroxybutyrate both in vitro and in vivo. Overall, this study suggests that targeting ß-hydroxybutyrate and its major rate-limiting enzyme ACAT1 may provide a new avenue for therapeutic intervention in CRC.

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.

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.

Cloning, Expression, and Functional Analysis of the Full-Length cDNA of Acetyl-CoA C-acetyltransferase (AACT) Genes Related to Terpenoid Synthesis in Platycodon grandiflorus.

Platycodon grandiflorus is a well-known and widely distributed traditional herbal medicine and functional food in Asia, with triterpenoids as the main bioactive component in its roots. Acetyl-CoA C-acetyltransferase (AACT) is the initiation enzyme in the mevalonate pathway and plays an important role in the biosynthesis of terpenoids. OBJECTIVE: The objective of this study was to clone and identify the PgAACT function in P. grandiflorus. METHODS: The full-length sequence of PgAACT genes was isolated and cloned from P. grandiflorus by polymerase chain reaction (PCR). The recombinant plasmid was constructed using the pET-32a vector and expressed in E. coli Transetta (DE3) cells. Subcellular localization of AACT was observed in the epidermal cells of N. tabacum. Quantitative reverse transcription-PCR (qRT-PCR) was used to identify the PgAACT gene transcription levels. After MeJA treatment, the changes in AACT gene expression were observed, and UHPLC-Q-Exactive Orbitrap MS/MS was used to detect the changes in P. grandiflorus saponins. RESULTS: In this study, two full-length cDNAs encoding AACT1 (PgAACT1) and AACT2 (PgAACT2) were isolated and cloned from P. grandiflorus. The deduced PgAACT1 and PgAACT2 proteins contain 408 and 416 amino acids, respectively. The recombinant vectors were constructed, and the protein expression was improved by optimizing the reaction conditions. Sodium dodecyl sulphate-polycrylamide gel electrophloresis and western blot analysis showed that the PgAACT genes were successfully expressed, with molecular weights of the recombinant proteins of 61 and 63 kDa, respectively. Subcellular localization showed that the PgAACT genes were localized in the cytoplasm. Tissue specificity analysis of P. grandiflorus from different habitats showed that PgAACT genes were expressed in the roots, stems, and leaves. After MeJA treatment, the expression level of PgAACT genes and the content of total saponins of P. grandiflorus were significantly increased, suggesting that PgAACT genes play an important role in regulating plant defense systems. CONCLUSION: Cloning, expression, and functional analysis of PgAACT1 and PgAACT2 will be helpful in understanding the role of these two genes in terpene biosynthesis.

Identification of two novel ACAT1 variant associated with beta-ketothiolase deficiency in a 9-month-old boy.

OBJECTIVES: Mitochondrial acetoacetyl-CoA thiolase (beta-ketothiolase, T2) is necessary for the catabolism of ketone bodies andisoleucine. T2 deficiency is an autosomal recessive metabolic disorder caused by variant in the ACAT1 gene. In this report, we describe two novel ACAT1 variant identified in a Chinese family. CASE PRESENTATION: The 9-month-old male proband was admitted to the pediatric intensive care unit for altered consciousness. At the time of admission, the patient had acidosis, drowsiness, and respiratory failure. Both urine organic acid analyses and LC-MS/MS suggested T2 deficiency. Novel compound heterozygous variant (c.871G>C and c.1016_1017del) in the ACAT1 gene were detected in the proband by WES and verified through direct sequencing. Family analysis demonstrated that the first variant was transmitted from his father and the second variant was from his mother, indicating autosomal recessive inheritance. This report is the first to describe the association of these variant with T2 deficiency based on genetic testing. Although these variant were identified in the patient's elder sister and elder brother, they continue to be asymptomatic. CONCLUSIONS: We identified two novel ACAT1 variants associated with T2 deficiency. The identification expands the spectrum of known variant linked to the disorder.

Integrated omics approach to unveil antifungal bacterial polyynes as acetyl-CoA acetyltransferase inhibitors.

Bacterial polyynes are highly active natural products with a broad spectrum of antimicrobial activities. However, their detailed mechanism of action remains unclear. By integrating comparative genomics, transcriptomics, functional genetics, and metabolomics analysis, we identified a unique polyyne resistance gene, masL (encoding acetyl-CoA acetyltransferase), in the biosynthesis gene cluster of antifungal polyynes (massilin A 1, massilin B 2, collimonin C 3, and collimonin D 4) of Massilia sp. YMA4. Crystallographic analysis indicated that bacterial polyynes serve as covalent inhibitors of acetyl-CoA acetyltransferase. Moreover, we confirmed that the bacterial polyynes disrupted cell membrane integrity and inhibited the cell viability of Candida albicans by targeting ERG10, the homolog of MasL. Thus, this study demonstrated that acetyl-CoA acetyltransferase is a potential target for developing antifungal agents.

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.

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.

Epigenetic inactivation of ACAT1 promotes epithelial-mesenchymal transition of clear cell renal cell carcinoma.

BACKGROUND: Acetyl-CoA acyltransferase 1 (ACAT1) is a key enzyme catalyzing the production of mitochondrial ketone bodies. We have shown that ACAT1 is down-regulated in kidney renal clear cell carcinoma (KIRC) previously. OBJECTIVE: To investigate the reasons for downregulation of ACAT1 in KIRC and explore the underlying mechanisms involved in metastatic inhibition regulated by ACAT1. METHODS: The Gene Expression Omnibus (GEO) database was queried for meta-analysis of ACAT1 mRNA expression in KIRC. The UALCAN website was used to compare the methylation levels of the ACAT1 promoter region in KIRC and normal tissues. RT-qPCR was used to quantitate ACAT1 transcription levels. The GCBI and Tarbase V.8 databases were used to predict miRNAs that may target the mRNA of ACAT1. The correlation between mRNA expression of ACAT1, MMP7 (matrix metallopeptidase 7), CDH1 (E-cadherin), EpCAM (epithelial cell adhesion molecule), and VIM (vimentin) was analyzed. Extracellular MMP7 protein was quantitated using an ELISA assay. RESULTS: The methylation level of the ACAT1 promoter region in KIRC was significantly higher than that in the normal kidney tissues. The ACAT1 mRNA expression in the KIRC cell lines was restored after treatment with 5-aza-dC (p < 0.05). MiR-21-5p is a conserved microRNA targeting ACAT1. It is expressed at a significantly higher level in KIRC than in normal tissues (p < 0.001). MiR-21-5p miRNA expression negatively correlates with ACAT1 mRNA expression. The expression of miR-21-5p is higher at the T3-T4 stages and in the histologic grades G3-G4. Patients with high miR-21-5p expression tended to have lower overall survival, suggesting that miR-21-5p could serve as a potentially valuable diagnostic biomarker for KIRC (AUC = 0.957; p < 0.001). A mimetic of miR-21-5p inhibited the expression of ACAT1 mRNA and protein. In addition, ACAT1 mRNA expression positively correlates with CDH1 and EpCAM but is negatively correlated with VIM. Overexpression of ACAT1 suppresses the secretion of MMP7 in KIRC cells. CONCLUSION: Expression of ACAT1 in KIRC is controlled at two levels, firstly by the hypermethylation of the ACAT1 promoter region and secondly by overexpression of miR-21-5p. Downregulation of ACAT1 expression correlates with epithelial-mesenchymal transition (EMT).

Cloning, Prokaryotic Expression, and Purification of Acetyl-CoA C-Acetyltransferase from Atractylodes lancea.

BACKGROUND: Cangzhu (Atractylodes lancea), a valuable and common traditional Chinese medicinal herb, is primarily used as an effective medicine with various health-promoting effects. The main pharmacological bioactive ingredients in the rhizome of A. lancea are terpenoids. Acetyl-CoA C-acetyltransferase (AACT) is the first enzyme in the terpenoid synthesis pathway and catalyzes two units of acetyl-CoA into acetoacetyl-CoA. OBJECTIVE: The objective of the present work was to clone and identify function of AlAACT from Atractylodes lancea. METHODS: A full-length cDNA clone of AlAACT was isolated using PCR and expressed in Escherichia coli. The expressed protein was purified using Ni-NTA agarose column using standard protocols. AlAACT was transiently expressed in N. benthamiana leaves to determine their subcellular location. The difference in growth between recombinant bacteria and control bacteria under different stresses was observed using the droplet plate experiment. RESULTS: In this study, a full-length cDNA of AACT (AlAACT) was cloned from A. lancea, which contains a 1,227 bp open reading frame and encodes a protein with 409 amino acids. Bioinformatic and phylogenetic analysis clearly suggested that AlAACT shared high similarity with AACTs from other plants. The recombinant protein pET32a(+)/AlAACT was successfully expressed in Escherichia coli BL21 (DE3) cells induced with 0.4 mM IPTG at 30°C as the optimized condition. The recombinant enzyme pET-32a-AlAACT was purified using the Ni-NTA column based on the His-tag, and the molecular weight was determined to be 62 kDa through SDS-PAGE and Western Blot analysis. The recombinant protein was eluted with 100, 300, and 500 mM imidazole; most of the protein was eluted with 300 mM imidazole. Under mannitol stress, the recombinant pET-32a- AlAACT protein showed a substantial advantage in terms of growth rates compared to the control. However, this phenomenon was directly opposite under NaCl abiotic stress. Subcellular localization showed that AlAACT localizes to the nucleus and cytoplasm. CONCLUSION: The expression and purification of recombinant enzyme pET-32a-AlAACT were successful, and the recombinant strain pET-32a-AlAACT in showed better growth in a drought stress. The expression of AlAACT-EGFP fusion protein revealed its localization in both nuclear and cytoplasm compartments. This study provides an important foundation for further research into the effects of terpenoid biosynthesis in A. lancea.

Association between ACAT1 rs1044925 and increased hypertension risk in Tongdao Dong.

Hypertension is a multifactorial disease that partially caused by genetic factors, including variation in genes related to lipid metabolism. ACAT1 gene is implicated in lipid metabolism for its encoding product, the enzyme acetyl-CoA acetyltransferase 1, catalyzing the synthesis of cholesteryl ester from cholesterol and playing an important role in the metabolism of cholesterol. Until now, there's little study on the relationship between ACAT1 variants and hypertension. Here, we report a link between ACAT1 rs1044925 and hypertension in Tongdao Dong population. Polymerase chain reaction-restriction fragment length polymorphism was used to detect the genotypes of the ACAT1 SNP rs1044925 in a total of 637 subjects, including 406 hypertensive patients and 231 normotensive controls. The genotypic and allelic frequencies of rs1044925 were significantly different between the normotensive and hypertensive subjects (P = .001). AC/CC genotypes of rs1044925 were associated with an increased risk of hypertension (AC/CC vs AA: adjusted odds ratio = 1.723, 95% confidence interval = 1.160-2.559, P = .007). However, the AC/CC genotypes showed no relationship with serum lipid levels. The results suggest that the C carriers of ACAT1 rs1044925 might increase the risk of hypertension in Tongdao Dong population, and the underlying mechanism needs to be further studied.

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.

Improvement of hyperlipidemia by aerobic exercise in mice through a regulatory effect of miR-21a-5p on its target genes.

Hyperlipidemia is a risk factor for cardiovascular disease, and miR-21a-5p plays an important role in the occurrence and progression of hyperlipidemia. Here, we aimed to investigate the mechanism of aerobic exercise improved hyperlipidemia through enhancing miR-21a-5p expression. In this study, high-fat/high-cholesterol diet mice received 8 weeks of aerobic exercise intervention, then we collected plasma and liver samples, we found that there had a notable improvement in weight gain, blood lipid level, and liver steatosis in hyperlipidemia mice after 8 weeks of aerobic exercise intervention. Besides, aerobic exercise significantly up-regulated the expression of miR-21a-5p and provoked favorable changes in the expression of target genes. Knockdown of miR-21a-5p resulted in dysregulation of lipid metabolism and increased expression of FABP7, HMGCR, ACAT1, and OLR1. While aerobic exercise could alleviate miR-21a-5p knock-down induced lipid metabolism disorder. Taken together, these results demonstrated that aerobic exercise improved hyperlipidemia through miR-21a-5p-induced inhibition of target genes FABP7, HMGCR, ACAT1, and OLR1.

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.

The unique molecular targets associated antioxidant and antifibrotic activity of curcumin in in vitro model of acute lung injury: A proteomic approach.

Bleomycin (BLM) injury is associated with the severity of acute lung injury (ALI) leading to fibrosis, a high-morbidity, and high-mortality respiratory disease of unknown etiology. BLM-induced ALI is marked by the activation of a potent fibrogenic cytokine transcription growth factor beta-1 (TGFß-1), which is considered a critical cytokine in the progression of alveolar injury. Previously, our work demonstrated that a diet-derived compound curcumin (diferuloylmethane), represents its antioxidative and antifibrotic application in TGF-ß1-mediated BLM-induced alveolar basal epithelial cells. However, curcumin-specific protein targets, as well as its mechanism using mass spectrometry-based proteomic approach, remain elusive. To elucidate the underlying mechanism, a quantitative proteomics approach and bioinformatics analysis were employed to identify the protein targets of curcumin in BLM or TGF-ß1-treated cells. With subsequent in vitro experiments, curcumin-related pathways and cellular processes were predicted and validated. The current study discusses two separate proteomics experiments using BLM and TGF-ß1-treated cells with the proteomics approach, various unique target proteins were identified, and proteomic analysis revealed that curcumin reversed the expressions of unique proteins like DNA topoisomerase 2-alpha (TOP2A), kinesin-like protein (KIF11), centromere protein F (CENPF), and so on BLM or TGF-ß1 injury. For the first time, the current study reveals that curcumin restores TGF-ß1 induced peroxisomes like PEX-13, PEX-14, PEX-19, and ACOX1. This was verified by subsequent in vitro assays. This study generated molecular evidence to deepen our understanding of the therapeutic role of curcumin at the proteomic level and may be useful to identify molecular targets for future drug discovery.

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.