RESUMEN
Ketosis in dairy cows is often accompanied by the dysregulation of lipid homeostasis in the liver. Acetyl-coenzyme A acetyltransferase 2 (ACAT2) is specifically expressed in the liver and is important for regulating lipid homeostasis in ketotic cows. Lentinan (LNT) has a wide range of pharmacological activities, and this study investigates the protective effects of LNT on ß-hydroxybutyrate (BHBA)-induced lipid metabolism disorder in bovine hepatocytes (BHECs) and elucidates the underlying mechanisms. BHECs were first pretreated with LNT to investigate the effect of LNT on BHBA-induced lipid metabolism disorder in BHECs. ACAT2 was then silenced or overexpressed to investigate whether this mediated the protective action of LNT against BHBA-induced lipid metabolism disorder in BHECs. Finally, BHECs were treated with LNT after silencing ACAT2 to investigate the interaction between LNT and ACAT2. LNT pretreatment effectively enhanced the synthesis and absorption of cholesterol, inhibited the synthesis of triglycerides, increased the expression of ACAT2, and elevated the contents of very low-density lipoprotein and low-density lipoprotein cholesterol, thereby ameliorating BHBA-induced lipid metabolism disorder in BHECs. The overexpression of ACAT2 achieved a comparable effect to LNT pretreatment, whereas the silencing of ACAT2 aggravated the effect of BHBA on inducing disorder in lipid metabolism in BHECs. Moreover, the protective effect of LNT against lipid metabolism disorder in BHBA-induced BHECs was abrogated upon silencing of ACAT2. Thus, LNT, as a natural protective agent, can enhance the regulatory capacity of BHECs in maintaining lipid homeostasis by upregulating ACAT2 expression, thereby ameliorating the BHBA-induced lipid metabolism disorder.
Asunto(s)
Ácido 3-Hidroxibutírico , Acetil-CoA C-Acetiltransferasa , Hepatocitos , Metabolismo de los Lípidos , Regulación hacia Arriba , Animales , Bovinos , Hepatocitos/metabolismo , Hepatocitos/efectos de los fármacos , Ácido 3-Hidroxibutírico/metabolismo , Ácido 3-Hidroxibutírico/farmacología , Metabolismo de los Lípidos/efectos de los fármacos , Acetil-CoA C-Acetiltransferasa/genética , Acetil-CoA C-Acetiltransferasa/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Trastornos del Metabolismo de los Lípidos/metabolismo , Trastornos del Metabolismo de los Lípidos/genética , Trastornos del Metabolismo de los Lípidos/tratamiento farmacológico , Trastornos del Metabolismo de los Lípidos/inducido químicamente , Triglicéridos/metabolismo , Enfermedades de los Bovinos/metabolismo , Enfermedades de los Bovinos/genética , Enfermedades de los Bovinos/tratamiento farmacológico , Cetosis/metabolismo , Cetosis/genética , Cetosis/inducido químicamenteRESUMEN
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.
Asunto(s)
Aciltransferasas , Tejido Adiposo , Acido Graso Sintasa Tipo I , Leucocitos Mononucleares , Lipasa , Trypanosoma cruzi , Humanos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/parasitología , Tejido Adiposo/parasitología , Tejido Adiposo/metabolismo , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/genética , Lipasa/genética , Lipasa/metabolismo , Acido Graso Sintasa Tipo I/genética , Acido Graso Sintasa Tipo I/metabolismo , Acetil-CoA C-Acetiltransferasa/genética , Acetil-CoA C-Acetiltransferasa/metabolismo , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/parasitología , Enfermedad de Chagas/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Carga de Parásitos , Expresión Génica , Células CultivadasRESUMEN
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.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , MicroARNs , ARN Largo no Codificante , Humanos , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Línea Celular Tumoral , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Movimiento Celular/genética , Acetil-CoA C-Acetiltransferasa/genética , Acetil-CoA C-Acetiltransferasa/metabolismoRESUMEN
Polyhydroxybutyrate (PHB) is a well-known biodegradable bioplastic synthesized by microorganisms and can be produced from volatile fatty acids (VFAs). Among VFAs acetate can be utilized by Halomonas sp. YLGW01 for growth and PHB production. In this study, Halomonas sp. JJY01 was developed through introducing acetyl-CoA acetyltransferase (atoAD) with LacIq-Ptrc promoter into Halomonas sp. YLGW01. The effect of expression of atoAD on acetate was investigated by comparison with acetate consumption and PHB production. Shake-flask study showed that Halomonas sp. JJY01 increased acetate consumption rate, PHB yield and PHB production (0.27 g/L/h, 0.075 g/g, 0.72 g/L) compared to the wild type strain (0.17 g/L/h, 0.016 g/g, 0.11 g/L). In 10 L fermenter scale fed-batch fermentation, the growth of Halomonas sp. JJY01 resulted in higher acetate consumption rate, PHB yield and PHB titer (0.55 g/L/h, 0.091 g/g, 4.6 g/L) than wild type strain (0.35 g/L/h, 0.067 h/h, 2.9 g/L). These findings demonstrate enhanced acetate utilization and PHB production through the introduction of atoAD in Halomonas strains.
Asunto(s)
Halomonas , Hidroxibutiratos , Hidroxibutiratos/metabolismo , Halomonas/genética , Halomonas/metabolismo , Acetil-CoA C-Acetiltransferasa/metabolismo , Polihidroxibutiratos , Acetatos/metabolismo , Poliésteres/metabolismoRESUMEN
Biofilm formation by Mycobacterium fortuitum causes serious threats to human health due to its increased contribution to nosocomial infections. In this study, the first comprehensive global proteome analysis of M. fortuitum was reported under planktonic and biofilm growth states. A label-free Q Exactive Quadrupole-Orbitrap tandem mass spectrometry analysis was performed on the protein lysates. The differentially abundant proteins were functionally characterized and re-annotated using Blast2GO and CELLO2GO. Comparative analysis of the proteins among two growth states provided insights into the phenotypic switch, and fundamental pathways associated with pathobiology of M. fortuitum biofilm, such as lipid biosynthesis and quorum-sensing. Interaction network generated by the STRING database revealed associations between proteins that endure M. fortuitum during biofilm growth state. Hypothetical proteins were also studied to determine their functional alliance with the biofilm phenotype. CARD, VFDB, and PATRIC analysis further showed that the proteins upregulated in M. fortuitum biofilm exhibited antibiotic resistance, pathogenesis, and virulence. Heatmap and correlation analysis provided the biomarkers associated with the planktonic and biofilm growth of M. fortuitum. Proteome data was validated by qPCR analysis. Overall, the study provides insights into previously unexplored biochemical pathways that can be targeted by novel inhibitors, either for shortened treatment duration or for eliminating biofilm of M. fortuitum and related nontuberculous mycobacterial pathogens. KEY POINTS: ⢠Proteomic analyses of M. fortuitum reveals novel biofilm markers. ⢠Acetyl-CoA acetyltransferase acts as the phenotype transition switch. ⢠The study offers drug targets to combat M. fortuitum biofilm infections.
Asunto(s)
Biopelículas , Redes y Vías Metabólicas , Mycobacterium fortuitum , Proteoma , Mycobacterium fortuitum/química , Mycobacterium fortuitum/metabolismo , Mycobacterium fortuitum/fisiología , Mycobacterium fortuitum/ultraestructura , Microscopía Electrónica de Rastreo , Proteoma/análisis , Acetil-CoA C-Acetiltransferasa/metabolismo , Percepción de QuorumRESUMEN
Cholesterol is essential for cellular function and is stored as cholesteryl esters (CEs). CEs biosynthesis is catalyzed by the enzymes acyl-CoA:cholesterol acyltransferase 1 and 2 (ACAT1 and ACAT2), with ACAT1 being the primary isoenzyme in most cells in humans. In Alzheimer's Disease, CEs accumulate in vulnerable brain regions. Therefore, ACATs may be promising targets for treating AD. F12511 is a high-affinity ACAT1 inhibitor that has passed phase 1 safety tests for antiatherosclerosis. Previously, we developed a nanoparticle system to encapsulate a large concentration of F12511 into a stealth liposome (DSPE-PEG2000 with phosphatidylcholine). Here, we injected the nanoparticle encapsulated F12511 (nanoparticle F) intravenously (IV) in wild-type mice and performed an HPLC/MS/MS analysis and ACAT enzyme activity measurement. The results demonstrated that F12511 was present within the mouse brain after a single IV but did not overaccumulate in the brain or other tissues after repeated IVs. A histological examination showed that F12511 did not cause overt neurological or systemic toxicity. We then showed that a 2-week IV delivery of nanoparticle F to aging 3xTg AD mice ameliorated amyloidopathy, reduced hyperphosphorylated tau and nonphosphorylated tau, and reduced neuroinflammation. This work lays the foundation for nanoparticle F to be used as a possible therapy for AD and other neurodegenerative diseases.
Asunto(s)
Enfermedad de Alzheimer , Humanos , Ratones , Animales , Ratones Transgénicos , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/patología , Liposomas , Distribución Tisular , Espectrometría de Masas en Tándem , Acetil-CoA C-Acetiltransferasa/metabolismoRESUMEN
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.
Asunto(s)
Neoplasias Colorrectales , Cetonas , Animales , Humanos , Ácido 3-Hidroxibutírico/farmacología , Espectrometría de Masas en Tándem , Neoplasias Colorrectales/patología , Proliferación Celular , Acetil-CoA C-Acetiltransferasa/genética , Acetil-CoA C-Acetiltransferasa/metabolismoRESUMEN
Thiolases are CoA-dependent enzymes that catalyze the thiolytic cleavage of 3-ketoacyl-CoA, as well as its reverse reaction, which is the thioester-dependent Claisen condensation reaction. Thiolases are dimers or tetramers (dimers of dimers). All thiolases have two reactive cysteines: (a) a nucleophilic cysteine, which forms a covalent intermediate, and (b) an acid/base cysteine. The best characterized thiolase is the Zoogloea ramigera thiolase, which is a bacterial biosynthetic thiolase belonging to the CT-thiolase subfamily. The thiolase active site is also characterized by two oxyanion holes, two active site waters, and four catalytic loops with characteristic amino acid sequence fingerprints. Three thiolase subfamilies can be identified, each characterized by a unique sequence fingerprint for one of their catalytic loops, which causes unique active site properties. Recent insights concerning the thiolase reaction mechanism, as obtained from recent structural studies, as well as from classical and recent enzymological studies, are addressed, and open questions are discussed.
Asunto(s)
Coenzima A , Cisteína , Coenzima A/química , Coenzima A/metabolismo , Cisteína/metabolismo , Modelos Moleculares , Acetil-CoA C-Acetiltransferasa/química , Acetil-CoA C-Acetiltransferasa/metabolismo , Dominio CatalíticoRESUMEN
Tuberculosis (TB) is one of the leading causes of human death caused by Mycobacterium tuberculosis (Mtb). Mtb can enter into a long-lasting persistence where it can utilize fatty acids as the carbon source. Hence, fatty acid metabolism pathway enzymes are considered promising and pertinent mycobacterial drug targets. FadA2 (thiolase) is one of the enzymes involved in Mtb's fatty acid metabolism pathway. FadA2 deletion construct (ΔL136-S150) was designed to produce soluble protein. The crystal structure of FadA2 (ΔL136-S150) at 2.9 Å resolution was solved and analysed for membrane-anchoring region. The four catalytic residues of FadA2 are Cys99, His341, His390 and Cys427, and they belong to four loops with characteristic sequence motifs, i.e., CxT, HEAF, GHP and CxA. FadA2 is the only thiolase of Mtb which belongs to the CHH category containing the HEAF motif. Analysing the substrate-binding channel, it has been suggested that FadA2 is involved in the ß-oxidation pathway, i.e., the degradative pathway, as the long-chain fatty acid can be accommodated in the channel. The catalysed reaction is favoured by the presence of two oxyanion holes, i.e., OAH1 and OAH2. OAH1 formation is unique in FadA2, formed by the NE2 of His390 present in the GHP motif and NE2 of His341 present in the HEAF motif, whereas OAH2 formation is similar to CNH category thiolase. Sequence and structural comparison with the human trifunctional enzyme (HsTFE-ß) suggests the membrane-anchoring region in FadA2. Molecular dynamics simulations of FadA2 with a membrane containing POPE lipid were conducted to understand the role of a long insertion sequence of FadA2 in membrane anchoring.
Asunto(s)
Mycobacterium tuberculosis , Humanos , Mycobacterium tuberculosis/metabolismo , Especificidad por Sustrato , Acetil-CoA C-Acetiltransferasa/química , Acetil-CoA C-Acetiltransferasa/metabolismoRESUMEN
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.
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Neoplasias de la Mama Triple Negativas , Humanos , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Línea Celular Tumoral , Acetiltransferasas/metabolismo , Acetilación , Movimiento Celular/genética , Proliferación Celular , Proteínas Represoras/metabolismo , Metiltransferasas/genética , Acetil-CoA C-Acetiltransferasa/genética , Acetil-CoA C-Acetiltransferasa/metabolismoRESUMEN
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.
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Neoplasias de la Próstata , Sarcoma , Humanos , Masculino , Acetil-CoA C-Acetiltransferasa/genética , Acetil-CoA C-Acetiltransferasa/metabolismo , Autofagia/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Especies Reactivas de OxígenoRESUMEN
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.
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Platycodon , Saponinas , Platycodon/genética , Platycodon/metabolismo , Acetil-CoA C-Acetiltransferasa/genética , Acetil-CoA C-Acetiltransferasa/metabolismo , ADN Complementario/genética , Regulación de la Expresión Génica de las Plantas , Escherichia coli/genética , Espectrometría de Masas en Tándem , Clonación Molecular , TerpenosRESUMEN
As an emerging pollutant, benzalkonium chlorides (BACs) potentially enriched in waste activated sludge (WAS). However, the microbial response mechanism under chronic effects of BACs on acidogenesis and methanogenesis in anaerobic digestion (AD) has not been clearly disclosed. This study investigated the AD (by-)products and microbial evolution under low to high BACs concentrations from bioreactor startup to steady running. It was found that BACs can lead to an increase of WAS hydrolysis and fermentation, but a disturbance to acidogenic bacteria also occurred at low BACs concentration. A noticeable inhibition to methanogenesis occurred when BAC concentration was up to 15 mg/g TSS. Metagenomic analysis revealed the key genes involved in acetic acid (HAc) biosynthesis (i.e. phosphate acetyltransferase, PTA), ß-oxidation pathway (acetyl-CoA C-acetyltransferase) and propionic acid (HPr) conversion was slightly promoted compared with control. Furthermore, BACs inhibited the acetotrophic methanogenesis (i.e. acetyl-CoA synthetase), especially BAC concentration was up to 15 mg/g TSS, thereby enhanced short chain fatty acids (SCFAs) accumulation. Overall, chronic stimulation of functional microorganisms with increasing concentrations of BACs impact WAS fermentation.
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Contaminantes Ambientales , Aguas del Alcantarillado , Acetilcoenzima A/metabolismo , Acetil-CoA C-Acetiltransferasa/metabolismo , Anaerobiosis , Compuestos de Benzalconio , Reactores Biológicos/microbiología , Ácidos Grasos Volátiles/metabolismo , Fermentación , Ligasas/metabolismo , Metano , Fosfato Acetiltransferasa/metabolismo , Propionatos , Aguas del Alcantarillado/microbiologíaRESUMEN
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.
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Acetil-CoA C-Acetiltransferasa , Espectrometría de Masas en Tándem , Acetil-CoA C-Acetiltransferasa/genética , Acetil-CoA C-Acetiltransferasa/metabolismo , Acetil-CoA C-Aciltransferasa/deficiencia , Acetil-CoA C-Aciltransferasa/genética , Acetil-CoA C-Aciltransferasa/metabolismo , Anciano , Errores Innatos del Metabolismo de los Aminoácidos , Niño , Cromatografía Liquida , Humanos , Lactante , MasculinoRESUMEN
AIM: To investigate cardiac signalling pathways connecting substrate utilization with left ventricular remodelling in a murine pressure overload model. METHODS: Cardiac hypertrophy was induced by transverse aortic constriction surgery in 20-week-old C57BL/6J mice treated with or without the sodium-glucose co-transporter 2 (SGLT2) inhibitor ertugliflozin (225 mg kg-1 chow diet) for 10 weeks. RESULTS: Ertugliflozin improved left ventricular function and reduced myocardial fibrosis. This occurred simultaneously with a fasting-like response characterized by improved glucose tolerance and increased ketone body concentrations. While cardiac insulin signalling was reduced in response to SGLT2 inhibition, AMP-activated protein kinase (AMPK) signalling was increased with induction of the fatty acid transporter cluster of differentiation 36 and phosphorylation of acetyl-CoA carboxylase (ACC). Further, enzymes responsible for ketone body catabolism (ß-hydroxybutyrate dehydrogenase, succinyl-CoA:3-oxoacid-CoA transferase and acetyl-CoA acetyltransferase 1) were induced by SGLT2 inhibition. Ertugliflozin led to more cardiac abundance of fatty acids, tricarboxylic acid cycle metabolites and ATP. Downstream mechanistic target of rapamycin (mTOR) pathway, relevant for protein synthesis, cardiac hypertrophy and adverse cardiac remodelling, was reduced by SGLT2 inhibition, with alleviation of endoplasmic reticulum (ER) stress and unfolded protein response (UPR) providing a potential mechanism for abundant reduced left ventricular apoptosis and fibrosis. CONCLUSION: SGLT2 inhibition reduced left ventricular fibrosis in a murine model of cardiac hypertrophy. Mechanistically, this was associated with reduced cardiac insulin and increased AMPK signalling as a potential mechanism for less cardiac mTOR activation with alleviation of downstream ER stress, UPR and apoptosis.
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Insulinas , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Proteínas Quinasas Activadas por AMP/metabolismo , Acetil-CoA C-Acetiltransferasa/metabolismo , Acetil-CoA Carboxilasa/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Apoptosis , Compuestos Bicíclicos Heterocíclicos con Puentes , Cardiomegalia/metabolismo , Cardiomegalia/patología , Coenzima A Transferasas/metabolismo , Estrés del Retículo Endoplásmico , Ácidos Grasos/metabolismo , Fibrosis , Glucosa/metabolismo , Hidroxibutirato Deshidrogenasa/metabolismo , Cetoácidos/metabolismo , Cetonas/metabolismo , Ratones , Ratones Endogámicos C57BL , Miocitos Cardíacos/metabolismo , Sirolimus/metabolismo , Sodio/metabolismo , Transportador 2 de Sodio-Glucosa/metabolismo , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacología , Inhibidores del Cotransportador de Sodio-Glucosa 2/uso terapéutico , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
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.
Asunto(s)
Acetil-CoA C-Acetiltransferasa , Antifúngicos , Acetil-CoA C-Acetiltransferasa/genética , Acetil-CoA C-Acetiltransferasa/metabolismo , Antifúngicos/farmacología , Bacterias/metabolismo , Candida albicans/genética , Candida albicans/metabolismo , Poliinos/metabolismo , Poliinos/farmacologíaRESUMEN
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).
Asunto(s)
Acetil-CoA C-Acetiltransferasa , Carcinoma de Células Renales , Transición Epitelial-Mesenquimal , Neoplasias Renales , MicroARNs , Acetil-CoA C-Acetiltransferasa/genética , Acetil-CoA C-Acetiltransferasa/metabolismo , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Proliferación Celular/genética , Epigénesis Genética , Molécula de Adhesión Celular Epitelial/genética , Molécula de Adhesión Celular Epitelial/metabolismo , Transición Epitelial-Mesenquimal/genética , Humanos , Neoplasias Renales/genética , Neoplasias Renales/patología , Metaloproteinasa 7 de la Matriz/genética , Metaloproteinasa 7 de la Matriz/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , ARN Mensajero/genéticaRESUMEN
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.
Asunto(s)
Atractylodes , Acetil-CoA C-Acetiltransferasa/genética , Acetil-CoA C-Acetiltransferasa/metabolismo , Atractylodes/genética , Atractylodes/metabolismo , Clonación Molecular , ADN Complementario/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Imidazoles/metabolismo , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TerpenosRESUMEN
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.
Asunto(s)
Acetil-CoA C-Acetiltransferasa/metabolismo , Proteínas Bacterianas/metabolismo , Cationes Monovalentes/metabolismo , Activación Enzimática , Potasio/metabolismo , Zoogloea/aislamiento & purificación , Acetilcoenzima A/química , Acetilcoenzima A/metabolismo , Acetil-CoA C-Acetiltransferasa/química , Acetil-CoA C-Acetiltransferasa/genética , Acilcoenzima A/química , Acilcoenzima A/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Biocatálisis , Cationes Monovalentes/química , Cristalografía por Rayos X , Cinética , Modelos Moleculares , Mutación , Potasio/química , Unión Proteica , Conformación Proteica , Ingeniería de Proteínas , Multimerización de Proteína , Especificidad por Sustrato , Zoogloea/enzimología , Zoogloea/genéticaRESUMEN
Glycine decarboxylase (GLDC) is a key enzyme of glycine cleavage system that converts glycine into one-carbon units. GLDC is commonly up-regulated and plays important roles in many human cancers. Whether and how GLDC is regulated by post-translational modifications is unknown. Here we report that mechanistic target of rapamycin complex 1 (mTORC1) signal inhibits GLDC acetylation at lysine (K) 514 by inducing transcription of the deacetylase sirtuin 3 (SIRT3). Upon inhibition of mTORC1, the acetyltransferase acetyl-CoA acetyltransferase 1 (ACAT1) catalyzes GLDC K514 acetylation. This acetylation of GLDC impairs its enzymatic activity. In addition, this acetylation of GLDC primes for its K33-linked polyubiquitination at K544 by the ubiquitin ligase NF-X1, leading to its degradation by the proteasomal pathway. Finally, we find that GLDC K514 acetylation inhibits glycine catabolism, pyrimidines synthesis and glioma tumorigenesis. Our finding reveals critical roles of post-translational modifications of GLDC in regulation of its enzymatic activity, glycine metabolism and tumorigenesis, and provides potential targets for therapeutics of cancers such as glioma.