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1.
Food Chem ; 334: 127567, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-32707362

RESUMO

Fruit acidity is an important determinant of peach organoleptic quality, but its regulatory mechanism remains elusive. Measurement of organic acids in ripe fruits of seventy-five peach cultivars revealed the predominant components malate and citrate, accompanied by quinate. Organic acid accumulation increased at early stages of fruit growth, but exhibited a more dramatic reduction in low-acid cultivar during later stages of fruit development compared to high-acid cultivars. Low-acid cultivars showed citrate degradation and less transport of malate into the vacuole due to up- and down-regulation of a GABA pathway gene GAD and a malate transporter gene ALMT9, respectively. The NAD-MDH1 gene might control the rate-limiting step in malate synthesis, while three genes, PDK, PK, and ADH, could affect citrate synthesis through the pyruvate-to-acetyl-CoA-to-citrate pathway. Altogether, these results suggested that malate accumulation is controlled at the level of metabolism and vacuolar storage, while metabolism is crucial for citrate accumulation in peach.


Assuntos
Ácidos Carboxílicos/metabolismo , Frutas/metabolismo , Malatos/metabolismo , Proteínas de Plantas/genética , Prunus persica/metabolismo , Acetilcoenzima A/metabolismo , Ácido Cítrico/metabolismo , Frutas/genética , Frutas/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/metabolismo , Prunus persica/genética , Prunus persica/crescimento & desenvolvimento , Ácido Pirúvico/metabolismo , Vacúolos/metabolismo
2.
Appl Environ Microbiol ; 86(23)2020 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-32948524

RESUMO

Acetogenic bacteria are a diverse group of anaerobes that use the reductive acetyl coenzyme A (acetyl-CoA) (Wood-Ljungdahl) pathway for CO2 fixation and energy conservation. The conversion of 2 mol CO2 into acetyl-CoA by using the Wood-Ljungdahl pathway as the terminal electron accepting process is the most prominent metabolic feature for these microorganisms. However, here, we describe that the fecal acetogen Clostridium bovifaecis strain BXX displayed poor metabolic capabilities of autotrophic acetogenesis, and acetogenic utilization of glucose occurred only with the supplementation of formate. Genome analysis of Clostridium bovifaecis revealed that it contains almost the complete genes of the Wood-Ljungdahl pathway but lacks the gene encoding formate dehydrogenase, which catalyzes the reduction of CO2 to formate as the first step of the methyl branch of the Wood-Ljungdahl pathway. The lack of a gene encoding formate dehydrogenase was verified by PCR, reverse transcription-PCR analysis, enzyme activity assay, and its formate-dependent acetogenic utilization of glucose on DNA, RNA, protein, and phenotype level, respectively. The lack of a formate dehydrogenase gene may be associated with the adaption to a formate-rich intestinal environment, considering the isolating source of strain BXX. The formate-dependent acetogenic growth of Clostridium bovifaecis provides insight into a unique metabolic feature of fecal acetogens.IMPORTANCE The acetyl-CoA pathway is an ancient pathway of CO2 fixation, which converts 2 mol of CO2 into acetyl-CoA. Autotrophic growth with H2 and CO2 via the acetyl-CoA pathway as the terminal electron accepting process is the most unique feature of acetogenic bacteria. However, the fecal acetogen Clostridium bovifaecis strain BXX displayed poor metabolic capabilities of autotrophic acetogenesis, and acetogenic utilization of glucose occurred only with the supplementation of formate. The formate-dependent acetogenic growth of Clostridium bovifaecis was associated with its lack of a gene encoding formate dehydrogenase, which may result from adaption to a formate-rich intestinal environment. This study gave insight into a unique metabolic feature of fecal acetogens. Because of the requirement of formate for the acetogenic growth of certain acetogens, the ecological impact of acetogens could be more complex and important in the formate-rich environment due to their trophic interactions with other microbes.


Assuntos
Ácido Acético/metabolismo , Proteínas de Bactérias , Clostridium/metabolismo , Formiato Desidrogenases/deficiência , Formiatos/metabolismo , Acetilcoenzima A/metabolismo , Clostridium/enzimologia , Clostridium/genética , Redes e Vias Metabólicas
3.
Proc Natl Acad Sci U S A ; 117(36): 22452-22461, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32820073

RESUMO

Carbon fixation via the Calvin cycle is constrained by the side activity of Rubisco with dioxygen, generating 2-phosphoglycolate. The metabolic recycling of phosphoglycolate was extensively studied in photoautotrophic organisms, including plants, algae, and cyanobacteria, where it is referred to as photorespiration. While receiving little attention so far, aerobic chemolithoautotrophic bacteria that operate the Calvin cycle independent of light must also recycle phosphoglycolate. As the term photorespiration is inappropriate for describing phosphoglycolate recycling in these nonphotosynthetic autotrophs, we suggest the more general term "phosphoglycolate salvage." Here, we study phosphoglycolate salvage in the model chemolithoautotroph Cupriavidus necator H16 (Ralstonia eutropha H16) by characterizing the proxy process of glycolate metabolism, performing comparative transcriptomics of autotrophic growth under low and high CO2 concentrations, and testing autotrophic growth phenotypes of gene deletion strains at ambient CO2 We find that the canonical plant-like C2 cycle does not operate in this bacterium, and instead, the bacterial-like glycerate pathway is the main route for phosphoglycolate salvage. Upon disruption of the glycerate pathway, we find that an oxidative pathway, which we term the malate cycle, supports phosphoglycolate salvage. In this cycle, glyoxylate is condensed with acetyl coenzyme A (acetyl-CoA) to give malate, which undergoes two oxidative decarboxylation steps to regenerate acetyl-CoA. When both pathways are disrupted, autotrophic growth is abolished at ambient CO2 We present bioinformatic data suggesting that the malate cycle may support phosphoglycolate salvage in diverse chemolithoautotrophic bacteria. This study thus demonstrates a so far unknown phosphoglycolate salvage pathway, highlighting important diversity in microbial carbon fixation metabolism.


Assuntos
Crescimento Quimioautotrófico/fisiologia , Glicolatos/metabolismo , Fotossíntese/fisiologia , Acetilcoenzima A/metabolismo , Proteínas de Bactérias/metabolismo , Ciclo do Carbono/fisiologia , Cupriavidus necator/genética , Cupriavidus necator/metabolismo , Malato Sintase/metabolismo , Malatos/metabolismo , Oxirredução
4.
Exerc Sport Sci Rev ; 48(4): 188-200, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32658040

RESUMO

We explore work from within the field of skeletal muscle and across the broader field of molecular biology, to propose that the link between exercise and skeletal muscle adaptation lies in the interplay between metabolism and epigenetics. Future investigations into such an interaction are crucial to advance our understanding of the beneficial effects of exercise on performance and health.


Assuntos
Metabolismo Energético , Epigênese Genética , Exercício Físico/fisiologia , Músculo Esquelético/fisiologia , Acetilcoenzima A/metabolismo , Acetilação , Adaptação Fisiológica , Ciclo do Ácido Cítrico , Metilação de DNA , Glicólise , Histonas/metabolismo , Humanos , Metabolismo dos Lipídeos , Músculo Esquelético/metabolismo , Transdução de Sinais
5.
Arch Biochem Biophys ; 691: 108507, 2020 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-32710884

RESUMO

Mammalian carnitine acetyltransferase (CrAT) is a mitochondrial enzyme that catalyzes the reversible transfer of an acetyl group from acetyl-CoA to carnitine. CrAT knockout studies have shown that this enzyme is critical to sustain metabolic flexibility, or the ability to switch between different fuel types, an underlying theme of the metabolic syndrome. These recent physiological findings imply that CrAT dysfunction, or its catalytic impairment, may lead to disease. To gain insight into the CrAT kinetic mechanism, we conducted stopped-flow experiments in various enzyme substrate/product conditions and analyzed full progress curves by global fitting. Simultaneous mixing of both substrates with CrAT produced relatively fast kinetics that follows an ordered bi bi mechanism. A great preference for ordered binding is supported by stopped-flow double mixing experiments such that premixed CrAT with acetyl-CoA or CoA demonstrated a biphasic decrease in initial rate that produces about a 100-fold attenuation in catalysis. Double mixing experiments also revealed that the CrAT initial rate is inhibited by 50% in approximately 8 s by either acetyl-CoA or CoA premixing. Analysis of available CrAT structures support a substrate conformational change between acetyl-CoA/CoA binary versus ternary complexes. Additional viscosity-based kinetic experiments yielded strong evidence that product release is the rate limiting step in the CrAT-catalyzed reaction.


Assuntos
Carnitina O-Acetiltransferase/química , Acetilcoenzima A/química , Acetilcoenzima A/metabolismo , Animais , Carnitina/química , Carnitina/metabolismo , Carnitina O-Acetiltransferase/metabolismo , Catálise , Domínio Catalítico , Coenzima A/química , Coenzima A/metabolismo , Columbidae , Cristalografia por Raios X , Cinética , Camundongos , Ligação Proteica
6.
Mol Pharmacol ; 98(2): 88-95, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32487734

RESUMO

Arylamine N-acetyltransferase 1 (NAT1) is a phase II xenobiotic-metabolizing enzyme that also has a role in cancer cell growth and metabolism. Recently, it was reported that NAT1 undergoes lysine acetylation, an important post-translational modification that can regulate protein function. In the current study, we use site-directed mutagenesis to identify K100 and K188 as major sites of lysine acetylation in the NAT1 protein. Acetylation of ectopically expressed NAT1 in HeLa cells was decreased by C646, an inhibitor of the protein acetyltransferases p300/CREB-binding protein (CBP). Recombinant p300 directly acetylated NAT1 in vitro. Acetylation of NAT1 was enhanced by the sirtuin (SIRT) inhibitor nicotinamide but not by the histone deacetylase inhibitor trichostatin A. Cotransfection of cells with NAT1 and either SIRT 1 or 2, but not SIRT3, significantly decreased NAT1 acetylation. NAT1 activity was evaluated in cells after nicotinamide treatment to enhance acetylation or cotransfection with SIRT1 to inhibit acetylation. The results indicated that NAT1 acetylation impaired its enzyme kinetics, suggesting decreased acetyl coenzyme A binding. In addition, acetylation attenuated the allosteric effects of ATP on NAT1. Taken together, this study shows that NAT1 is acetylated by p300/CBP in situ and is deacetylated by the sirtuins SIRT1 and 2. It is hypothesized that post-translational modification of NAT1 by acetylation at K100 and K188 may modulate NAT1 effects in cells. SIGNIFICANCE STATEMENT: There is growing evidence that arylamine N-acetyltransferase 1 has an important cellular role in addition to xenobiotic metabolism. Here, we show that NAT1 is acetylated at K100 and K188 and that changes in protein acetylation equilibrium can modulate its activity in cells.


Assuntos
Arilamina N-Acetiltransferase/química , Arilamina N-Acetiltransferase/metabolismo , Proteína de Ligação a CREB/genética , Proteína p300 Associada a E1A/genética , Isoenzimas/química , Isoenzimas/metabolismo , Sirtuína 1/genética , Sirtuína 2/genética , Acetilcoenzima A/metabolismo , Acetilação/efeitos dos fármacos , Arilamina N-Acetiltransferase/genética , Benzoatos/farmacologia , Proteína de Ligação a CREB/metabolismo , Cristalografia por Raios X , Proteína p300 Associada a E1A/metabolismo , Células HeLa , Humanos , Ácidos Hidroxâmicos/farmacologia , Isoenzimas/genética , Lisina/química , Lisina/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Niacinamida/farmacologia , Conformação Proteica , Pirazóis/farmacologia , Sirtuína 1/metabolismo , Sirtuína 2/metabolismo , Transfecção
7.
Life Sci ; 256: 117975, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32565251

RESUMO

Our goal is to understand how loss of circulating estrogens and estrogen replacement affect brain physiology and function, particularly in brain regions involved in cognitive processes. We recently conducted a large metabolomics study characterizing the effects of rodent models of menopause and treatment with estrogen receptor (ER) agonists on neurochemical targets in hippocampus, frontal cortex, and striatum. Here we characterize effects on levels of several key enzymes involved in glucose utilization and energy production, specifically phosphofructokinase, glyceraldehyde 3-phosphate dehydrogenase, and pyruvate dehydrogenase. We also evaluated effects on levels of ß-actin and α-tubulin, choline acetyltransferase (ChAT) activity, and levels of ATP citrate lyase. All experiments were conducted in young adult rats. Experiment 1 compared the effects of ovariectomy (OVX), a model of surgical menopause, and 4-vinylcyclohexene diepoxide (VCD)-treatments, a model of transitional menopause, with tissues collected at proestrus and at diestrus. Experiment 2 used a separate cohort of rats to evaluate the same targets in OVX and VCD-treated rats treated with estradiol or with selective ER agonists. Differences in the expression of metabolic enzymes between cycling animals and models of surgical and transitional menopause were detected. These differences were model-, region- and time- dependent, and were modulated by selective ER agonists. Collectively, the findings demonstrate that loss of ovarian function and ER agonist treatments have differing effects in OVX vs. VCD-treated rats. Differences may help to explain differences in the effects of estrogen treatments on brain function and cognition in women who have experienced surgical vs. transitional menopause.


Assuntos
Acetilcoenzima A/metabolismo , Encéfalo/metabolismo , Colina O-Acetiltransferase/metabolismo , Estrogênios/farmacologia , Menopausa/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Cicloexenos/toxicidade , Estradiol/sangue , Feminino , Menopausa/efeitos dos fármacos , Ovariectomia/efeitos adversos , Ratos , Ratos Sprague-Dawley , Compostos de Vinila/toxicidade
8.
PLoS Genet ; 16(6): e1008863, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32559195

RESUMO

Inactivation of the Rb tumor suppressor causes context-dependent increases in cell proliferation or cell death. In a genetic screen for factors that promoted Rb mutant cell death in Drosophila, we identified Psid, a regulatory subunit of N-terminal acetyltransferase B (NatB). We showed that NatB subunits were required for elevated EGFR/MAPK signaling and Rb mutant cell survival. We showed that NatB regulates the posttranscriptional levels of the highly conserved pathway components Grb2/Drk, MAPK, and PP2AC but not that of the less conserved Sprouty. Interestingly, NatB increased the levels of positive pathway components Grb2/Drk and MAPK while decreased the levels of negative pathway component PP2AC, which were mediated by the distinct N-end rule branch E3 ubiquitin ligases Ubr4 and Cnot4, respectively. These results suggest a novel mechanism by which NatB and N-end rule pathways modulate EGFR/MAPK signaling by inversely regulating the levels of multiple conserved positive and negative pathway components. As inactivation of Psid blocked EGFR signaling-dependent tumor growth, this study raises the possibility that NatB is potentially a novel therapeutic target for cancers dependent on deregulated EGFR/Ras signaling.


Assuntos
Proteínas Sanguíneas/metabolismo , Proteínas de Drosophila/metabolismo , Receptores ErbB/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Acetiltransferase N-Terminal B/metabolismo , Neoplasias/genética , Receptores de Peptídeos de Invertebrados/metabolismo , Acetilcoenzima A/metabolismo , Acetilação , Alelos , Animais , Animais Geneticamente Modificados , Apoptose/genética , Proteínas Sanguíneas/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Modelos Animais de Doenças , Proteínas de Drosophila/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Masculino , Acetiltransferase N-Terminal B/genética , Neoplasias/patologia , Proteína do Retinoblastoma/genética , Mutações Sintéticas Letais , Fatores de Transcrição/genética
9.
Nat Commun ; 11(1): 3148, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561715

RESUMO

Macroautophagy ("autophagy") is the main lysosomal catabolic process that becomes activated under nutrient-depleted conditions, like amino acid (AA) starvation. The mechanistic target of rapamycin complex 1 (mTORC1) is a well-conserved negative regulator of autophagy. While leucine (Leu) is a critical mTORC1 regulator under AA-starved conditions, how Leu regulates autophagy is poorly understood. Here, we describe that in most cell types, including neurons, Leu negatively regulates autophagosome biogenesis via its metabolite, acetyl-coenzyme A (AcCoA). AcCoA inhibits autophagy by enhancing EP300-dependent acetylation of the mTORC1 component raptor, with consequent activation of mTORC1. Interestingly, in Leu deprivation conditions, the dominant effects on autophagy are mediated by decreased raptor acetylation causing mTORC1 inhibition, rather than by altered acetylation of other autophagy regulators. Thus, in most cell types we examined, Leu regulates autophagy via the impact of its metabolite AcCoA on mTORC1, suggesting that AcCoA and EP300 play pivotal roles in cell anabolism and catabolism.


Assuntos
Autofagia/fisiologia , Leucina/metabolismo , Proteína Regulatória Associada a mTOR/metabolismo , Acetilcoenzima A/metabolismo , Acetilação , Animais , Autofagossomos , Linhagem Celular , Proteína p300 Associada a E1A/metabolismo , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Cultura Primária de Células , Inanição/metabolismo
10.
Biochim Biophys Acta Proteins Proteom ; 1868(9): 140462, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32485238

RESUMO

Malic enzymes participate in key metabolic processes, the MaeB-like malic enzymes carry a catalytic inactive phosphotransacetylase domain whose function remains elusive. Here we show that acetyl-CoA directly binds and inhibits MaeB-like enzymes with a saturable profile under physiological relevant acetyl-CoA concentrations. A MaeB-like enzyme from the nitrogen-fixing bacterium Azospirillum brasilense, namely AbMaeB1, binds both acetyl-CoA and unesterified CoASH in a way that inhibition of AbMaeB1 by acetyl-CoA is relieved by increasing CoASH concentrations. Hence, AbMaeB1 senses the acetyl-CoA/CoASH ratio. We revisited E. coli MaeB regulation to determine the inhibitory constant for acetyl-CoA. Our data support that the phosphotransacetylase domain of MaeB-like enzymes senses acetyl-CoA to dictate the fate of carbon distribution at the phosphoenol-pyruvate / pyruvate / oxaloacetate metabolic node.


Assuntos
Acetilcoenzima A/metabolismo , Coenzima A/metabolismo , Malato Desidrogenase/metabolismo , Malatos/metabolismo , NADP/metabolismo , Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Malato Desidrogenase/genética , Fosfato Acetiltransferase/metabolismo
11.
Nucleic Acids Res ; 48(10): 5294-5305, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32369169

RESUMO

The broad host range bacteriophage Mu employs a novel 'methylcarbamoyl' modification to protect its DNA from diverse restriction systems of its hosts. The DNA modification is catalyzed by a phage-encoded protein Mom, whose mechanism of action is a mystery. Here, we characterized the co-factor and metal-binding properties of Mom and provide a molecular mechanism to explain 'methylcarbamoyl'ation of DNA by Mom. Computational analyses revealed a conserved GNAT (GCN5-related N-acetyltransferase) fold in Mom. We demonstrate that Mom binds to acetyl CoA and identify the active site. We discovered that Mom is an iron-binding protein, with loss of Fe2+/3+-binding associated with loss of DNA modification activity. The importance of Fe2+/3+ is highlighted by the colocalization of Fe2+/3+ with acetyl CoA within the Mom active site. Puzzlingly, acid-base mechanisms employed by >309,000 GNAT members identified so far, fail to support methylcarbamoylation of adenine using acetyl CoA. In contrast, free-radical chemistry catalyzed by transition metals like Fe2+/3+ can explain the seemingly challenging reaction, accomplished by collaboration between acetyl CoA and Fe2+/3+. Thus, binding to Fe2+/3+, a small but unprecedented step in the evolution of Mom, allows a giant chemical leap from ordinary acetylation to a novel methylcarbamoylation function, while conserving the overall protein architecture.


Assuntos
Aciltransferases/química , Aciltransferases/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Acetilcoenzima A/metabolismo , Bacteriófago mu/fisiologia , Domínio Catalítico , Escherichia coli/genética , Escherichia coli/virologia , Ferro/metabolismo , Conformação Proteica
12.
Mol Cell ; 79(1): 30-42.e4, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32473093

RESUMO

Autophagy is activated by prolonged fasting but cannot overcome the ensuing hepatic lipid overload, resulting in fatty liver. Here, we describe a peroxisome-lysosome metabolic link that restricts autophagic degradation of lipids. Acyl-CoA oxidase 1 (Acox1), the enzyme that catalyzes the first step in peroxisomal ß-oxidation, is enriched in liver and further increases with fasting or high-fat diet (HFD). Liver-specific Acox1 knockout (Acox1-LKO) protected mice against hepatic steatosis caused by starvation or HFD due to induction of autophagic degradation of lipid droplets. Hepatic Acox1 deficiency markedly lowered total cytosolic acetyl-CoA levels, which led to decreased Raptor acetylation and reduced lysosomal localization of mTOR, resulting in impaired activation of mTORC1, a central regulator of autophagy. Dichloroacetic acid treatment elevated acetyl-CoA levels, restored mTORC1 activation, inhibited autophagy, and increased hepatic triglycerides in Acox1-LKO mice. These results identify peroxisome-derived acetyl-CoA as a key metabolic regulator of autophagy that controls hepatic lipid homeostasis.


Assuntos
Acetilcoenzima A/metabolismo , Acil-CoA Oxidase/fisiologia , Autofagia , Ácidos Graxos/química , Fígado Gorduroso/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Peroxissomos/química , Acetilação , Animais , Proteína 5 Relacionada à Autofagia/fisiologia , Dieta Hiperlipídica/efeitos adversos , Jejum , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Feminino , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Oxirredução , Peroxissomos/metabolismo , Proteína Regulatória Associada a mTOR/genética , Proteína Regulatória Associada a mTOR/metabolismo
13.
Nat Commun ; 11(1): 2358, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32398649

RESUMO

Sphingosine kinase1 (SphK1) is an acetyl-CoA dependent acetyltransferase which acts on cyclooxygenase2 (COX2) in neurons in a model of Alzheimer's disease (AD). However, the mechanism underlying this activity was unexplored. Here we show that N-acetyl sphingosine (N-AS) is first generated by acetyl-CoA and sphingosine through SphK1. N-AS then acetylates serine 565 (S565) of COX2, and the N-AS-acetylated COX2 induces the production of specialized pro-resolving mediators (SPMs). In a mouse model of AD, microglia show a reduction in N-AS generation, leading to decreased acetyl-S565 COX2 and SPM production. Treatment with N-AS increases acetylated COX2 and N-AS-triggered SPMs in microglia of AD mice, leading to resolution of neuroinflammation, an increase in microglial phagocytosis, and improved memory. Taken together, these results identify a role of N-AS in the dysfunction of microglia in AD.


Assuntos
Doença de Alzheimer/imunologia , Anti-Inflamatórios/farmacologia , Encéfalo/imunologia , Microglia/imunologia , Esfingosina/análogos & derivados , Acetilcoenzima A/metabolismo , Acetilação , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Animais , Anti-Inflamatórios/uso terapêutico , Encéfalo/patologia , Linhagem Celular , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Modelos Animais de Doenças , Humanos , Masculino , Memória/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Microglia/patologia , Mutagênese , Neurônios , Fagocitose/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Presenilina-1/genética , Cultura Primária de Células , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/metabolismo , Esfingosina/metabolismo
14.
Life Sci ; 252: 117661, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32305523

RESUMO

AIMS: Hydrogen sulfide (H2S) as a novel gasotransmitter can be endogenously produced in liver by cystathionine gamma-lyase (CSE). The dysfunctions of CSE/H2S system have been linked to various liver diseases. Acetyl-CoA is the key intermediate from the metabolism of lipid. This study examined the roles of H2S in hepatic acetyl-CoA and lipid metabolism. MATERIALS AND METHODS: Both in vitro cell model and in vivo animal model of lipid accumulation were used in this study. Western blotting and real-time PCR were used for analysis of protein and mRNA expression. Acetyl-CoA was analyzed by a coupled enzyme assay, and lipid accumulation was observed with Oil Red O staining. KEY FINDINGS: Incubation of human liver carcinoma (HepG2) cells with a mixture of free fatty acids (FFAs) or high glucose reduced CSE expression and H2S production, promoted intracellular accumulation of acetyl-CoA and lipid. Supply of exogenous NaHS or cysteine reduced acetyl-CoA contents and lipid accumulation, while blockage of CSE activity promoted intracellular lipid accumulation. Furthermore, H2S blocked FFAs-induced transcriptions of de novo lipogenesis, inflammation, and fibrosis-related genes. In vivo, knockout of CSE gene stimulated more hepatic acetyl-CoA and lipid accumulation in mice induced by high-fat choline-deficient diet. The expressions of lipogenesis, inflammation, and fibrosis-related genes were significantly higher in liver tissues from CSE knockout mice when compared with wild-type mice. SIGNIFICANCE: CSE/H2S system is indispensable for maintaining the homeostasis of acetyl-CoA and lipid accumulation and protecting from the development of inflammation and fibrosis in liver under excessive caloric ingestion.


Assuntos
Acetilcoenzima A/metabolismo , Cistationina gama-Liase/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Sulfeto de Hidrogênio/metabolismo , Hepatopatia Gordurosa não Alcoólica/fisiopatologia , Animais , Cistationina gama-Liase/genética , Modelos Animais de Doenças , Células Hep G2 , Humanos , Metabolismo dos Lipídeos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
15.
Nature ; 579(7800): 586-591, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32214246

RESUMO

Consumption of fructose has risen markedly in recent decades owing to the use of sucrose and high-fructose corn syrup in beverages and processed foods1, and this has contributed to increasing rates of obesity and non-alcoholic fatty liver disease2-4. Fructose intake triggers de novo lipogenesis in the liver4-6, in which carbon precursors of acetyl-CoA are converted into fatty acids. The ATP citrate lyase (ACLY) enzyme cleaves cytosolic citrate to generate acetyl-CoA, and is upregulated after consumption of carbohydrates7. Clinical trials are currently pursuing the inhibition of ACLY as a treatment for metabolic diseases8. However, the route from dietary fructose to hepatic acetyl-CoA and lipids remains unknown. Here, using in vivo isotope tracing, we show that liver-specific deletion of Acly in mice is unable to suppress fructose-induced lipogenesis. Dietary fructose is converted to acetate by the gut microbiota9, and this supplies lipogenic acetyl-CoA independently of ACLY10. Depletion of the microbiota or silencing of hepatic ACSS2, which generates acetyl-CoA from acetate, potently suppresses the conversion of bolus fructose into hepatic acetyl-CoA and fatty acids. When fructose is consumed more gradually to facilitate its absorption in the small intestine, both citrate cleavage in hepatocytes and microorganism-derived acetate contribute to lipogenesis. By contrast, the lipogenic transcriptional program is activated in response to fructose in a manner that is independent of acetyl-CoA metabolism. These data reveal a two-pronged mechanism that regulates hepatic lipogenesis, in which fructolysis within hepatocytes provides a signal to promote the expression of lipogenic genes, and the generation of microbial acetate feeds lipogenic pools of acetyl-CoA.


Assuntos
Acetatos/metabolismo , Açúcares da Dieta/metabolismo , Frutose/metabolismo , Microbioma Gastrointestinal/fisiologia , Lipogênese , Fígado/metabolismo , ATP Citrato (pro-S)-Liase/deficiência , ATP Citrato (pro-S)-Liase/genética , ATP Citrato (pro-S)-Liase/metabolismo , Acetato-CoA Ligase/deficiência , Acetato-CoA Ligase/genética , Acetato-CoA Ligase/metabolismo , Acetilcoenzima A/metabolismo , Animais , Ácido Cítrico/metabolismo , Açúcares da Dieta/administração & dosagem , Açúcares da Dieta/farmacologia , Ácidos Graxos/metabolismo , Frutose/administração & dosagem , Frutose/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Marcação por Isótopo , Lipogênese/efeitos dos fármacos , Lipogênese/genética , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Camundongos , Especificidade por Substrato
16.
Nucleic Acids Res ; 48(8): 4115-4138, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32182340

RESUMO

Epigenetic regulation of gene expression is tightly controlled by the dynamic modification of histones by chemical groups, the diversity of which has largely expanded over the past decade with the discovery of lysine acylations, catalyzed from acyl-coenzymes A. We investigated the dynamics of lysine acetylation and crotonylation on histones H3 and H4 during mouse spermatogenesis. Lysine crotonylation appeared to be of significant abundance compared to acetylation, particularly on Lys27 of histone H3 (H3K27cr) that accumulates in sperm in a cleaved form of H3. We identified the genomic localization of H3K27cr and studied its effects on transcription compared to the classical active mark H3K27ac at promoters and distal enhancers. The presence of both marks was strongly associated with highest gene expression. Assessment of their co-localization with transcription regulators (SLY, SOX30) and chromatin-binding proteins (BRD4, BRDT, BORIS and CTCF) indicated systematic highest binding when both active marks were present and different selective binding when present alone at chromatin. H3K27cr and H3K27ac finally mark the building of some sperm super-enhancers. This integrated analysis of omics data provides an unprecedented level of understanding of gene expression regulation by H3K27cr in comparison to H3K27ac, and reveals both synergistic and specific actions of each histone modification.


Assuntos
Elementos Facilitadores Genéticos , Epigênese Genética , Código das Histonas , Regiões Promotoras Genéticas , Espermatogênese/genética , Acetilcoenzima A/metabolismo , Acetilação , Acil Coenzima A/metabolismo , Animais , Evolução Biológica , Crotonatos/metabolismo , Genômica , Histonas/química , Histonas/metabolismo , Lisina/metabolismo , Masculino , Metabolômica , Camundongos Endogâmicos C57BL , Proteômica , Transcrição Genética , Leveduras/metabolismo , Leveduras/fisiologia
17.
Proc Natl Acad Sci U S A ; 117(13): 7516-7523, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32170009

RESUMO

Among CO2-fixing metabolic pathways in nature, the linear Wood-Ljungdahl pathway (WLP) in phylogenetically diverse acetate-forming acetogens comprises the most energetically efficient pathway, requires the least number of reactions, and converts CO2 to formate and then into acetyl-CoA. Despite two genes encoding glycine synthase being well-conserved in WLP gene clusters, the functional role of glycine synthase under autotrophic growth conditions has remained uncertain. Here, using the reconstructed genome-scale metabolic model iSL771 based on the completed genome sequence, transcriptomics, 13C isotope-based metabolite-tracing experiments, biochemical assays, and heterologous expression of the pathway in another acetogen, we discovered that the WLP and the glycine synthase pathway are functionally interconnected to fix CO2, subsequently converting CO2 into acetyl-CoA, acetyl-phosphate, and serine. Moreover, the functional cooperation of the pathways enhances CO2 consumption and cellular growth rates via bypassing reducing power required reactions for cellular metabolism during autotrophic growth of acetogens.


Assuntos
Aminoácido Oxirredutases/metabolismo , Aminometiltransferase/metabolismo , Processos Autotróficos/fisiologia , Complexos Multienzimáticos/metabolismo , Acetilcoenzima A/metabolismo , Aminoácido Oxirredutases/genética , Aminometiltransferase/genética , Proteínas de Bactérias/metabolismo , Ciclo do Carbono , Dióxido de Carbono/metabolismo , Monóxido de Carbono/metabolismo , Clostridium/metabolismo , Redes e Vias Metabólicas , Complexos Multienzimáticos/genética , Família Multigênica , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo
18.
Nature ; 579(7798): 279-283, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32132708

RESUMO

Although it is well-established that reductions in the ratio of insulin to glucagon in the portal vein have a major role in the dysregulation of hepatic glucose metabolism in type-2 diabetes1-3, the mechanisms by which glucagon affects hepatic glucose production and mitochondrial oxidation are poorly understood. Here we show that glucagon stimulates hepatic gluconeogenesis by increasing the activity of hepatic adipose triglyceride lipase, intrahepatic lipolysis, hepatic acetyl-CoA content and pyruvate carboxylase flux, while also increasing mitochondrial fat oxidation-all of which are mediated by stimulation of the inositol triphosphate receptor 1 (INSP3R1). In rats and mice, chronic physiological increases in plasma glucagon concentrations increased mitochondrial oxidation of fat in the liver and reversed diet-induced hepatic steatosis and insulin resistance. However, these effects of chronic glucagon treatment-reversing hepatic steatosis and glucose intolerance-were abrogated in Insp3r1 (also known as Itpr1)-knockout mice. These results provide insights into glucagon biology and suggest that INSP3R1 may represent a target for therapies that aim to reverse nonalcoholic fatty liver disease and type-2 diabetes.


Assuntos
Glucagon/farmacologia , Gluconeogênese/efeitos dos fármacos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Fígado/efeitos dos fármacos , Acetilcoenzima A/metabolismo , Tecido Adiposo/efeitos dos fármacos , Animais , Diabetes Mellitus Tipo 2/fisiopatologia , Ativação Enzimática/efeitos dos fármacos , Glucagon/sangue , Receptores de Inositol 1,4,5-Trifosfato/genética , Lipase/metabolismo , Lipólise/efeitos dos fármacos , Lipólise/genética , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Hepatopatia Gordurosa não Alcoólica/fisiopatologia , Oxirredução/efeitos dos fármacos
19.
Pharmacol Rep ; 72(1): 225-237, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32016856

RESUMO

BACKGROUND: Hyperactivation of blood platelets is an essential factor in the pathomechanism of diabetes-evoked angiopathies. The aim of this work was to investigate whether blood platelets hyperactivation resulting from type 2 diabetic hyperglycaemia-increased pyruvate dehydrogenase complex activity and excessive acetyl-CoA accumulation may be brought to the normal range by the enzyme inhibitors. METHODS: Platelets were isolated from the blood of 9 type 2 diabetic patients and 10 healthy donors. Effects of 3-bromopyruvate and 3-nitropropionate on pyruvate dehydrogenase complex (PDHC) and succinate dehydrogenase activities, as well as levels of acetyl-CoA, ATP, thiobarbituric acid reactive species and aggregation were assessed in non-activated and thrombin-activated platelets. RESULTS: In type 2 diabetic patients fasting plasma glucose and fructosamine levels were 61 and 64% higher, respectively, than in the healthy group (p < 0.001). In non-activated diabetic platelets PDHC activity, PDHC-E2, acetyl-CoA and ATP levels were 66, 70, 68 and 60%, higher, respectively, than in platelets from healthy controls (p < 0.01). 3-bromopyruvate (0.1 mM) decreased pyruvate dehydrogenase activity in healthy and diabetic platelets by 42% and 59%, respectively. Similar inhibitory effects were observed for acetyl-CoA and ATP levels, aggregation and TBARS accumulation rates. Succinate dehydrogenase activity was inhibited by 3-nitropropionate (10 mM) to 38 and 41% of control values in healthy and diabetic platelets, respectively, but affected neither function nor acetyl-CoA metabolism in platelets of both groups. CONCLUSIONS: These data indicate that inhibition of pyruvate dehydrogenase excessive activity in diabetic platelets by 3-bromopyruvate may normalise their functional parameters through adjustment of acetyl-CoA/ATP levels to control values. Platelets from blood of diabetic patients display higher activities of pyruvate dehydrogenase complex (PDHC), higher levels of dihydrolipoate transacetylase (DLAT, E2 subunit of PDHC) as well as higher levels of acetyl-CoA yielding greater ATP/ADP accumulation than in platelets of normoglycemic subjects. Therefore, in diabetic platelets, thrombin caused higher release of ATP/ADP triggering excessive production of reactive oxygen species (ROS) and stronger aggregation compared to control platelets. In diabetic platelets, relative excess of DLAT in PDHC made them highly susceptible to 3-bromopyruvate (3BrP) inhibition. Resulting limitation of acetyl-CoA provision by 3-BrP normalised activity of diabetic platelets.


Assuntos
Plaquetas/efeitos dos fármacos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Complexo Piruvato Desidrogenase/antagonistas & inibidores , Piruvatos/farmacologia , Acetilcoenzima A/metabolismo , Trifosfato de Adenosina/metabolismo , Adulto , Estudos de Casos e Controles , Diabetes Mellitus Tipo 2/fisiopatologia , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Nitrocompostos/farmacologia , Propionatos/farmacologia , Succinato Desidrogenase/metabolismo
20.
J Appl Microbiol ; 129(2): 345-355, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32091657

RESUMO

AIMS: Paclitaxel is a type of broad-spectrum anticancer drug in short supply. The price of acetyl-CoA (17 709 677·4 USD mol-1 ), which is the acetyl group donor for the enzymatic synthesis of the intermediate, baccatin Ⅲ, is still the bottleneck of the mass production of paclitaxel. This study reports a novel acetyl group donor, which could substantially reduce the cost of production. METHODS AND RESULTS: In this study, a substrate spectrum with 14 kinds of representative acetyl-donor substitutes predicted by computer-aided methods was tested in a 10-deacetylbaccatin Ⅲ-10-O-acetyltransferase (DBAT) heterogeneous-expressed open-whole-cell catalytic system. The results of computer prediction and experimental analysis revealed the rule of the acetyl-donor compounds based on this substrate spectrum. N-acetyl-d-glucosamine (30·95 USD mol-1 , about 572 202-fold cheaper than acetyl-CoA) is selected as a suitable substitute under the rule. The yield when using N-acetyl-d-glucosamine as acetyl donor in open-whole-cell catalytic system was 2·13-fold of that when using acetyl-CoA. In the in vivo system, the yield increased 24·17%, which may indicate its cooperation with acetyl-CoA. CONCLUSION: The success of open-whole-cell synthesis and in vivo synthesis of baccatin Ⅲ by adding N-acetyl-d-glucosamine as acetyl substrate demonstrates that it is a useful substrate to improve the yield of baccatin Ⅲ. SIGNIFICANCE AND IMPACT OF THE STUDY: All these findings provided a potential acetyl-donor substitute for acetyl-CoA, as well as a low cost and efficient method of preparing paclitaxel through baccatin Ⅲ semi-synthesis.


Assuntos
Acetilglucosamina/metabolismo , Alcaloides/biossíntese , Acetilcoenzima A/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Alcaloides/economia , Antineoplásicos Fitogênicos/biossíntese , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/economia , Biocatálise , Paclitaxel/biossíntese , Paclitaxel/química , Paclitaxel/economia , Especificidade por Substrato , Taxoides/economia
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