Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 69
Filtrar
Mais filtros










Filtros aplicados

Base de dados
Tipo de estudo
Intervalo de ano de publicação
1.
ACS Chem Biol ; 13(3): 685-693, 2018 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-29336543

RESUMO

Histone deacetylase 11 (HDAC11) is a sole member of the class IV HDAC subfamily with negligible intrinsic deacetylation activity. Here, we report in vitro profiling of HDAC11 deacylase activities, and our data unequivocally show that the enzyme efficiently removes acyl moieties spanning 8-18 carbons from the side chain nitrogen of the lysine residue of a peptidic substrate. Additionally, N-linked lipoic acid and biotin are removed by the enzyme, although with lower efficacy. Catalytic efficiencies toward dodecanoylated and myristoylated peptides were 77 700 and 149 000 M-1 s-1, respectively, making HDAC11 the most proficient fatty-acid deacylase of the HDAC family. Interestingly, HDAC11 is strongly inhibited by free myristic, palmitic, and stearic acids with inhibition constants of 6.5, 0.9, and 1.6 µM, respectively. At the same time, its deacylase activity is stimulated more than 2.5-fold by both palmitoyl-coenzyme A and myristoyl-coenzyme A, pointing toward metabolic control of the enzymatic activity by fatty-acid metabolites. Our data reveal novel enzymatic activity of HDAC11 that can, in turn, facilitate the uncovering of additional biological functions of the enzyme as well as the design of isoform-specific HDAC inhibitors.


Assuntos
Acetil-CoA Hidrolase/metabolismo , Desenho de Fármacos , Histona Desacetilases/metabolismo , Acetil-CoA Hidrolase/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Ácidos Graxos/farmacologia , Histona Desacetilases/efeitos dos fármacos , Lisina/metabolismo , Peptídeos/metabolismo , Especificidade por Substrato
2.
FEMS Yeast Res ; 15(3)2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25852051

RESUMO

Acetyl-coenzyme A (acetyl-CoA) is not only an essential intermediate in central carbon metabolism, but also an important precursor metabolite for native or engineered pathways that can produce many products of commercial interest such as pharmaceuticals, chemicals or biofuels. In the yeast Saccharomyces cerevisiae, acetyl-CoA is compartmentalized in the cytosol, mitochondrion, peroxisome and nucleus, and cannot be directly transported between these compartments. With the acetyl-carnitine or glyoxylate shuttle, acetyl-CoA produced in peroxisomes or the cytoplasm can be transported into the cytoplasm or the mitochondria. However, whether acetyl-CoA generated in the mitochondria can be exported to the cytoplasm is still unclear. Here, we investigated whether the transfer of acetyl-CoA from the mitochondria to the cytoplasm can occur using a pyruvate decarboxylase negative, non-fermentative yeast strain. We found that mitochondrial Ach1 can convert acetyl-CoA in this compartment into acetate, which crosses the mitochondrial membrane before being converted into acetyl-CoA in the cytosol. Based on our finding we propose a model in which acetate can be used to exchange acetyl units between mitochondria and the cytosol. These results will increase our fundamental understanding of intracellular transport of acetyl units, and also help to develop microbial cell factories for many kinds of acetyl-CoA derived products.


Assuntos
Ácido Acético/metabolismo , Acetilcoenzima A/metabolismo , Acetil-CoA Hidrolase/metabolismo , Coenzima A-Transferases/metabolismo , Citosol/química , Mitocôndrias/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Mitocôndrias/metabolismo , Piruvato Descarboxilase/deficiência , Saccharomyces cerevisiae/metabolismo
3.
PLoS Genet ; 11(3): e1005029, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25768301

RESUMO

The formation of DNA double-strand breaks (DSBs) must take place during meiosis to ensure the formation of crossovers, which are required for accurate chromosome segregation, therefore avoiding aneuploidy. However, DSB formation must be tightly regulated to maintain genomic integrity. How this regulation operates in the context of different chromatin architectures and accessibility, and how it is linked to metabolic pathways, is not understood. We show here that global histone acetylation levels undergo changes throughout meiotic progression. Moreover, perturbations to global histone acetylation levels are accompanied by changes in the frequency of DSB formation in C. elegans. We provide evidence that the regulation of histone acetylation requires CRA-1, a NatB domain-containing protein homologous to human NAA25, which controls the levels of acetyl-Coenzyme A (acetyl-CoA) by antagonizing ACER-1, a previously unknown and conserved acetyl-CoA hydrolase. CRA-1 is in turn negatively regulated by XND-1, an AT-hook containing protein. We propose that this newly defined protein network links acetyl-CoA metabolism to meiotic DSB formation via modulation of global histone acetylation.


Assuntos
Acetilcoenzima A/metabolismo , Acetil-CoA Hidrolase/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Recombinação Genética , Acetilação , Animais , Quebras de DNA de Cadeia Dupla , Histonas/metabolismo , Cromossomo X/metabolismo
4.
PLoS One ; 9(5): e96370, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24823794

RESUMO

Acetyl Coenzyme A-dependent N-, O- and N,O-acetylation of aromatic amines and hydrazines by arylamine N-acetyltransferases is well characterised. Here, we describe experiments demonstrating that human arylamine N-acetyltransferase Type 1 and its murine homologue (Type 2) can also catalyse the direct hydrolysis of acetyl Coenzyme A in the presence of folate. This folate-dependent activity is exclusive to these two isoforms; no acetyl Coenzyme A hydrolysis was found when murine arylamine N-acetyltransferase Type 1 or recombinant bacterial arylamine N-acetyltransferases were incubated with folate. Proton nuclear magnetic resonance spectroscopy allowed chemical modifications occurring during the catalytic reaction to be analysed in real time, revealing that the disappearance of acetyl CH3 from acetyl Coenzyme A occurred concomitantly with the appearance of a CH3 peak corresponding to that of free acetate and suggesting that folate is not acetylated during the reaction. We propose that folate is a cofactor for this reaction and suggest it as an endogenous function of this widespread enzyme. Furthermore, in silico docking of folate within the active site of human arylamine N-acetyltransferase Type 1 suggests that folate may bind at the enzyme's active site, and facilitate acetyl Coenzyme A hydrolysis. The evidence presented in this paper adds to our growing understanding of the endogenous roles of human arylamine N-acetyltransferase Type 1 and its mouse homologue and expands the catalytic repertoire of these enzymes, demonstrating that they are by no means just xenobiotic metabolising enzymes but probably also play an important role in cellular metabolism. These data, together with the characterisation of a naphthoquinone inhibitor of folate-dependent acetyl Coenzyme A hydrolysis by human arylamine N-acetyltransferase Type 1/murine arylamine N-acetyltransferase Type 2, open up a range of future avenues of exploration, both for elucidating the developmental role of these enzymes and for improving chemotherapeutic approaches to pathological conditions including estrogen receptor-positive breast cancer.


Assuntos
Acetil-CoA Hidrolase/metabolismo , Acetiltransferases/metabolismo , Arilamina N-Acetiltransferase/metabolismo , Ácido Fólico/metabolismo , Isoenzimas/metabolismo , Acetilação , Animais , Humanos , Hidrólise , Camundongos , Ressonância Magnética Nuclear Biomolecular
5.
Artigo em Inglês | MEDLINE | ID: mdl-24192375

RESUMO

Neisseria meningitidis is the causative microorganism of many human diseases, including bacterial meningitis; together with Streptococcus pneumoniae, it accounts for approximately 80% of bacterial meningitis infections. The emergence of antibiotic-resistant strains of N. meningitidis has created a strong urgency for the development of new therapeutics, and the high-resolution structural elucidation of enzymes involved in cell metabolism represents a platform for drug development. Acetyl-CoA hydrolase is involved in multiple functions in the bacterial cell, including membrane synthesis, fatty-acid and lipid metabolism, gene regulation and signal transduction. Here, the first recombinant protein expression, purification and crystallization of a hexameric acetyl-CoA hydrolase from N. meningitidis are reported. This protein was crystallized using the hanging-drop vapour-diffusion technique at pH 8.5 and 290 K using ammonium phosphate as a precipitant. Optimized crystals diffracted to 2.0 Šresolution at the Australian Synchrotron and belonged to space group P2(1)3 (unit-cell parameters a = b = c = 152.2 Å), with four molecules in the asymmetric unit.


Assuntos
Acetil-CoA Hidrolase/química , Acetil-CoA Hidrolase/isolamento & purificação , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Neisseria meningitidis/enzimologia , Cristalização , Eletroforese em Gel de Poliacrilamida , Humanos , Difração de Raios X
6.
J Lipid Res ; 54(8): 2049-59, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23709691

RESUMO

Acyl-CoA thioesterase 12 (ACOT12) is the major enzyme known to hydrolyze the thioester bond of acetyl-CoA in the cytosol in the liver. ACOT12 contains a catalytic thioesterase domain at the N terminus and a steroidogenic acute regulatory protein-related lipid transfer (START) domain at the C terminus. We investigated the effects of lipids (phospholipids, sphingolipids, fatty acids, and sterols) on ACOT12 thioesterase activity and found that the activity was inhibited by phosphatidic acid (PA) in a noncompetitive manner. In contrast, the enzymatic activity of a mutant form of ACOT12 lacking the START domain was not inhibited by the lipids. These results suggest that the START domain is important for regulation of ACOT12 activity by PA. We also found that PA could bind to thioesterase domain, but not to the START domain, and had no effect on ACOT12 dissociation. ACOT12 is detectable in the liver but not in hepatic cell lines such as HepG2, Hepa-1, and Fa2N-4. ACOT12 mRNA and protein levels in rat primary hepatocytes decreased following treatment with insulin. These results suggest that cytosolic acetyl-CoA levels in the liver are controlled by lipid metabolites and hormones, which result in allosteric enzymatic and transcriptional regulation of ACOT12.


Assuntos
Acetil-CoA Hidrolase/genética , Acetil-CoA Hidrolase/metabolismo , Citoplasma/enzimologia , Transcrição Genética/genética , Acetil-CoA Hidrolase/antagonistas & inibidores , Acetil-CoA Hidrolase/deficiência , Animais , Insulina/farmacologia , Lipídeos/biossíntese , Fígado/enzimologia , Fígado/metabolismo , Dados de Sequência Molecular , Ácidos Fosfatídicos/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Transcrição Genética/efeitos dos fármacos
7.
Biochim Biophys Acta ; 1823(8): 1389-94, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22705155

RESUMO

BACKGROUND: Several important functions for a successful spermatogenesis are dependent on Sertoli cells (SCs). Besides their unique characteristics as support cells, they produce essential cofactors and metabolites, and are responsible for nurturing the developing germ cells. The continuous production of lipids, phospholipids and proteins by germ cells must require high amounts of metabolic precursors. Thus, we hypothesized that hSCs could produce acetate in a hormonally-regulated manner. METHODS: hSC-enriched primary cultures were maintained in the absence of insulin or in the presence of 17ß-estradiol (E2) or 5α-dihydrotestosterone (DHT). Acetate production was determined by 1H-NMR. mRNA gene expression levels of Acetyl CoA hydrolase (ACoA Hyd) and Acetyl CoA synthase (ACoA Synt) were determined by RT-PCR. RESULTS: hSCs produced high amounts of acetate suggesting that this metabolite should play a key role on the progression of spermatogenesis, namely as a metabolic precursor for the synthesis of cellular constituents. In addition, acetate metabolism proved to be under strict hormonal regulation. In the presence of E2 or DHT, hSCs produced different amounts of acetate. While E2 treatment increased acetate production, increasing ACoA Hyd gene transcript levels, DHT-treated cells showed decreased acetate production, differently modulating the ratio ACoA Hyd/ACoA Synt. Surprisingly, insulin-deprivation completely suppressed acetate production/export and significantly decreased the ACoA Hyd gene transcript levels. GENERAL SIGNIFICANCE: Taken together, these results suggest that, although hSCs are primarily described as lactate producers, the elevated production of acetate deserves special attention, in order to clarify the mechanisms behind its hormonal regulation and its role on a successful spermatogenesis.


Assuntos
Acetatos/metabolismo , Estradiol/fisiologia , Insulina/fisiologia , Células de Sertoli/metabolismo , Acetil-CoA Hidrolase/genética , Acetil-CoA Hidrolase/metabolismo , Androgênios/farmacologia , Androgênios/fisiologia , Células Cultivadas , Di-Hidrotestosterona/farmacologia , Estradiol/farmacologia , Expressão Gênica , Humanos , Insulina/deficiência , Masculino
8.
J Biol Chem ; 287(21): 17186-97, 2012 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-22474284

RESUMO

Insect stage trypanosomes use an "acetate shuttle" to transfer mitochondrial acetyl-CoA to the cytosol for the essential fatty acid biosynthesis. The mitochondrial acetate sources are acetate:succinate CoA-transferase (ASCT) and an unknown enzymatic activity. We have identified a gene encoding acetyl-CoA thioesterase (ACH) activity, which is shown to be the second acetate source. First, RNAi-mediated repression of ASCT in the ACH null background abolishes acetate production from glucose, as opposed to both single ASCT and ACH mutants. Second, incorporation of radiolabeled glucose into fatty acids is also abolished in this ACH/ASCT double mutant. ASCT is involved in ATP production, whereas ACH is not, because the ASCT null mutant is ∼1000 times more sensitive to oligomycin, a specific inhibitor of the mitochondrial F(0)/F(1)-ATP synthase, than wild-type cells or the ACH null mutant. This was confirmed by RNAi repression of the F(0)/F(1)-ATP synthase F(1)ß subunit, which is lethal when performed in the ASCT null background but not in the wild-type cells or the ACH null background. We concluded that acetate is produced from both ASCT and ACH; however, only ASCT is responsible, together with the F(0)/F(1)-ATP synthase, for ATP production in the mitochondrion.


Assuntos
Acetatos/metabolismo , Acetilcoenzima A/metabolismo , Acetil-CoA Hidrolase/metabolismo , Trifosfato de Adenosina/biossíntese , Coenzima A-Transferases/metabolismo , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/enzimologia , Acetilcoenzima A/genética , Acetil-CoA Hidrolase/genética , Coenzima A-Transferases/genética , Ácidos Graxos/genética , Ácidos Graxos/metabolismo , Glucose/genética , Glucose/metabolismo , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Mutação , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Proteínas de Protozoários/genética , Trypanosoma brucei brucei/genética
9.
Am J Physiol Regul Integr Comp Physiol ; 298(5): R1435-43, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20237302

RESUMO

To examine the regulation of hepatic acetogenesis in neonatal swine, carnitine palmitoyltransferase I (CPT I) activity was measured in the presence of varying palmitoyl-CoA (substrate) and malonyl-CoA (inhibitor) concentrations, and [1-(14)C]-palmitate oxidation was simultaneously measured. Accumulation rates of (14)C-labeled acetate, ketone bodies, and citric acid cycle intermediates within the acid-soluble products were determined using radio-HPLC. Measurements were conducted in mitochondria isolated from newborn, 24-h (fed or fasted), and 5-mo-old pigs. Acetate rather than ketone bodies was the predominant radiolabeled product, and its production increased twofold with increasing fatty acid oxidation during the first 24-h suckling period. The rate of acetogenesis was directly proportional to CPT I activity. The high activity of CPT I in 24-h-suckling piglets was not attributable to an increase in CPT I gene expression, but rather to a large decrease in the sensitivity of CPT I to malonyl-CoA inhibition, which offset a developmental decrease in affinity of CPT I for palmitoyl-CoA. Specifically, the IC(50) for malonyl-CoA inhibition and K(m) value for palmitoyl-CoA measured in 24-h-suckling pigs were 1.8- and 2.7-fold higher than measured in newborn pigs. The addition of anaplerotic carbon from malate (10 mM) significantly reduced (14)C accumulation in acetate (P < 0.003); moreover, the reduction was much greater in newborn (80%) than in 24-h-fed (72%) and 5-mo-old pigs (55%). The results demonstrate that acetate is the primary product of hepatic mitochondrial beta-oxidation in Sus scrofa and that regulation during early development is mediated primarily via kinetic modulation of CPT I.


Assuntos
Acetatos/metabolismo , Carnitina O-Palmitoiltransferase/metabolismo , Ácidos Graxos/metabolismo , Fígado/enzimologia , Sus scrofa/metabolismo , Acetil-CoA Hidrolase/metabolismo , Fatores Etários , Animais , Animais Lactentes , Radioisótopos de Carbono , Carnitina O-Palmitoiltransferase/genética , Ácido Cítrico/metabolismo , Ciclo do Ácido Cítrico/fisiologia , Ativação Enzimática/fisiologia , Regulação Enzimológica da Expressão Gênica , Corpos Cetônicos/metabolismo , Malonil Coenzima A/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias Hepáticas/enzimologia , Oxirredução , Palmitatos/farmacocinética , Palmitoil Coenzima A/metabolismo , RNA Mensageiro/metabolismo
10.
Fungal Genet Biol ; 46(6-7): 473-85, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19298859

RESUMO

Saccharomyces cerevisiae and Neurospora crassa mutants defective in the so-called acetyl-CoA hydrolases Ach1p and Acu-8, respectively, display a severe growth defect on acetate, which is most strongly pronounced under acidic conditions. Acetyl-CoA hydrolysis is an energy wasting process and therefore denoted as a biochemical conundrum. Acetyl-CoA hydrolases show high sequence identity to the CoA-transferase CoaT from Aspergillus nidulans. Therefore, we extensively re-characterised the yeast enzyme. Ach1p showed highest specific activity for the CoASH transfer from succinyl-CoA to acetate and only a minor acetyl-CoA-hydrolase activity. Complementation of an ach1 mutant with the coaT gene reversed the growth defect on acetate confirming the in vivo function of Ach1p as a CoA-transferase. Our results imply that Ach1p is involved in mitochondrial acetate detoxification by a CoASH transfer from succinyl-CoA to acetate. Thereby, Ach1p does not perform the energy wasting hydrolysis of acetyl-CoA but conserves energy by the detoxification of mitochondrial acetate.


Assuntos
Ácido Acético/metabolismo , Acetil-CoA Hidrolase/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Acetilcoenzima A/metabolismo , Acetil-CoA Hidrolase/genética , Acetil-CoA Hidrolase/isolamento & purificação , Acetil-CoA Hidrolase/metabolismo , Cinética , Mitocôndrias/química , Mitocôndrias/enzimologia , Mitocôndrias/genética , Mitocôndrias/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato
11.
Eukaryot Cell ; 7(10): 1733-41, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18689527

RESUMO

Acetyl coenzyme A (acetyl-CoA) is the central intermediate of the pathways required to metabolize nonfermentable carbon sources. Three such pathways, i.e., gluconeogenesis, the glyoxylate cycle, and beta-oxidation, are required for full virulence in the fungal pathogen Candida albicans. These processes are compartmentalized in the cytosol, mitochondria, and peroxosomes, necessitating transport of intermediates across intracellular membranes. Acetyl-CoA is trafficked in the form of acetate by the carnitine shuttle, and we hypothesized that the enzymes that convert acetyl-CoA to/from acetate, i.e., acetyl-CoA hydrolase (ACH1) and acetyl-CoA synthetase (ACS1 and ACS2), would regulate alternative carbon utilization and virulence. We show that C. albicans strains depleted for ACS2 are unviable in the presence of most carbon sources, including glucose, acetate, and ethanol; these strains metabolize only fatty acids and glycerol, a substantially more severe phenotype than that of Saccharomyces cerevisiae acs2 mutants. In contrast, deletion of ACS1 confers no phenotype, though it is highly induced in the presence of fatty acids, perhaps explaining why acs2 mutants can utilize fatty acids. Strains lacking ACH1 have a mild growth defect on some carbon sources but are fully virulent in a mouse model of disseminated candidiasis. Both ACH1 and ACS2 complement mutations in their S. cerevisiae homolog. Together, these results show that acetyl-CoA metabolism and transport are critical for growth of C. albicans on a wide variety of nutrients. Furthermore, the phenotypic differences between mutations in these highly conserved genes in S. cerevisiae and C. albicans support recent findings that significant functional divergence exists even in fundamental metabolic pathways between these related yeasts.


Assuntos
Acetilcoenzima A/metabolismo , Candida albicans/metabolismo , Carbono/metabolismo , Acetato-CoA Ligase/genética , Acetato-CoA Ligase/metabolismo , Acetil-CoA Hidrolase/genética , Acetil-CoA Hidrolase/metabolismo , Animais , Candida albicans/enzimologia , Candida albicans/genética , Candida albicans/patogenicidade , Candidíase/microbiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Teste de Complementação Genética , Camundongos , Mutação , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Virulência
12.
Mol Cell Endocrinol ; 265-266: 113-20, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17207922

RESUMO

Stimulation of receptors and subsequent signal transduction results in the activation of arachidonic acid (AA) release. Once AA is released from phospholipids or others esters, it may be metabolized via the cycloxygenase or the lipoxygenase pathways. How the cells drive AA to these pathways is not elucidated yet. It is reasonable to speculate that each pathway will have different sources of free AA triggered by different signal transduction pathways. Several reports have shown that AA and its lipoxygenase-catalyzed metabolites play essential roles in the regulation of steroidogenesis by influencing cholesterol transport from the outer to the inner mitochondrial membrane, the rate-limiting step in steroid hormone biosynthesis. Signals that stimulate steroidogenesis also cause the release of AA from phospholipids or other esters by mechanisms that are not fully understood. This review focuses on the enzymes of AA release that impact on steroidogenesis.


Assuntos
Glândulas Suprarrenais/enzimologia , Ácido Araquidônico/metabolismo , Células Intersticiais do Testículo/enzimologia , Tioléster Hidrolases/metabolismo , Acetil-CoA Hidrolase/metabolismo , Animais , Colesterol/metabolismo , Humanos , Masculino , Mitocôndrias/enzimologia , Esteroides/biossíntese
13.
Acta Biochim Pol ; 53(3): 553-61, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16951743

RESUMO

A cDNA encoding human cytosolic acetyl-CoA hydrolase (CACH) was isolated from a human liver cDNA library, sequenced and functionally expressed in insect cells. The human CACH cDNA encodes a 555-amino-acid sequence that is 81.4%/78.7% identical to those of the mouse/rat homologue, suggesting a conserved role for this enzyme in the human and rodent livers. Bioinformatical study further reveals a high degree of similarity among the human and rodent CACHs as follows: First, the gene is composed of 15 exons ranging in size from 56 to 157 bp. Second, the protein consists of two thioesterase regions and a C-terminal steroidogenic acute regulatory protein-related lipid transfer (START) domain. Third, the promoter region is GC-rich and contains GC boxes, but lacks both TATA and CCAAT boxes, the typical criteria of housekeeping genes. A consensus peroxisome proliferator responsive element (PPRE) present in the rodent CACH promoter regions supports marked CACH induction in rat liver by peroxisome proliferator (PP).


Assuntos
Acetil-CoA Hidrolase/genética , Acetil-CoA Hidrolase/isolamento & purificação , Citosol/enzimologia , Expressão Gênica , Acetil-CoA Hidrolase/química , Acetil-CoA Hidrolase/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Biblioteca Gênica , Humanos , Fígado/enzimologia , Camundongos , Dados de Sequência Molecular , Ratos , Proteínas Recombinantes , Mapeamento por Restrição , Homologia de Sequência de Aminoácidos , Spodoptera
14.
Biochim Biophys Acta ; 1761(1): 17-23, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16476568

RESUMO

Acetate has been found as an endogenous metabolite of beta-oxidation of fatty acids in liver. In order to investigate the regulation of acetate generation in liver mitochondria, we attempted to purify a mitochondrial acetyl-CoA hydrolase in rat liver. This acetyl-CoA-hydrolyzing activity in isolated mitochondria was induced by the treatment of rats with di(2-ehtylhexyl)phthalate (DEHP), a peroxisome proliferator which induces expression of several peroxisomal and mitochondrial enzymes involved in beta-oxidation of fatty acids. The purified enzyme was 43-kDa in molecular mass by SDS/PAGE. Internal amino acid sequencing of this enzyme revealed that it was identical with mitochondrial 3-ketoacyl-CoA thiolase, suggesting that this enzyme has two kinds of activities, 3-ketoacyl-CoA thiolase and acetyl-CoA hydrolase activities. Kinetic studies clearly indicated that this enzyme had the both activities and each activity was inhibited by the substrates of the other activity, that is, 3-ketoacyl-CoA thiolase activity was inhibited by acetyl-CoA, on the other hand, acetyl-CoA hydrolase activity was inhibited by acetoacetyl-CoA in a competitive manner. These findings suggested that acetate generation in liver mitochondria is a side reaction of this known enzyme, 3-ketoacyl-CoA thiolase, and this enzyme may regulate its activities depending on each substrate level.


Assuntos
Acetatos/metabolismo , Acetil-CoA C-Aciltransferase/metabolismo , Acetil-CoA Hidrolase/metabolismo , Mitocôndrias Hepáticas/metabolismo , Acetil-CoA Hidrolase/isolamento & purificação , Acil Coenzima A/metabolismo , Animais , Cromatografia em Gel , Dietilexilftalato/farmacologia , Ativação Enzimática , Cinética , Masculino , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/enzimologia , Ratos , Ratos Sprague-Dawley
15.
Plant Mol Biol ; 55(5): 645-62, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15604707

RESUMO

Acetyl Coenzyme A (acetyl CoA) is required in the mitochondria to fuel the operation of the Krebs cycle and within the cytosolic, peroxisomal and plastidial compartments wherein it acts as the immediate precursor for a wide range of anabolic functions. Since this metabolite is impermeable to membranes it follows that discrete pathways both for its synthesis and for its utilization must be present in each of these organelles and that the size of the various compartmented pools are independently regulated. To determine the specific role of acetyl CoA in the mitochondria we exploited a transgenic approach to introduce a yeast acetyl CoA hydrolase (EC 3.1.2.1.) into this compartment in tobacco plants. Despite the facts that the introduced enzyme was correctly targeted and that there were marked reductions in the levels of citrate and malate and an increase in the acetate content of the transformants, the transgenic plants surprisingly exhibited increased acetyl CoA levels. The lines were further characterised by a severe growth retardation, abnormal leaf colouration and a dramatic reduction in photosynthetic activity correlated with a marked reduction in the levels of transcripts of photosynthesis and in the content of photosynthetic pigments. The altered rate of photosynthesis in the transgenics was also reflected by a modified carbon partitioning in leaves of these lines, however, further studies revealed that this was most likely caused by a decreased source to sink transport of carbohydrate. In summary these results suggest that the content of acetyl CoA is under tight control and that alterations in the level of this central metabolite have severe metabolic and developmental consequences in tobacco.


Assuntos
Acetil-CoA Hidrolase/genética , Regulação Enzimológica da Expressão Gênica , Mitocôndrias/enzimologia , Fotossíntese/fisiologia , Tabaco/genética , Acetato-CoA Ligase/genética , Acetil-CoA Hidrolase/metabolismo , Transporte Biológico , Metabolismo dos Carboidratos , Regulação da Expressão Gênica de Plantas , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Ácidos Glicéricos/metabolismo , Glicina/metabolismo , Transferases Intramoleculares/genética , Fenótipo , Fotossíntese/genética , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Complexo Piruvato Desidrogenase/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Ribulose-Bifosfato Carboxilase/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Amido/metabolismo , Sacarose/metabolismo , Fatores de Tempo , Tabaco/crescimento & desenvolvimento , Tabaco/fisiologia , Transcrição Genética/genética , Uridina Difosfato Glucose/metabolismo
16.
Biochem J ; 383(Pt. 3): 517-27, 2004 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-15233626

RESUMO

3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (HMGS; EC 2.3.3.10) is the second enzyme in the cytoplasmic mevalonate pathway of isoprenoid biosynthesis, and catalyses the condensation of acetyl-CoA with acetoacetyl-CoA (AcAc-CoA) to yield S-HMG-CoA. In this study, we have first characterized in detail a plant HMGS, Brassica juncea HMGS1 (BjHMGS1), as a His6-tagged protein from Escherichia coli. Native gel electrophoresis analysis showed that the enzyme behaves as a homodimer with a calculated mass of 105.8 kDa. It is activated by 5 mM dithioerythreitol and is inhibited by F-244 which is specific for HMGS enzymes. It has a pH optimum of 8.5 and a temperature optimum of 35 degrees C, with an energy of activation of 62.5 J x mol(-1). Unlike cytosolic HMGS from chicken and cockroach, cations like Mg2+, Mn2+, Zn2+ and Co2+ did not stimulate His6-BjHMGS1 activity in vitro; instead all except Mg2+ were inhibitory. His6-BjHMGS1 has an apparent K(m-acetyl-CoA) of 43 microM and a V(max) of 0.47 micromol x mg(-1) x min(-1), and was inhibited by one of the substrates (AcAc-CoA) and by both products (HMG-CoA and HS-CoA). Site-directed mutagenesis of conserved amino acid residues in BjHMGS1 revealed that substitutions R157A, H188N and C212S resulted in a decreased V(max), indicating some involvement of these residues in catalytic capacity. Unlike His6-BjHMGS1 and its soluble purified mutant derivatives, the H188N mutant did not display substrate inhibition by AcAc-CoA. Substitution S359A resulted in a 10-fold increased specific activity. Based on these kinetic analyses, we generated a novel double mutation H188N/S359A, which resulted in a 10-fold increased specific activity, but still lacking inhibition by AcAc-CoA, strongly suggesting that His-188 is involved in conferring substrate inhibition on His6-BjHMGS1. Substitution of an aminoacyl residue resulting in loss of substrate inhibition has never been previously reported for any HMGS.


Assuntos
Hidroximetilglutaril-CoA Sintase/genética , Mostardeira/enzimologia , Acetil-CoA Hidrolase/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Arabidopsis/química , Arginina/genética , Proteínas Aviárias/química , Cátions/metabolismo , Galinhas/genética , Dicroísmo Circular/métodos , Baratas/genética , Ácidos Graxos Insaturados/metabolismo , Regulação Enzimológica da Expressão Gênica/genética , Histidina/biossíntese , Histidina/química , Histidina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Hidroximetilglutaril-CoA Sintase/biossíntese , Hidroximetilglutaril-CoA Sintase/química , Hidroximetilglutaril-CoA Sintase/metabolismo , Proteínas de Insetos/química , Cinética , Lactonas/metabolismo , Camundongos , Dados de Sequência Molecular , Peso Molecular , Mutação/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas Recombinantes/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Alinhamento de Sequência/métodos , Temperatura
17.
Exp Anim ; 52(2): 99-107, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12806884

RESUMO

The primary aim of the present study was to define central and peripheral physiological differences between dietary obesity-susceptible (DOS) and obesity-resistant (DOR) outbred Sprague Dawley (SD) rats when given a moderate high fat diet containing 32.34% of energy as a fat. After a 9-week feeding period, the DOS-SD rats consumed significantly more feed (11.1%) and had higher abdominal (39.9%) and epididymal (27.5%) fat pads than the DOR-SD rats. In addition, serum leptin and insulin levels were significantly increased in the DOS-SD rats compared with those in the DOR-SD rats. However, we did not observe significant differences in serum triglyceride, cholesterol and glucose. No differences in hypothalamic OB-Ra and Rb mRNA expressions were found between the two groups. In contrast, arcuate NPY immunohistochemical expression was much higher in the DOS-SD rats than in the DOR-SD rats, though NPY expression in the supraoptic and paraventricular nuclei was not different between the two phenotypes. In peripheral tissues, the DOS-SD rats showed noticeably increased acetyl CoA carboxylase (ACC) mRNA expression in the liver, not epididymal fat. However, Western blot of peroxisomal proliferator activated factor gamma (PPAR gamma) in the liver and epididymal fat was not different between the two phenotypes of SD rats. It was concluded that different body weight phenotypes within outbred SD population responded differently to the development of dietary induced obesity via altered anabolic features in the hypothalamus and liver.


Assuntos
Dieta , Predisposição Genética para Doença , Obesidade/etiologia , Acetil-CoA Hidrolase/genética , Animais , Sequência de Bases , Colesterol/sangue , Primers do DNA , Imuno-Histoquímica , Insulina/sangue , Leptina/sangue , Masculino , Obesidade/genética , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Receptores de Superfície Celular/genética , Receptores para Leptina , Triglicerídeos/sangue
18.
J Chromatogr B Analyt Technol Biomed Life Sci ; 790(1-2): 239-44, 2003 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-12767336

RESUMO

An overview of the purification of an oligomeric enzyme, an extramitochondrial acetyl-coenzyme A hydrolase from rat liver, is presented. The enzyme has been purified to homogeneity using two successive size-exclusion chromatography runs, first for the monomeric and second for the oligomeric form of the enzyme. The sequential gel-filtration steps efficiently removed the contaminants of any molecular size, first of different size from that of the monomeric form of the enzyme (K(av)=0.47 on Superdex 200) and second of different size from that of the oligomeric form (K(av)=0.33), allowing us to purify the enzyme in high purity. This strategy provides an excellent model for purifying many other oligomeric proteins including key enzymes or allosteric enzymes regulating metabolism.


Assuntos
Acetil-CoA Hidrolase/isolamento & purificação , Cromatografia em Gel/métodos , Fígado/enzimologia , Acetil-CoA Hidrolase/química , Animais , Biopolímeros , Citosol/enzimologia , Ratos
19.
J Biol Chem ; 278(19): 17203-9, 2003 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-12606555

RESUMO

Acetyl-CoA hydrolase (Ach1p), catalyzing the hydrolysis of acetyl-CoA, is presumably involved in regulating intracellular acetyl-CoA or CoASH pools; however, its intracellular functions and distribution remain to be established. Using site-directed mutagenesis analysis, we demonstrated that the enzymatic activity of Ach1p is dependent upon its putative acetyl-CoA binding sites. The ach1 mutant causes a growth defect in acetate but not in other non-fermentable carbon sources, suggesting that Ach1p is not involved in mitochondrial biogenesis. Overexpression of Ach1p, but not constructs containing acetyl-CoA binding site mutations, in ach1-1 complemented the defect of acetate utilization. By subcellular fractionation, most of the Ach1p in yeast was distributed with mitochondria and little Ach1p in the cytoplasm. By immunofluorescence microscopy, we show that Ach1p and acetyl-CoA binding site-mutated constructs, but not its N-terminal deleted construct, are localized in mitochondria. Moreover, the onset of pseudohyphal development in homozygote ach1-1 diploids was abolished. We infer that Ach1p may be involved in a novel acetyl-CoA biogenesis and/or acetate utilization in mitochondria and thereby indirectly affect pseudohyphal development in yeast.


Assuntos
Acetil-CoA Hidrolase/metabolismo , Proteínas Fúngicas/metabolismo , Saccharomyces cerevisiae/enzimologia , Acetilcoenzima A/metabolismo , Acetil-CoA Hidrolase/genética , Sequência de Aminoácidos , Proteínas Fúngicas/genética , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Alinhamento de Sequência
20.
Acta Biochim Pol ; 49(4): 937-45, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12545200

RESUMO

A cytosolic acetyl-CoA hydrolase (CACH) cDNA has been isolated from mouse liver cDNA library and sequenced. Recombinant expression of the cDNA in insect cells resulted in overproduction of active acetyl-CoA hydrolyzing enzyme protein. The mouse CACH cDNA encoded a 556-amino-acid sequence that was 93.5% identical to rat CACH, suggesting a conserved role for this enzyme in the mammalian liver. Database searching shows no homology to other known proteins, but reveals homological cDNA sequences showing two single-nucleotide polymorphisms (SNPs) in the CACH coding region. The discovery of mouse CACH cDNA is an important step towards genetic studies on the functional analysis of this enzyme by gene-knockout and transgenic approaches.


Assuntos
Acetil-CoA Hidrolase/genética , Acetil-CoA Hidrolase/metabolismo , Citosol/enzimologia , Metabolismo dos Lipídeos , Acetil-CoA Hidrolase/química , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Expressão Gênica , Fígado/enzimologia , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Spodoptera/citologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA