RESUMEN
Acyl-CoA synthetases (ACSs) play an essential role in fatty acid metabolism. ACS3 is an arachidonate-preferring enzyme expressed in a wide range of human tissues including brain, heart, placenta, prostate, skeletal muscle, testis and thymus. As an initial step to understanding the transcriptional regulation of the human ACS3 gene, we analyzed the genomic organization and transcription units of the human ACS3 gene. Sequence analysis of genomic clones demonstrates that the human ACS3 gene spans at least 80.6 kb and contains 17 exons. The human ACS3 gene was mapped between the sequence-tagged site markers D2S360 and WI-21901. Sequence inspection of the 5'-flanking region revealed potential DNA elements including CCAAT, AP-1, Oct-1, GATAs, SRY, CdxA, Nkx-2.5, c-Myb, HSF2, NF-AT, AP-2, NF-Y, and p300. A minimal promoter region required for the expression of the human ACS3 gene in melanoma G361 cells was determined.
Asunto(s)
Coenzima A Ligasas/genética , Genes/genética , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Transcripción Genética/genética , Región de Flanqueo 5'/genética , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Humanos Par 2/genética , ADN/química , ADN/genética , ADN/aislamiento & purificación , Exones , Femenino , Expresión Génica , Células HL-60 , Células HeLa , Humanos , Intrones , Células K562 , Luciferasas/genética , Luciferasas/metabolismo , Masculino , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Distribución Tisular , Células Tumorales CultivadasRESUMEN
Acyl-CoA synthetase 4 (ACS4) is an arachidonate-preferring isozyme of ACS family predominantly expressed in steroidogenic tissues. Isolation and characterization of genomic clones encoding human ACS4 revealed that the genomic organization of the gene. The human ACS4 gene spans approximately 90 kb and consists of 16 exons. Sequence inspection of the 5'-flanking region revealed potential DNA elements including GATAs, p300, AP-4, SRY, CREB and MyoD. A minimal promoter region required for the expression of ACS4 in HeLa S3 cells was determined. The human ACS4 gene was mapped between the STS markers, WI-17685 and CHLC.GATA81B07 on Xq22-23 region.
Asunto(s)
Coenzima A Ligasas/genética , Exones , Intrones , Transcripción Genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN , Células HeLa , Humanos , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
There is increasing evidence that lipid peroxidation is involved in many of the pathophysiologies associated with cardiovascular diseases, such as atherosclerosis and the long-term complications of diabetes. Among the products which originate from the peroxidation of cellular membrane lipids, 4-hydroxy-2-nonenal (HNE) is believed to be largely responsible for the cytopathological effects observed during oxidative stress in vivo. Here we found that HNE dramatically inhibited the expression of adhesion molecules induced by inflammatory stimuli in human aortic endothelial cells. The inhibition was found to be accompanied by a significant reduction of NF-kappaB activation followed by nuclear localization. This and the observation that the HNE treatment of the cells resulted in a rapid reduction of intracellular glutathione levels suggest that redox regulation of NF-kappaB may be involved in the modulation of the endothelial response by reactive aldehydes.
Asunto(s)
Aldehídos/metabolismo , Endotelio Vascular/metabolismo , Aldehídos/toxicidad , Células Cultivadas , Quimiocina CCL2/metabolismo , Selectina E/metabolismo , Endotelio Vascular/efectos de los fármacos , Glutatión/metabolismo , Humanos , Inflamación/etiología , Inflamación/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Lipopolisacáridos/toxicidad , FN-kappa B/metabolismo , Estrés Oxidativo , Factor de Necrosis Tumoral alfa/farmacología , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
ACS3 is a recently identified acyl-CoA synthetase (ACS) isozyme that preferentially utilizes laurate, myristate, arachidonate, and eicosapentaenoate among saturated and unsaturated long chain fatty acids. The ACS3 purified from COS cells transfected with the ACS3 cDNA was separated by SDS-PAGE into two major forms of 79 and 80 kDa. We report here that alternative translation initiation from ACS3 mRNA gives rise to these two isoforms of ACS3. In vitro mutagenesis of the ACS3 cDNA revealed that the translation of the 80-kDa and 79-kDa isoforms started from the first and second in-frame AUGs, respectively. The two isoforms of ACS3 expressed in COS cells exhibited similar levels of ACS activities toward palmitate and myristate. Immunocytochemistry of intact COS cells transfected with various ACS3 expression vectors suggested that the two forms are localized in the extranuclear compartment, where they exhibit a reticular pattern. In rat cerebrum, the 80-kDa isoform of ACS3 was detected mainly in the microsomal fraction. Only a trace amount of the 79-kDa isoform was detected in rat cerebrum, whereas both forms were detected in rat glioma cell line KEG1 cells.
Asunto(s)
Coenzima A Ligasas/genética , Coenzima A Ligasas/metabolismo , Biosíntesis de Proteínas , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Animales , Encéfalo/metabolismo , Células COS/metabolismo , ADN Complementario , Ácidos Grasos/metabolismo , Inmunohistoquímica , Isoenzimas/genética , Metionina , Mutación , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Ácido Nucleico , Fracciones Subcelulares , Especificidad por Sustrato , TransfecciónAsunto(s)
Cromosomas Humanos Par 2/genética , Coenzima A Ligasas/genética , ADN Complementario/genética , Isoenzimas/genética , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bandeo Cromosómico , Mapeo Cromosómico , Cartilla de ADN/genética , Humanos , Células Híbridas , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , RatasRESUMEN
We report herein the cDNA cloning of a novel rat acyl-CoA synthetase (ACS) that preferentially uses arachidonate and eicosapentaenoate. This newly identified ACS (designated ACS4) contains 670 amino acids and is 68% identical to rat ACS3, a previously characterized ACS that is highly expressed in brain. ACS4 was overproduced in Escherichia coli and the resulting enzyme was purified to homogeneity. The purified enzyme utilizes arachidonate and eicosapentaenoate most preferentially among C8-C22 saturated fatty acids and C14-C22 unsaturated fatty acids. Kinetic analyses revealed that the enzyme has a high affinity for arachidonate and eicosapentaenoate and low affinity for palmitate. ACS4 transcripts are detectable in a wide range of tissues, with the highest level in adrenal gland. Immunoreactivity to ACS4 was detected in the zona fasciculata and reticularis of adrenal gland, in the corpus luteum and stromal luteinized cells in ovary, and in the Leydig cells of testis.
Asunto(s)
Glándulas Suprarrenales/enzimología , Ácido Araquidónico/metabolismo , Coenzima A Ligasas/metabolismo , Ovario/enzimología , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Testículo/enzimología , Secuencia de Aminoácidos , Animales , Northern Blotting , Coenzima A Ligasas/química , ADN Complementario , Femenino , Masculino , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Two types of acyl-CoA synthetase (ACS), designated ACS1 and ACS2, are structurally similar isozymes with different tissue distributions. The two enzymes are organized into the following five regions: an NH2 terminus; two luciferase-like regions; a linker connecting the luciferase-like regions; a COOH terminus. Under the control of a lac promoter, rat ACS1 and ACS2 were overproduced in Escherichia coli and purified to homogeneity. The specific activities of the purified ACS1 and ACS2 were 26.2 mumol.min-1.mg-1 and 7.4 mumol.min-1.mg-1, respectively, and the most efficiently utilized saturated fatty acids were those with 10-18 carbon atoms. Among unsaturated fatty acids with 16-22 carbon atoms, the most preferred substrates were palmitoleate, oleate and linoleate for ACS1, and, for ACS2, oleate, arachidonate, eicosapentaenoate and docosahexaenoate. To determine the functionally important regions in the ACS isozymes, we constructed five ACS1 mutants lacking each of the five regions. Introduction of these mutants into E. coli revealed that all five regions in ACS1 are required for functional expression of the enzyme in E. coli; deletion of any one of the five regions almost completely abolished the enzyme activity.