Assuntos
Membrana Celular/enzimologia , Oxirredutases Intramoleculares/metabolismo , Sequência de Aminoácidos , Animais , DNA Complementar/genética , Humanos , Oxirredutases Intramoleculares/química , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/isolamento & purificação , Dados de Sequência Molecular , Mapeamento Físico do Cromossomo , Prostaglandina-E Sintases , Homologia de Sequência , Compostos de Sulfidrila/farmacologiaRESUMO
NeuroD-related factor (NDRF)/NeuroD2 is a basic helix-loop-helix (bHLH) protein that plays important roles in neuronal development. To elucidate the NDRF transcription network, we used mouse cDNA microarray analysis combined with a tetracycline-regulatable expression system in P19 embryonal carcinoma cells. Five genes were identified to be up-regulated in the presence of NDRF protein. RNA hybridization analysis confirmed that brain-lipid-binding protein (BLBP) and inhibitor of differentiation 1 (Id1) genes were among the five genes that were rapidly and significantly up-regulated after induction of NDRF. When a dominant negative form of NDRF protein was expressed during retinoic acid-induced neuronal differentiation of P19 cells, the BLBP gene, but not the Id1 gene, was potently repressed. Immunohistochemical analysis revealed that both NDRF and Id1 immunoreactivities were observed in some granule cells of the cerebellum in the postnatal period. These results suggest that NDRF or its related bHLH proteins may act upstream of these genes in a subset of developing neurons.
Assuntos
Regulação da Expressão Gênica , Neurônios/metabolismo , Neuropeptídeos/biossíntese , Neuropeptídeos/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Tetraciclina/farmacologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Northern Blotting , Western Blotting , Encéfalo/embriologia , Diferenciação Celular , Linhagem Celular Tumoral , DNA Complementar/metabolismo , Doxiciclina/farmacologia , Genes Dominantes , Imuno-Histoquímica , Proteína 1 Inibidora de Diferenciação , Camundongos , Neurônios/citologia , Hibridização de Ácido Nucleico , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , RNA/metabolismo , RNA Mensageiro/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Tempo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ativação Transcricional , Transfecção , Tretinoína/farmacologia , Regulação para CimaRESUMO
The amino acid sequence of membrane-associated prostaglandin (PG) E synthase-2 (mPGE synthase-2), which has a broad specificity in its thiol requirement for a catalytic activity, has the consensus region from 104Leu to 120Leu found in glutaredoxin and of thioredoxin. The sequence of Cys-x-x-Cys in the consensus region is the active site for thioredoxin and mPGE synthase-2 also has this amino acid sequence (110Cys-x-x-113Cys). The mutation from 110Cys to Ser or the double mutation from 110Cys and 113Cys to Ser caused loss of PGE synthase activity, whereas the single mutation from 113Cys to Ser did not affect the enzyme activity. These results indicate that 110Cys, but not 113Cys, is the essential amino acid in the active site of mPGE synthase-2. 110Cys is an important amino acid in PGE synthase activity and plays the critical role as Cys at the same position in redoxin. Moreover, we found that the reduced form of lipoic acid (dihydrolipoic acid) serves as one of the natural activators of mPGE synthase-2 in the cells.
Assuntos
Cisteína/química , Oxirredutases Intramoleculares/química , Oxirredutases , Ácido Tióctico/análogos & derivados , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Ditiotreitol/farmacologia , Relação Dose-Resposta a Droga , Glutarredoxinas , Humanos , Cinética , Leucina/química , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Oxirredução , Estresse Oxidativo , Prostaglandina-E Sintases , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas/química , Serina/química , Ácido Tióctico/química , Tiorredoxinas/químicaRESUMO
Membrane-associated prostaglandin E synthase (mPGE synthase) was previously purified to apparent homogeneity from the microsomal fraction of bovine heart (Watanabe, K., et al., Biochim. Biophys. Acta 1439, 406--414, 1999). The N-terminal 22-amino acid sequence of the purified enzyme was identical to that of the 88th to 109th amino acids deduced from the monkey (AB046026) or human (AK024100) cDNA that encodes a hypothetical protein with unknown function. The primary structure has the consensus region of glutaredoxin and of thioredoxin. We constructed an expression plasmid, using the vector (pTrc-HisA) and the monkey cDNA for the 290-amino-acid polypeptide. The recombinant protein with a M(r) of 33 kDa exhibited PGE synthase activity and was purified to apparent homogeneity by nickel-chelating column chromatography. The V(max) and K(m) values for PGH(2) of the purified recombinant mPGE synthase were about 3.3 mumol/min center dot mg of protein and 28 muM, respectively. The recombinant enzyme was activated by various SH-reducing reagents, i.e., dithiothreitol, glutathione (GSH), and beta-mercaptoethanol, in order of decreasing effectiveness. Moreover, the mRNA distribution was high in the heart and brain, but the mRNA was not expressed in the seminal vesicles. These results indicate that the recombinant mPGE synthase is identical to the enzyme purified from the microsomal fraction of bovine heart, and is a novel type of mPGE synthase based on the primary structure, a broad specificity of thiol requirement, and tissue distribution.