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1.
J Cell Sci ; 116(Pt 17): 3601-10, 2003 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-12893813

RESUMO

Precise body and organ sizes in the adult animal are ensured by a range of signaling pathways. In a screen to identify genes affecting hindgut morphogenesis in Drosophila, we identified a P-element insertion in dRheb, a novel, highly conserved member of the Ras superfamily of G-proteins. Overexpression of dRheb in the developing fly (using the GAL4:UAS system) causes dramatic overgrowth of multiple tissues: in the wing, this is due to an increase in cell size; in cultured cells, dRheb overexpression results in accumulation of cells in S phase and an increase in cell size. Using a loss-of-function mutation we show that dRheb is required in the whole organism for viability (growth) and for the growth of individual cells. Inhibition of dRheb activity in cultured cells results in their arrest in G1 and a reduction in size. These data demonstrate that dRheb is required for both cell growth (increase in mass) and cell cycle progression; one explanation for this dual role would be that dRheb promotes cell cycle progression by affecting cell growth. Consistent with this interpretation, we find that flies with reduced dRheb activity are hypersensitive to rapamycin, an inhibitor of the growth regulator TOR. In cultured cells, the effect of overexpressing dRheb was blocked by the addition of rapamycin. These results imply that dRheb is involved in TOR signaling.


Assuntos
Drosophila/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Neuropeptídeos/metabolismo , Sequência de Aminoácidos , Animais , Diferenciação Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Tamanho Celular/efeitos dos fármacos , Tamanho Celular/fisiologia , Células Cultivadas , Drosophila/citologia , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/metabolismo , Fase G1/fisiologia , Dados de Sequência Molecular , Proteínas Monoméricas de Ligação ao GTP/genética , Neuropeptídeos/genética , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Quinases , Proteína Enriquecida em Homólogo de Ras do Encéfalo , Fase S/fisiologia , Transdução de Sinais/fisiologia , Sirolimo/farmacologia , Serina-Treonina Quinases TOR , Asas de Animais/citologia , Asas de Animais/metabolismo
2.
J Biol Chem ; 277(2): 1058-65, 2002 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-11694513

RESUMO

Protein l-isoaspartate-(d-aspartate) O-methyltransferases (EC ), present in a wide variety of prokaryotic and eukaryotic organisms, can initiate the conversion of abnormal l-isoaspartyl residues that arise spontaneously with age to normal l-aspartyl residues. In addition, the mammalian enzyme can recognize spontaneously racemized d-aspartyl residues for conversion to l-aspartyl residues, although no such activity has been seen to date for enzymes from lower animals or prokaryotes. In this work, we characterize the enzyme from the hyperthermophilic archaebacterium Pyrococcus furiosus. Remarkably, this methyltransferase catalyzes both l-isoaspartyl and d-aspartyl methylation reactions in synthetic peptides with affinities that can be significantly higher than those of the human enzyme, previously the most catalytically efficient species known. Analysis of the common features of l-isoaspartyl and d-aspartyl residues suggested that the basic substrate recognition element for this enzyme may be mimicked by an N-terminal succinyl peptide. We tested this hypothesis with a number of synthetic peptides using both the P. furiosus and the human enzyme. We found that peptides devoid of aspartyl residues but containing the N-succinyl group were in fact methyl esterified by both enzymes. The recent structure determined for the l-isoaspartyl methyltransferase from P. furiosus complexed with an l-isoaspartyl peptide supports this mode of methyl-acceptor recognition. The combination of the thermophilicity and the high affinity binding of methyl-accepting substrates makes the P. furiosus enzyme useful both as a reagent for detecting isomerized and racemized residues in damaged proteins and for possible human therapeutic use in repairing damaged proteins in extracellular environments where the cytosolic enzyme is not normally found.


Assuntos
Adenosina/análogos & derivados , Proteínas Arqueais/metabolismo , Etionina/análogos & derivados , Oligopeptídeos/metabolismo , Proteína D-Aspartato-L-Isoaspartato Metiltransferase/metabolismo , Pyrococcus furiosus/enzimologia , Adenosina/metabolismo , Alanina/química , Alanina/metabolismo , Proteínas Arqueais/genética , Desoxiadenosinas/metabolismo , Estabilidade Enzimática , Etionina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Metilação , Estrutura Molecular , Proteína D-Aspartato-L-Isoaspartato Metiltransferase/antagonistas & inibidores , Proteína D-Aspartato-L-Isoaspartato Metiltransferase/genética , Pyrococcus furiosus/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , S-Adenosil-Homocisteína/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Especificidade por Substrato , Temperatura , Tionucleosídeos/metabolismo
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