RESUMO
We have identified new members of the histone deacetylase enzyme family in Drosophila melanogaster. dHDAC6 is a class II deacetylase with two active sites, and dSIR2 is an NAD-dependent histone deacetylase. These proteins, together with two class I histone deacetylases, dHDAC1 and dHDAC3, have been expressed and characterized as epitope-tagged recombinant proteins in Schneider SL2 cells. All these proteins have in vitro deacetylase activity and are able to deacetylate core histone H4 at all four acetylatable lysine residues (5, 8, 12, and 16). Recombinant dHDAC6 and dSIR2 are both insensitive to TSA and HC toxin and resistant, relative to dHDAC1 and dHDAC3, to inhibition by sodium butyrate. Indirect immunofluorescence microscopy of stably transfected SL2 lines reveals that dHDAC1 and dSIR2 are nuclear, dHDAC6 is cytosolic, and dHDAC3 is detectable in both cytosol and nucleus. dHDAC6 and dSIR2 elute from Superose 6 columns with apparent molecular weights of 90 and 200 kDa, respectively. In contrast, dHDAC1 and dHDAC3elute at 800 and 700 kDa, respectively, suggesting that they are components of multiprotein complexes. Consistent with this, recombinant dHDAC1 coimmunoprecipitates with components of the Drosophila NuRD complex and dHDAC3 with an as yet unknown 45-kDa protein.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/enzimologia , Histona Desacetilases/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae , Transativadores/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Técnica Indireta de Fluorescência para Anticorpo , Expressão Gênica , Células HeLa , Desacetilase 6 de Histona , Inibidores de Histona Desacetilases , Histona Desacetilases/genética , Histonas/metabolismo , Humanos , Proteínas de Insetos/metabolismo , Lisina/metabolismo , Dados de Sequência Molecular , Sirtuína 1 , Sirtuína 2 , Sirtuínas , Transativadores/antagonistas & inibidores , Transativadores/genéticaRESUMO
The steady-state level of histone acetylation in eukaryotes is established and maintained by multiple histone acetyltransferases (HATs) and histone deacetylases (HDACs) and affects both the structure and the function of chromatin. Histone deacetylases play a key role in the regulation of transcription, and form a highly conserved protein family in many eukaryotic species. Here we describe the cloning, sequencing and genetic mapping of two histone deacetylase genes in Drosophila melanogaster: dHDAC1 is essentially identical to the previously cloned D. melanogaster d-Rpd3 gene and dHDAC3, a novel gene, is orthologous to the human and the chicken (Gallus gallus) HDAC3 genes. The predicted amino acid sequence (438 aa) of dHDAC3 shows 58.1% identity with dHDAC1/d-Rpd3, the only previously known member of the HDAC family in this organism. The map positions on polytene chromosomes for dHDAC1 and dHDAC3 were determined as 64C1-6 and 83A3-4 respectively. A search for other dHDAC3-like genes failed to find other potential paralogues in D. melanogaster, but identified significant homologies with bacterial and fungal genes encoding enzymes that metabolise acetyl groups, and with genes for other hydrolyases such as carboxypeptidase. In addition, histone deacetylase activity in D. melanogaster nuclear extracts can be inhibited by high concentrations of zinc and activated by low concentrations, which is identical to the properties of bovine carboxypeptidase A. On the basis of sequence and functional similarities, we suggest that histone deacetylases are metal-substituted enzymes.
Assuntos
DNA Complementar/genética , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Histona Desacetilases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Núcleo Celular/química , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/enzimologia , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar/química , DNA Complementar/isolamento & purificação , Drosophila melanogaster/química , Ativação Enzimática/efeitos dos fármacos , Histona Desacetilases/efeitos dos fármacos , Histona Desacetilases/metabolismo , Isoenzimas/genética , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Zinco/farmacologiaRESUMO
Intravenous hyperalimentation in chronically ill patients has become increasingly common in hospitalized patients. Total parenteral nutrition includes supply of carbohydrates, amino acids, and lipids. The safety of a new emulsion of safflower oil (Liposyn 10%) infused by peripheral vein was evaluated in 15 normal male volunteers. All subjects tolerated Liposyn infusion, with a low incidence of side effects, when given either as a single infusion or multiple daily infusions to provide 4% of daily caloric requirements in the form of linoleic acid for 5 consecutive days. In large doses, the lipid infusion was accompanied by a decrease in Lee-White clotting time in most subjects, and 1.5 to 2 U/ml of heparin added to the emulsion reversed this effect. Such mini doses of heparin also accelerated the breakdown and disappearance of triglycerides, with a resultant increase in serum free fatty acids and cholesterol. These data suggest that safflower oil emulsion can be used as a source of essential fatty acids for intravenous alimentation. It is also suggested that patients receiving lipid infusion should receive heparin to minimize risks associated with hypercoagulability of blood.