RESUMO
Polyglutamine diseases include at least nine neurodegenerative disorders, each caused by a CAG repeat expansion in a different gene. Accumulation of mutant polyglutamine-containing proteins occurs in patients, and evidence from cell culture and animal experiments suggests the nucleus as a site of pathogenesis. To understand the consequences of nuclear accumulation, we created a cell culture system with nuclear-targeted polyglutamine. In our system, cell death can be mitigated by overexpression of full-length cAMP response element binding protein (CREB)-binding protein (CBP) or its amino-terminal portion alone. CBP is one of several histone acetyltransferases sequestered by polyglutamine inclusions. We found histone acetylation to be reduced in cells expressing mutant polyglutamine. Reversal of this hypoacetylation, which can be achieved either by overexpression of CBP or its amino terminus or by treatment with deacetylase inhibitors, reduced cell loss. These findings suggest that nuclear accumulation of polyglutamine can lead to altered protein acetylation in neurons and indicate a novel therapeutic strategy for polyglutamine disease.
Assuntos
Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , Peptídeos/antagonistas & inibidores , Animais , Proteína de Ligação a CREB , Morte Celular/efeitos dos fármacos , Linhagem Celular , Camundongos , Neurônios Motores/efeitos dos fármacos , Proteínas Nucleares/genética , Peptídeos/toxicidade , Receptores Androgênicos/genética , Transativadores/genética , TransfecçãoRESUMO
Proteins with expanded polyglutamine repeats cause Huntington's disease and other neurodegenerative diseases. Transcriptional dysregulation and loss of function of transcriptional co-activator proteins have been implicated in the pathogenesis of these diseases. Huntington's disease is caused by expansion of a repeated sequence of the amino acid glutamine in the abnormal protein huntingtin (Htt). Here we show that the polyglutamine-containing domain of Htt, Htt exon 1 protein (Httex1p), directly binds the acetyltransferase domains of two distinct proteins: CREB-binding protein (CBP) and p300/CBP-associated factor (P/CAF). In cell-free assays, Httex1p also inhibits the acetyltransferase activity of at least three enzymes: p300, P/CAF and CBP. Expression of Httex1p in cultured cells reduces the level of the acetylated histones H3 and H4, and this reduction can be reversed by administering inhibitors of histone deacetylase (HDAC). In vivo, HDAC inhibitors arrest ongoing progressive neuronal degeneration induced by polyglutamine repeat expansion, and they reduce lethality in two Drosophila models of polyglutamine disease. These findings raise the possibility that therapy with HDAC inhibitors may slow or prevent the progressive neurodegeneration seen in Huntington's disease and other polyglutamine-repeat diseases, even after the onset of symptoms.
Assuntos
Inibidores Enzimáticos/metabolismo , Glutamina/metabolismo , Inibidores de Histona Desacetilases , Proteínas do Tecido Nervoso/metabolismo , Doenças Neurodegenerativas/metabolismo , Proteínas Nucleares/metabolismo , Peptídeos/metabolismo , Proteínas de Saccharomyces cerevisiae , Transativadores/metabolismo , Acetilação , Acetiltransferases/metabolismo , Animais , Animais Geneticamente Modificados , Proteína de Ligação a CREB , Modelos Animais de Doenças , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteína p300 Associada a E1A , Regulação da Expressão Gênica , Glutationa Transferase/metabolismo , Histona Acetiltransferases , Histona Desacetilases/metabolismo , Histonas/metabolismo , Proteína Huntingtina , Doença de Huntington/enzimologia , Doença de Huntington/metabolismo , Doença de Huntington/prevenção & controle , Degeneração Neural , Proteínas do Tecido Nervoso/química , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/enzimologia , Proteínas Nucleares/química , Células PC12 , Estrutura Terciária de Proteína , Ratos , Sequências Repetitivas de Aminoácidos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Complexo Correpressor Histona Desacetilase e Sin3RESUMO
Huntington's Disease (HD) is caused by an expansion of a polyglutamine tract within the huntingtin (htt) protein. Pathogenesis in HD appears to include the cytoplasmic cleavage of htt and release of an amino-terminal fragment capable of nuclear localization. We have investigated potential consequences to nuclear function of a pathogenic amino-terminal region of htt (httex1p) including aggregation, protein-protein interactions, and transcription. httex1p was found to coaggregate with p53 in inclusions generated in cell culture and to interact with p53 in vitro and in cell culture. Expanded httex1p represses transcription of the p53-regulated promoters, p21(WAF1/CIP1) and MDR-1. httex1p was also found to interact in vitro with CREB-binding protein (CBP) and mSin3a, and CBP to localize to neuronal intranuclear inclusions in a transgenic mouse model of HD. These results raise the possibility that expanded repeat htt causes aberrant transcriptional regulation through its interaction with cellular transcription factors which may result in neuronal dysfunction and cell death in HD.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Doença de Huntington/genética , Proteínas do Tecido Nervoso/fisiologia , Proteínas Nucleares/fisiologia , Proteínas Repressoras/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Animais , Células Cultivadas , Humanos , Proteína Huntingtina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos TransgênicosRESUMO
The MDH3 isozyme of Saccharomyces cerevisiae was purified from a haploid strain containing disruptions in genomic loci encoding the mitochondrial MDH1 and nonmitochondrial MDH2 isozymes. Partial amino acid sequence analysis of the purified enzyme was conducted and used to plan polymerase chain reaction techniques to clone the MDH3 gene. The isolated gene was found to encode a 343-residue polypeptide with a molecular weight of 37,200. The deduced amino acid sequence was closely related to those of MDH1 (50% residue identity) and of MDH2 (43% residue identity). The MDH3 sequence was found to contain a carboxyl-terminal SKL tripeptide, characteristic of many peroxisomal enzymes, and immunochemical analysis was used to confirm organellar localization of the MDH3 isozyme. Levels of MDH3 were determined to be elevated in cells grown with acetate as a carbon source, and under these conditions, MDH3 contributed approximately 10% of the total cellular malate dehydrogenase activity. Disruption of the chromosomal MDH3 locus produced a reduction in cellular growth rates on acetate, consistent with the presumed function of this isozyme in the glyoxylate pathway of yeast. Combined disruption of MDH1, MDH2, and MDH3 loci in a haploid strain resulted in the absence of detectable cellular malate dehydrogenase activity.
Assuntos
Genes Fúngicos , Isoenzimas/genética , Malato Desidrogenase/genética , Mitocôndrias/enzimologia , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Cromossomos Fúngicos , Clonagem Molecular , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , Escherichia coli/genética , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Cinética , Malato Desidrogenase/isolamento & purificação , Malato Desidrogenase/metabolismo , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Reação em Cadeia da Polimerase/métodos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Homologia de Sequência de AminoácidosRESUMO
The structure of the tricarboxylic acid cycle enzyme malate dehydrogenase is highly conserved in various organisms. To test the extent of functional conservation, the rat mitochondrial enzyme and the enzyme from Escherichia coli were expressed in a strain of Saccharomyces cerevisiae containing a disruption of the chromosomal MDH1 gene encoding yeast mitochondrial malate dehydrogenase. The authentic precursor form of the rat enzyme, expressed using a yeast promoter and a multicopy plasmid, was found to be efficiently targeted to yeast mitochondria and processed to a mature active form in vivo. Mitochondrial levels of the polypeptide and malate dehydrogenase activity were found to be similar to those for MDH1 in wild-type yeast cells. Efficient expression of the E. coli mdh gene was obtained with multicopy plasmids carrying gene fusions encoding either a mature form of the procaryotic enzyme or a precursor form with the amino terminal mitochondrial targeting sequence from yeast MDH1. Very low levels of mitochondrial import and processing of the precursor form were obtained in vivo and activity could be demonstrated for only the expressed precursor fusion protein. Results of in vitro import experiments suggest that the percursor form of the E. coli protein associates with yeast mitochondria but is not efficiently internalized. Respiratory rates measured for isolated yeast mitochondria containing the mammalian or procaryotic enzyme were, respectively, 83 and 62% of normal, suggesting efficient delivery of NADH to the respiratory chain. However, expression of the heterologous enzymes did not result in full complementation of growth phenotypes associated with disruption of the yeast MDH1 gene.
Assuntos
Malato Desidrogenase/metabolismo , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Animais , Transporte Biológico , Clonagem Molecular , Escherichia coli/enzimologia , Expressão Gênica , Teste de Complementação Genética , Técnicas In Vitro , Malato Desidrogenase/genética , Mitocôndrias/enzimologia , Dados de Sequência Molecular , Consumo de Oxigênio , Ratos , Especificidade da EspécieRESUMO
The nucleotide sequence was determined for a 1.5-kilobase genomic fragment containing the mitochondrial malate dehydrogenase gene (MDH1) of Saccharomyces cerevisiae. The open-reading frame encodes a precursor form of the mature enzyme containing an amino-terminal extension of 17 amino acid residues. In vitro translation experiments confirm that the initial translation product of MDH1 is larger than the mature polypeptide. Transcription of MDH1 initiates at several sites from 83 to 97 nucleotides 5' of the translational start site. Alignment of the amino acid sequence for the mature yeast enzyme with those for mammalian mitochondrial and for Escherichia coli malate dehydrogenases reveals polypeptides of very similar sizes with identical amino acids at 54% and 48% of the residue positions, respectively. The amino acid sequences of the yeast and mammalian mitochondrial targeting sequences are similar but less related than the mature polypeptides. The yeast MDH1 gene is shown to reside on chromosome XI.