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1.
PLoS One ; 10(8): e0136247, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26302492

RESUMO

Skeletal muscle atrophy is a consequence of several physiological and pathophysiological conditions including muscle disuse, aging and diseases such as cancer and heart failure. In each of these conditions, the predominant mechanism contributing to the loss of skeletal muscle mass is increased protein turnover. Two important mechanisms which regulate protein stability and degradation are lysine acetylation and ubiquitination, respectively. However our understanding of the skeletal muscle proteins regulated through acetylation and ubiquitination during muscle atrophy is limited. Therefore, the purpose of the current study was to conduct an unbiased assessment of the acetylation and ubiquitin-modified proteome in skeletal muscle during a physiological condition of muscle atrophy. To induce progressive, physiologically relevant, muscle atrophy, rats were cast immobilized for 0, 2, 4 or 6 days and muscles harvested. Acetylated and ubiquitinated peptides were identified via a peptide IP proteomic approach using an anti-acetyl lysine antibody or a ubiquitin remnant motif antibody followed by mass spectrometry. In control skeletal muscle we identified and mapped the acetylation of 1,326 lysine residues to 425 different proteins and the ubiquitination of 4,948 lysine residues to 1,131 different proteins. Of these proteins 43, 47 and 50 proteins were differentially acetylated and 183, 227 and 172 were differentially ubiquitinated following 2, 4 and 6 days of disuse, respectively. Bioinformatics analysis identified contractile proteins as being enriched among proteins decreased in acetylation and increased in ubiquitination, whereas histone proteins were enriched among proteins increased in acetylation and decreased in ubiquitination. These findings provide the first proteome-wide identification of skeletal muscle proteins exhibiting changes in lysine acetylation and ubiquitination during any atrophy condition, and provide a basis for future mechanistic studies into how the acetylation and ubiquitination status of these identified proteins regulates the muscle atrophy phenotype.


Assuntos
Progressão da Doença , Proteínas Musculares/biossíntese , Músculo Esquelético/metabolismo , Proteoma/genética , Acetilação , Sequência de Aminoácidos/genética , Animais , Expressão Gênica , Humanos , Músculo Esquelético/fisiopatologia , Processamento de Proteína Pós-Traducional/genética , Ratos , Transdução de Sinais , Ubiquitina/genética
2.
J Biol Chem ; 288(50): 35940-51, 2013 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-24163369

RESUMO

Genome-scale mapping suggests that the function of DNA methylation varies with genomic context beyond transcriptional repression. However, the use of DNA-demethylating agents (e.g. 5-aza-2'-deoxycytidine (5aza-dC)) to study epigenetic regulation often focuses on gene activation and ignores repression elicited by 5aza-dC. Here, we show that repression of NEK2, which encodes the never in mitosis A (NIMA)-related kinase, by 5aza-dC is context-specific as NEK2 transcript levels were reduced in HCT116 colon cancer cells but not in isogenic p53(-/-) cells. Bisulfite sequencing showed that DNA methylation was restricted to the distal region of the NEK2 promoter. Demethylation by 5aza-dC was associated with increased accessibility to micrococcal nuclease, i.e. nucleosome depletion. Conversely, methyltransferase accessibility protocol for individual templates (MAPit) methylation footprinting showed that nucleosome occupancy and DNA methylation at the distal promoter were significantly increased in p53(-/-) cells, suggesting dynamic regulation of chromatin structure at this region by p53 in HCT116 cells. Stabilization of endogenous p53 by doxorubicin or ectopic expression of p53, but not a p53 DNA-binding mutant, decreased NEK2 expression. Chromatin immunoprecipitation demonstrated direct and specific association of p53 with the distal NEK2 promoter, which was enhanced by doxorubicin. Luciferase reporters confirmed that this region is required for p53-mediated repression of NEK2 promoter activity. Lastly, modulation of p53 abundance altered nucleosome occupancy and DNA methylation at its binding region. These results identify NEK2 as a novel p53-repressed gene, illustrate that its repression by 5aza-dC is specific and associated with nucleosome reorganization, and provide evidence that identification of partially methylated regions can reveal novel p53 target genes.


Assuntos
Metilação de DNA , Regiões Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinases/genética , Proteína Supressora de Tumor p53/metabolismo , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Sequência de Bases , Sítios de Ligação , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Proliferação de Células/efeitos dos fármacos , DNA/metabolismo , Metilação de DNA/efeitos dos fármacos , Decitabina , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Células HCT116 , Humanos , Quinases Relacionadas a NIMA , Nucleossomos/efeitos dos fármacos , Nucleossomos/genética , Nucleossomos/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética
3.
J Endocrinol ; 210(2): 199-207, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21565854

RESUMO

The activation of proopiomelanocortin (POMC) neurons in different regions of the brain, including the arcuate nucleus of the hypothalamus (ARC) and the nucleus of the solitary tract curtails feeding and attenuates body weight. In this study, we compared the effects of delivery of a recombinant adeno-associated viral (rAAV) construct encoding POMC to the ARC with delivery to the ventral tegmental area (VTA). F344×Brown Norway rats were high-fat (HF) fed for 14 days after which self-complementary rAAV constructs expressing either green fluorescent protein or the POMC gene were injected using coordinates targeting either the VTA or the ARC. Corresponding increased POMC levels were found at the predicted injection sites and subsequent α-melanocyte-stimulating hormone levels were observed. Food intake and body weight were measured for 4 months. Although caloric intake was unaltered by POMC overexpression, weight gain was tempered with POMC overexpression in either the VTA or the ARC compared with controls. There were parallel decreases in adipose tissue reserves. In addition, levels of oxygen consumption and brown adipose tissue uncoupling protein 1 were significantly elevated with POMC treatment in the VTA. Interestingly, tyrosine hydroxylase levels were increased in both the ARC and VTA with POMC overexpression in either the ARC or the VTA. In conclusion, these data indicate a role for POMC overexpression within the VTA reward center to combat HF-induced obesity.


Assuntos
Gorduras na Dieta , Ingestão de Alimentos/fisiologia , Obesidade/genética , Pró-Opiomelanocortina/genética , Área Tegmentar Ventral/metabolismo , Tecido Adiposo Marrom/metabolismo , Animais , Núcleo Arqueado do Hipotálamo/metabolismo , Peso Corporal/fisiologia , Técnicas de Transferência de Genes , Canais Iônicos/genética , Canais Iônicos/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Obesidade/metabolismo , Obesidade/terapia , Consumo de Oxigênio/fisiologia , Pró-Opiomelanocortina/metabolismo , Ratos , Proteína Desacopladora 1
4.
Expert Opin Drug Metab Toxicol ; 2(3): 399-418, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16863442

RESUMO

The human cytochrome P450 (CYP) superfamily of enzymes regulate hepatic phase 1 drug metabolism and subsequently play a significant role in pharmacokinetics, drug discovery and drug development. Alternative splicing of the cytochrome CYP gene transcripts enhances gene diversity and may play a role in transcriptional regulation of certain CYP proteins. Tissue-specific alternative splicing of CYPs is significant for its potential to add greater dimension to differential drug metabolism in hepatic and extrahepatic tissues, such as the brain, and to our understanding of the CYP family. This review provides an overview of tissue-specific splicing patterns, splicing types, regulation and the functional diversities between liver and splice variant CYP proteins and further explores the relevance of tissue-specific alternative splicing of CYPs in the nervous system.


Assuntos
Processamento Alternativo , Encéfalo/enzimologia , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação Enzimológica da Expressão Gênica , Fígado/enzimologia , Animais , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP2D6/genética , Sistema Enzimático do Citocromo P-450/genética , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Polimorfismo de Nucleotídeo Único
5.
Tex Med ; 101(10): 62-70, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17094519

RESUMO

Although the role of environmental health hazards in cancer and other diseases is increasingly appreciated, most physicians have had little or no training in environmental health or in integrating exposure histories into their clinical practice. As part of the Texas Medical Association Physician Oncology Education Program, we mailed a questionnaire to 350 Texas primary care physicians (PCPs) to evaluate their attitudes, practice, training, and preferred sources for information regarding environmental health issues. Of the respondents, 86.1% reported that they had never received specific training in environmental health history-taking and 91.7% indicated a desire to learn more about environmental health hazards. The data also revealed that patients regularly raise questions about environmental topics that PCPs do not routinely discuss. Our findings identify a need for more environmental health education for Texas PCPs, and we suggest several possible mechanisms by which this might be accomplished.


Assuntos
Saúde Ambiental , Atenção Primária à Saúde , Atitude do Pessoal de Saúde , Estudos de Coortes , Coleta de Dados , Educação Médica Continuada , Saúde Ambiental/educação , Conhecimentos, Atitudes e Prática em Saúde , Humanos , Anamnese , Oncologia/educação , Fatores de Risco , Sociedades Médicas , Inquéritos e Questionários , Texas , Fatores de Tempo
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