Dan Ryder

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… Show more

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. 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. Show less

Abstract

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, encoding the NIMA-related kinase, by 5aza-dC is context specific, as NEK2 transcript levels were reduced in HCT116 colon… Show more

Abstract

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, encoding the 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 (MNase), i.e. nucleosome depletion. Conversely, 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 (doxo) 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 doxo. Luciferase reporters confirmed 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. Show less

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… Show more

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. Show less

Cytochrome P450 (P450) is a super-family of drug metabolizing enzymes. P450 enzymes have dual function; they can metabolize drugs to pharmacologically inactive metabolites facilitating their excretion or biotransform them to pharmacologically active metabolites which may have longer half-life than the parent drug. The variable pharmacological response to psychoactive drugs typically seen in population groups is often not accountable by considering dissimilarities in hepatic metabolism… Show more

Cytochrome P450 (P450) is a super-family of drug metabolizing enzymes. P450 enzymes have dual function; they can metabolize drugs to pharmacologically inactive metabolites facilitating their excretion or biotransform them to pharmacologically active metabolites which may have longer half-life than the parent drug. The variable pharmacological response to psychoactive drugs typically seen in population groups is often not accountable by considering dissimilarities in hepatic metabolism. Metabolism in brain specific nuclei may play a role in pharmacological modulation of drugs acting on the CNS and help explain some of the diverse response to these drugs seen in patient population. P450 enzymes are also present in brain where drug metabolism can take place and modify therapeutic action of drugs at the site of action. We have earlier demonstrated an intrinsic difference in the biotransformation of alprazolam (ALP) in brain and liver, relatively more α-hydroxy alprazolam (α-OHALP) is formed in brain as compared to liver. In the present study we show that recombinant CYP3A43 metabolizes ALP to both α-OHALP and 4-hydroxy alprazolam (4-OHALP) while CYP3A4 metabolizes ALP predominantly to its inactive metabolite, 4-OHALP. The expression of CYP3A43 mRNA in human brain samples correlates with formation of relatively higher levels of α-OH ALP indicating that individuals who express higher levels of CYP3A43 in the brain would generate larger amounts of α-OHALP. Further, the expression of CYP3A43 was relatively higher in brain as compared to liver across different ethnic populations. Since CYP3A enzymes play a prominent role in the metabolism of drugs, the higher expression of CYP3A43 would generate metabolite profile of drugs differentially in human brain and thus impact the pharmacodynamics of psychoactive drugs at the site of action. Show less

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… Show more

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. Show less

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… Show more

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. Show less