Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of Sachharomyces cerevisiae cultures following L-carnitine supplementation

ABSTRACT: Microarray analysis of the effect of L-carnitine supplementation on global expression of Sachharomyces cerevisiae cultures in logarithmic growing conditions and after exposure to H2O2 induced oxidative stress; L-Carnitine plays a well documented role in eukaryotic energy homeostasis by acting as a shuttling molecule for activated acyl residues across intracellular membranes. This activity is supported by carnitine acyl-transferases and transporters, and is referred to as the carnitine shuttle. However, several pleiotropic and often beneficial effects of carnitine in humans have been reported that appear to be unrelated to the shuttling activity, but little conclusive evidence regarding the molecular networks that would be affected by carnitine exist. We have recently demonstrated a protective role of carnitine in oxidative stress in yeast that is independent of the carnitine shuttle. A DNA microarray-based global gene expression analysis identified Cyc3p, a cytochrome c heme lyase, as being important for carnitine's protective impact in oxidative stress conditions. These findings establish a direct genetic link to a carnitine-related phenotype that is independent of the shuttle system. The data suggest that the yeast Saccharomyces cerevisiae should provide a useful model for further elucidation of carnitine's physiological roles. Experiment Overall Design: Yeast cultures was grown to mid-log phase with and without carnitine supplementation to a final concentration of 100 mg/L. A second set was grown to mid log phase, with and without carnitine supplementation and exposed to 0.4 mM H2O2 for 30 min. The experiments were performed using biological duplicate.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Jaco Franken

PROVIDER: E-GEOD-16346 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Poly(ADP-ribose) Polymerase 1 (PARP-1) is a nuclear enzyme that catalyse the transfer of ADP-ribose units from NAD+ to several target proteins involved in cellular stress responses. Using the cell line WRL68, we have previously shown that the activation of PARP-1 induced by oxidative stress after H2O2 treatment lead to depletion of cellular NAD+ and ATP, which promote cell death. In this work, we used LC-MS/MS based phosphoproteomics to show that the oxidative damage induced by H2O2 treatment modified the phosphorylation of several proteins in WRL68 cells, most of them related to transcription process. Interestingly, genetic or pharmacological inhibition of PARP-1 reduced the phosphorylation of YAP1, a transcriptional coactivator involved in cell survival, in WRL68 cells after H2O2 treatment. Inhibition of PARP-1 increased cell viability after oxidative treatment and diminished the degradation of YAP1; the elimination of YAP1 by gene silencing abrogated the protective effect of PARP-1 inhibition, indicating that YAP1 is important for the survival of WRL68 cells after oxidative damage. In the same way, the supplementation of NAD+, substrate of PARP-1, on WRL68 cells suffering oxidative damage reduced the phosphorylation of YAP1, suggesting that the loss of cellular NAD+ caused by PARP-1 activation after oxidative treatment is responsible for the phosphorylation of YAP1. Finally, PARP-1 silencing after oxidative treatment diminished the activation of AMPK, a master regulator of cell metabolism implicated in the restoration of energy balance during metabolic stress. Since NAD+ supplementation reduced the phosphorylation of some AMPK substrates in WRL68 cells after oxidative treatment, we hypothesize that the loss of cellular NAD+ after PARP-1 activation may induce a metabolic stress that activates AMPK. In summary, we showed a new crucial role of PARP-1 in the response to oxidative stress in WRL68 cells; PARP-1 activation reduces the cell viability by promoting the phosphorylation and degradation of YAP1, which is mediated by the energy stress caused by PARP-1 activation.

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Transcription profiling of Sachharomyces cerevisiae cultures following L-carnitine supplementation

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