The H2O2 Stimulon in Saccharomyces cerevisiae

Abstract

The changes in gene expression underlying the yeast adaptive stress response to H 2 O 2 were analyzed by comparative two-dimensional gel electrophoresis of total cell proteins. The synthesis of at least 115 proteins is stimulated by H 2 O 2 , whereas 52 other proteins are repressed by this treatment. We have identified 71 of the stimulated and 44 of the repressed targets. The kinetics and dose-response parameters of the H 2 O 2 genomic response were also analyzed. Identification of these proteins and their mapping into specific cellular processes give a distinct picture of the way in which yeast cells adapt to oxidative stress. Aerobic organisms have to maintain a reduced cellular redox environment in the face of the prooxidative conditions characteristic of aerobic life. The incomplete reduction of oxygen to water during respiration leads to the formation of redox-active oxygen intermediates (ROI) 1 such as the superoxide anion radical (O 2 Ϫ ), hydrogen peroxide (H 2 O 2 ), and the hydroxyl radical (OH ⅐ ) (for review, see Refs. 1-3). ROI are also produced during the ␤-oxidation of fatty acids, and upon exposure to radiation, light, metals, and redox active drugs. Oxidative stress results from abnormally high levels of ROI which perturb the cell redox status and leads to damage to lipids, proteins, DNA, and eventually cell death. Living organisms constantly sense and adapt to such redox perturbations by the induction of batteries of genes or stimulons whose products act to maintain the cellular redox environment (4 (9,10). Yeast has the same defense mechanisms as higher eukaryotes (for review, see Refs. 11 and 12) and offers the power of genome-wide experimental approaches owing to the availability of the complete sequence of its genome. It therefore represents an ideal eukaryotic model in which to study the cellular redox control and ROI metabolism. We recently established a general method to identify yeast proteins based on two-dimensional gel electrophoresis (13). We used this genome-wide experimental approach to characterize proteins whose expression is altered upon exposure to low doses of H 2 O 2 . Such an oxidative stress challenge results in a dramatic genomic response involving at least 167 proteins. Identification of these proteins and their mapping into cellular processes give a global view of the ubiquitous cellular changes elicited by H 2 O 2 and provides the framework for understanding the mechanisms of cellular redox homeostasis and H 2 O 2 metabolism. ura3-52 lys2-801 amber ade2-101 ochre trp1-⌬1 leu2-⌬1) was used for the analysis of the H 2 O 2 response. The strain S288C (15) was used for protein spot identification. Strains were grown at 30°C in a medium containing 0.67% yeast nitrogen base without amino acids (Difco), 2% glucose, buffered to pH 5.8 with 1% succinate and 0.6% NaOH. For YPH98, uracil, adenine, lysine, tryptophan and leucine (30 mg/liter) were added to the culture medium. MATERIALS AND METHODS Strains and Growth Conditions-The yeast strain YPH98 (14) (MATaIdentification of P...

Regulatory crosstalk of the metabolic network

“…This process is very fast; GAPDH is inactivated within seconds and PPP metabolites increase almost immediately [24]. A few minutes later, a genetic response is induced, by which the PPP is upregulated and glycolytic enzymes are downregulated [70]. After the transcriptional reconfiguration of the pathway is complete ($30-60 minutes), the GAPDH block is released and the metabolic flux is re-established [71,72].…”

Disruption of Yeast Forkhead-associated Cell Cycle Transcription by Oxidative Stress

“…Oxidative stress in yeast has been studied by exposing cells to agents that are already reactive, typically HP, or drugs that cause the intracellular accumulation of reactive oxygen species (Godon et al, 1998;Dumond et al, 2000;Gasch et al, 2000). Menadione (MD) is such a drug, generating reactive superoxide ions, which can be further oxidized to give HP (Monks et al, 1992;Shackelford et al, 2000).…”

“…They pose a threat to organisms by damaging a variety of cellular macromolecules, including DNA, membrane lipids, and proteins and are implicated with carcinogenesis, aging, and numerous degenerative diseases (Finkel and Holbrook, 2000;Neumann et al, 2003). The major ROS derived from oxygen are superoxide ions, hydrogen peroxide (HP), and hydroxy radicals, ordered here by increasing reactivity (reviewed in Shackelford et al, 2000).Oxidative stress in yeast has been studied by exposing cells to agents that are already reactive, typically HP, or drugs that cause the intracellular accumulation of reactive oxygen species (Godon et al, 1998;Dumond et al, 2000;Gasch et al, 2000). Menadione (MD) is such a drug, generating reactive superoxide ions, which can be further oxidized to give HP (Monks et al, 1992;Shackelford et al, 2000).…”

Assessing factors for reliable quantitative proteomics based on two‐dimensional gel electrophoresis

“…The first dimension separation can be performed with either carrier ampholites or immobilized pH gradients. Proteins may be stained with chemical dyes (such as Coomassie Blue [12][13] or silver nitrate [14]), labelled with radioactive isotopes ( 3 H, 14 C, 35 S, 32 P) [15][16][17], or revealed by fluorography. Using phosphor screen technology, one can label differently the proteome in two different conditions by growing cells on two isotope media, such as 3 H and 35 S [18], or 3 H and 14 C [19], and compare the relative values of each signal.…”

“…Similarly, the difference gel electrophoresis (DIGE [20]) is based on the differential incorporation of two fluorescent cyanine dyes. Various software packages have been developed for protein quantification [21], and have been used to study a large range of biological processes in various species, such as physiological changes due to environmental stress [15][16][17]22] or to development [12,14]. In genetics, proteins have been used to reveal variability between species [23] or genotypes [12][13][24][25][26].…”