Hydrogen peroxide-induced carbonylation of key metabolic enzymes in Saccharomyces cerevisiae: the involvement of the oxidative stress response regulators Yap1 and Skn7

H(2)O(2) induces a specific protein oxidation in yeast cells, and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (Tdh) is a major target. Using a 2D-gel system to study protein carbonylation, it is shown in this work that both Tdh2p and Tdh3p isozymes were oxidized during exposure to H(2)O(2). In addition, we identified two other proteins carbonylated and inactivated: Cu,Zn-superoxide dismutase and phosphoglycerate mutase. The oxidative inactivation of Cu,Zn-superoxide dismutase decreases the antioxidant capacity of yeast cells and probably contributes to H(2)O(2)-induced cell death. Cyclophilin 1 was also carbonylated, but CPH1 gene disruption did not affect peroxide stress sensitivity. The correlation between H(2)O(2) sensitivity and the accumulation of oxidized proteins was evaluated by assaying protein carbonyls in mutants deficient in the stress response regulators Yap1p and Skn7p. The results show that the high sensitivity of yap1delta and skn7delta mutants to H(2)O(2) was correlated with an increased induction of protein carbonylation. In wild-type cells, the acquisition of stress resistance by pre-exposure to a sublethal H(2)O(2) stress was associated with a lower accumulation of oxidized proteins. However, pre-exposure of yap1delta and skn7delta cells to 0.4 mM H(2)O(2) decreased protein carbonylation induced by 1.5 mM H(2)O(2), indicating that the adaptive mechanism involved in the protection of proteins from carbonylation is Yap1p- and Skn7p-independent.     

The Skn7 response regulator of Saccharomyces cerevisiae interacts with Hsf1 in vivo and is required for the induction of heat shock genes by oxidative stress

The Skn7 response regulator has previously been shown to play a role in the induction of stress-responsive genes in yeast, e.g., in the induction of the thioredoxin gene in response to hydrogen peroxide. The yeast Heat Shock Factor, Hsf1, is central to the induction of another set of stress-inducible genes, namely the heat shock genes. These two regulatory trans-activators, Hsf1 and Skn7, share certain structural homologies, particularly in their DNA-binding domains and the presence of adjacent regions of coiled-coil structure, which are known to mediate protein-protein interactions. Here, we provide evidence that Hsf1 and Skn7 interact in vitro and in vivo and we show that Skn7 can bind to the same regulatory sequences as Hsf1, namely heat shock elements. Furthermore, we demonstrate that a strain deleted for the SKN7 gene and containing a temperature-sensitive mutation in Hsf1 is hypersensitive to oxidative stress. Our data suggest that Skn7 and Hsf1 cooperate to achieve maximal induction of heat shock genes in response specifically to oxidative stress. We further show that, like Hsf1, Skn7 can interact with itself and is localized to the nucleus under normal growth conditions as well as during oxidative stress.     

A proteome analysis of the cadmium response in Saccharomyces cerevisiae

Cadmium is very toxic at low concentrations, but the basis for its toxicity is not clearly understood. We analyzed the proteomic response of yeast cells to acute cadmium stress and identified 54 induced and 43 repressed proteins. A striking result is the strong induction of 9 enzymes of the sulfur amino acid biosynthetic pathway. Accordingly, we observed that glutathione synthesis is strongly increased in response to cadmium treatment. Several proteins with antioxidant properties were also induced. The induction of nine proteins is dependent upon the transactivator Yap1p, consistent with the cadmium hypersensitive phenotype of the YAP1-disrupted strain. Most of these proteins are also overexpressed in a strain overexpressing Yap1p, a result that correlates with the cadmium hyper-resistant phenotype of this strain. Two of these Yap1p-dependent proteins, thioredoxin and thioredoxin reductase, play an important role in cadmium tolerance because strains lacking the corresponding genes are hypersensitive to this metal. Altogether, our data indicate that the two cellular thiol redox systems, glutathione and thioredoxin, are essential for cellular defense against cadmium.