Genetic Analysis of Glutathione Peroxidase in Oxidative Stress Response of Saccharomyces cerevisiae

Abstract

Three glutathione peroxidase homologs (YKL026C, YBR244W, and YIR037W/HYR1) were found in the Saccharomyces Genome Database. We named them GPX1, GPX2, and GPX3, respectively, and we investigated the function of each gene product. The gpx3Δ mutant was hypersensitive to peroxides, whereas null mutants of the GPX1 and GPX2 did not show any obvious phenotypes. Glutathione peroxidase activity decreased approximately 57 and 93% in the gpx3Δ and gpx1Δ/gpx2Δ/gpx3Δ mutants, respectively, compared with that of wild type. Expression of the GPX3 gene was not induced by any stresses tested, whereas that of the GPX1 gene was induced by glucose starvation. The GPX2 gene expression was induced by oxidative stress, which was dependent upon the Yap1p. The TSA1 (thiol-specific antioxidant) gene encodes thioredoxin peroxidase that can reduce peroxides by using thioredoxin as a reducing power. Disruption of the TSA1 gene enhanced the basal expression level of the Yap1p target genes such as GSH1, GLR1, and GPX2 and that resulted in increases of total glutathione level and activities of glutathione reductase and glutathione peroxidase. However, expression of the TSA1 gene did not increase in the gpx1Δ/gpx2Δ/gpx3Δ mutant. Therefore, de novo synthesis and recycling of glutathione were increased in the tsa1Δ mutant to maintain the catalytic cycle of glutathione peroxidase reaction efficiently as a backup system for thioredoxin peroxidase. Three glutathione peroxidase homologs (YKL026C, YBR244W, and YIR037W/HYR1) were found in the Saccharomyces Genome Database. We named them GPX1, GPX2, and GPX3, respectively, and we investigated the function of each gene product. The gpx3Δ mutant was hypersensitive to peroxides, whereas null mutants of the GPX1 and GPX2 did not show any obvious phenotypes. Glutathione peroxidase activity decreased approximately 57 and 93% in the gpx3Δ and gpx1Δ/gpx2Δ/gpx3Δ mutants, respectively, compared with that of wild type. Expression of the GPX3 gene was not induced by any stresses tested, whereas that of the GPX1 gene was induced by glucose starvation. The GPX2 gene expression was induced by oxidative stress, which was dependent upon the Yap1p. The TSA1 (thiol-specific antioxidant) gene encodes thioredoxin peroxidase that can reduce peroxides by using thioredoxin as a reducing power. Disruption of the TSA1 gene enhanced the basal expression level of the Yap1p target genes such as GSH1, GLR1, and GPX2 and that resulted in increases of total glutathione level and activities of glutathione reductase and glutathione peroxidase. However, expression of the TSA1 gene did not increase in the gpx1Δ/gpx2Δ/gpx3Δ mutant. Therefore, de novo synthesis and recycling of glutathione were increased in the tsa1Δ mutant to maintain the catalytic cycle of glutathione peroxidase reaction efficiently as a backup system for thioredoxin peroxidase. glutathione peroxidase thioredoxin peroxidase lipid hydroperoxide tert-butyl hydroperoxide Yap1p response element base pairs polymerase chain reaction stress response element heat shock protein All aerobic organisms use molecular oxygen for respiration or oxidation of nutrients to acquire the energy efficiently. Molecular oxygen is reduced to H2O through acceptance of four electrons. During the reduction of molecular oxygen, several reactive oxygen species are formed, i.e. acceptance of one, two, and three electrons to form, respectively, superoxide anion radical (O⨪2), hydrogen peroxide (H2O2), and hydroxyl radical (HO⋅). These reactive oxygen species attack almost all cell components, DNA, protein, and lipid membrane, and they sometimes cause lethal damage to the cells. Among the reactive oxygen species, HO⋅ as well as perhydroxyl radical (HOO⋅) can extract bis-allylic hydrogen atom of unsaturated fatty acid (LH) to form lipid alkyl radical (L⋅) (1Aikens J. Dix T.A. J. Biol. Chem. 1991; 266: 15091-15098Abstract Full Text PDF PubMed Google Scholar). The L⋅ is oxidized by molecular oxygen to generate a lipid peroxy radical (LOO⋅), and the LOO⋅ thus formed reacts with LH to give lipid hydroperoxide (LOOH) and L⋅. A radical chain reaction is then propagated. LOOH also belongs to the reactive oxygen species, and the occurrence of the LOOHs in biological membranes may be one of the major oxidative damages to the cells. Because reactive oxygen species are commonplace in aerobic organisms, they have enzymatic as well as non-enzymatic defense systems. For example, superoxide dismutase catalyzes disproportions of O⨪2to O2 and H2O2, and H2O2 thus formed is decomposed to H2O and O2 by catalase. H2O2 as well as LOOH are reduced to H2O and corresponding alcohol by glutathione peroxidase (GPx).1 Ascorbate can also work as a reductant for ascorbate peroxidase in plants (2Asada K. Scandalios J.G. Oxidative Stress and the Molecular Biology of Antioxidant Defenses. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1997: 715-735Google Scholar). β-Carotene and tocopherol function as radical scavengers. Glut