Superoxide Dismutase 1-mediated Production of Ethanol- and DNA-derived Radicals in Yeasts Challenged with Hydrogen Peroxide

Abstract

Peroxiredoxins are receiving increasing attention as defenders against oxidative damage and sensors of hydrogen peroxide-mediated signaling events. In the yeast Saccharomyces cerevisiae, deletion of one or more isoforms of the peroxiredoxins is not lethal but compromises genome stability by mechanisms that remain under scrutiny. Here, we show that cytosolic peroxiredoxin-null cells (tsa1⌬tsa2⌬) are more resistant to hydrogen peroxide than wildtype (WT) cells and consume it faster under fermentative conditions. Also, tsa1⌬tsa2⌬ cells produced higher yields of the 1-hydroxyethyl radical from oxidation of the glucose metabolite ethanol, as proved by spin-trapping experiments. A major role for Fenton chemistry in radical formation was excluded by comparing WT and tsa1⌬tsa2⌬ cells with respect to their levels of total and chelatable metal ions and of radical produced in the presence of chelators. The main route for 1-hydroxyethyl radical formation was ascribed to the peroxidase activity of Cu,Znsuperoxide dismutase (Sod1), whose expression and activity increased ϳ5-and 2-fold, respectively, in tsa1⌬tsa2⌬ compared with WT cells. Accordingly, overexpression of human Sod1 in WT yeasts led to increased 1-hydroxyethyl radical production. Relevantly, tsa1⌬tsa2⌬ cells challenged with hydrogen peroxide contained higher levels of DNA-derived radicals and adducts as monitored by immuno-spin trapping and incorporation of 14 C from glucose into DNA, respectively. The results indicate that part of hydrogen peroxide consumption by tsa1⌬tsa2⌬ cells is mediated by induced Sod1, which oxidizes ethanol to the 1-hydroxyethyl radical, which, in turn, leads to increased DNA damage. Overall, our studies provide a pathway to account for the hypermutability of peroxiredoxin-null strains.Living organisms are constantly exposed to oxygen-and nitrogen-derived reactive species that are produced by normal metabolic activity and in response to external stimuli. To protect themselves against the toxicity of these species, aerobic organisms have evolved a range of defense mechanisms (1). Among these, a family of cysteine-based peroxidases, currently named peroxiredoxins, has attracted considerable attention because of its ubiquity and versatility. These enzymes have been shown to detoxify hydrogen peroxide, organic peroxides and peroxynitrite through oxidation of their reactive cysteine residues, which are recycled back by reducing equivalents provided by thioredoxin and other thiol-electron donors (reviewed in Refs. 2-5). In addition to detoxifying peroxides, specific peroxiredoxins have been shown to act as molecular chaperones (6, 7) and to play roles in regulating hydrogen peroxide-mediated cell signaling events (3,8,9).Many organisms have multiple peroxiredoxins. Six peroxiredoxins have been identified in human cells, and five in the yeast Saccharomyces cerevisiae. Peroxiredoxin isoforms are distributed to different locations within the cell, and two of the yeast peroxiredoxins, Tsa1 and Tsa2, are cytosolic (10). Tsa1 was the first peroxiredox...

Loss of the Thioredoxin Reductase Trr1 Suppresses the Genomic Instability of Peroxiredoxin tsa1 Mutants

“…The endogenous concentrations of H 2 O 2 and alkyl hydroperoxides in wild-type and tsa1 Δ strains have so far not been precisely determined, because available ROS sensors were not selective or sensitive enough [19]. However, several laboratories have detected a significant increased intracellular ROS levels in tsa1 Δ strains [16], [20]–[23]. The conversion of this excess of endogenous H 2 O 2 to hydroxyl radical through the Fenton reaction, believed to occur at or near DNA, could induce a variety of types of DNA damage [24].…”

“…The endogenous concentrations of H 2 O 2 and alkyl hydroperoxides in wild-type and tsa1 Δ strains have so far not been precisely determined because of the lack of the selective, quantitative and sensitive ROS probes [19]. Nevertheless, several laboratories report an increased intracellular ROS in tsa1 Δ strains [16], [20]–[23]. Importantly, Can R mutation rate of a tsa1 Δ strain decreases substantially under anaerobiosis, further suggesting that the increase of intracellular H 2 O 2 concentration in tsa1 Δ cells is the primary cause of the genome instability of these cells under aerobiosis.…”

Metabolic and Environmental Conditions Determine Nuclear Genomic Instability in Budding Yeast Lacking Mitochondrial DNA

“…High-energy radicals could also stem from reaction between metabolic byproducts, driven by an enzyme; in peroxiredoxin tsa mutants, the reaction of ethanol with hydrogen peroxide leads to high-energy radicals (1-ethoxyhydroxyl). This reaction is catalyzed by the peroxidase activity of superoxide dismutase Sod1, when this cytoplasmic enzyme is mislocalized to the nucleus (Ogusucu et al 2009). In a similar way, Sod2, a nuclear-encoded mitochondrial superoxide dismutase, could potentially drive such a reaction; mislocalized Sod2 (misimported in the mitochondria because of low ΔΨ in rho0 cells) could induce nuclear DNA damage in the presence of H 2 O 2 and ethanol.…”

Loss of Yeast Peroxiredoxin Tsa1p Induces Genome Instability through Activation of the DNA Damage Checkpoint and Elevation of dNTP Levels

“…First, tsa1 Δ cells produce significantly more ROS [4], which cause DNA and protein damage [27],[42],[43]. Second, loss of TSA1 results in increased formation of Rad52-YFP foci, an indicator of DNA DSBs [23].…”

“…Although both an aberrantly high level of ROS and an imbalance in free radical contents, which is caused by compensational activation of other antioxidants such as Sod1p [27], could underlie the mutator phenotype of tsa1 Δ cells, additional events subsequent to the disruption of TSA1 might also be influential in the induction of genome instability. In this study we asked whether perturbation of dNTP pools might contribute to the mutator phenotype observed in tsa1 Δ cells.…”